Introduction

COVID-19 pandemic spread in almost all, all over the globe countries .Millions have been affected and millions died. The situation necessitate international efforts of medical internists epidemiologists, immunologist, and vaccine technologist .Four main preventive vaccine versions were becoming in hand for human mass vaccination within one year after the pandemic spread. So far concerning immunology of the disease. Scientists early in the pandemic starts to assess humoral immunity suitable for sero-diagnosis and sero-therapy of the disease as well as tempting and still tempting to developing the COVID-19 valid preventive vaccines. Currently, cell and lymphocyte immunologists take the forefront position in COVID-19 research. They articulate cellular immunology, flow cytometery, single cell RNA sequencing, mass transcriptomics, proteomics and cell-cell inter- actomics for tracing lymphocyte immunotypes and the allied cell type interactions .As well as their roles in; the pathogenesis, immune- pathogenesis, and disease outcome prediction.

Aiming

The objective of the present special issue “IMMUNE CELLS  IN COVID-19” published in IJPC was focusing onto the; ontogeny, biology, molecular biology, immuno-typing of immune cells and their roles in covuid-19.In which the latest information were collected, analyzed and formatted as series of opinion papers.

Scope

Lymphoid, lympho-myeloid and myeloid cell lineages for lymphocytes, monocytes and granulocytes as well as their mature forms are forming the backbone of human and mammalian immune system. The function of the immune system is mainly to recognize and destroy foreign invaders via a tripartite responses as; innate, immune cross-roads and adaptive immune responses. Immune cells the subject of the present special issue were assigned to these three immune system arms.

List of Contents

Section One: Innate Immunity

1. Immunology of mononuclear phagocytes system in pulmonary patho-type of COVID-19.*

Section Two: Immune Cross-roads

1. The interplay of NKT cells in severe sars-cov-2 human infections*
2. TH17 cells and the intercellular functions in severe, critical, deceased and vaccine of COVID-19*

Section Three: Adaptive Immunity

1. B Cell immunology of COVID-19*.
2. Regulatory Lymphocytes in COVID-19*.
3. 6-MAIT functions in homeostasis,covid-19 infected an covid-19

*The authorship;

IBRAHIM MS SHNAWA, College of Biotechnology, University of Qasim and Hilla University, College Babylon, Iraq.

Abstract

Mononuclear Phagocyte System MPS has an essential role in all stages of human SARS-COV-2 pulmonary infections. The objective of the present opinion paper was to through a light on the forefront achievements on the immunology of MPS in COVID-19. Single cell mRNA sequencing, single nucleus RNA sequencing, PCR, transcriptomics, flow cytometery, histo and gross pathology were the main assays tempted for assessments through an in-vitro, ex-vivo and in-vivo experimental settings. Monocyte, macrophage, alveolar macrophage, dendritic cells via an increment or decrement shift in number or function could probe the disease severity. MPS cells are either primer infected and lead to serial cellular events ended with severity. Or the epithelial cells found in the micro-environmental continuum with the APS were infected leading to MPS cell infection followed by cell-cell cross-talks, positive loop feedback mechanisms with T lymphocyte and/or MPS cells interferon axis functions. The overall immune response patterns of the lung in severe COVID-19 were; hyper-inflammation, immune impairment, hypoxia and severity terminated with death if not managed at earliest. An immune six point severity index was proposed as a diagnostic battery to be of use in an advance immunology laboratory was suggested. Molecular immune concept of circuit was briefed. MPS immune functions in pulmonary COVID-19 hold the position of double sward beneficial in some functional aspects and deleterious in others.

Keywords

Alveolar, Asymptomatic, Cytometery, Dendritic cell, Flow, Infection, Macrophages, Monocyte

Introduction

Mononuclear phagocyte system MPS take part in the functions of the human immune system both in health and disease. As a system is composed of circulating monocyte and tissue resident forms, the tissue resident forms got different names in different tissue microenvironment as; Glial in brain, alveolar in lungs, Kupffer in liver, osteoclast in bone, dendritic in spleen and other lymph glands and blood stream and Langerhans in skin. Some of which undergoes phase transition as that of glial cells in central nervous system. MPS performed immune functions both in the natural (innate) and adaptive immune responses. In other word they take part in immune cross-roads functions. In general MPS interplay immune functions in viral diseases and have special immune potentials in COVID-19. In health, MPS performed; phagocytosis, antigen presentation, shaping the adaptive immune responses, production of cytokines  and chemokines. While in disease state MPS played a role in the infectious inflammatory processes and in immune tissue  injuries due to an excessive cytokine production insitue in the affected tissue microenvironment [1-3]. In the present opinion tempts were made  to review the immunology of mononuclear phagocyte cell system in pulmonary COVID-19.

Cellular Immunology of MPS

MPS cells originated from the pluripotent stem cells in bone marrow in human adults. From the stem cells, lympho-myeloid progenitor cell line was developed which then differentiated to pro- monoblast, mono-blast, pro-monocyte then to monocyte in blood stream. From blood stream migrate to tissue compartments. During such migration they undergo morphologic and functional changes that fits to the target tissue compartment or organ while migration within blood vessels would not accompanied by morphologic, functional and/or mitogenic changes.

MPS cells are of large sizes and have multiple secondary lysozymes. They are characterized  by  active  endoplasmic reticulum and active Golgi apparatus which means  have  both  active biosynthetic pathways and active secretions with an evident acclimatization to their microenvironment. In lungs MPS express oxidative metabolism, their half-life were ranged between 60 to90 days in various organ/systems. During the inflammatory stimuli, therein  there  are  increments  in  the  in  their  development   in bone marrow and disseminated to various tissues through blood stream. On surface of MPCS cells there are numbers of markers   like:  MHCI,  MHCII,  C1,  C2,  FC,  CD11,  CD18,  CD13,   CD16,

CD17, CD31, and IA. The biophysical characteristics of MPS are; stimulated by bacterial lipo-polysachaaride LPS and mitogenic lectins, adhere to glass, recognize the antigens by TLR receptor. They secrete; hydrolytic enzymes, enzyme  inhibitors,  cytokines,  fat derived factors, complement components, and mirobicidal materials. When activated lymphocyte produce both of; macrophage inhibitory factor MIF it affects inhibition in spread of macrophages and   macrophage   stimulating   factors   which   stimulate   them for pinocytosis, phagocytosis, and induce the appearance of immune associated antigen Ia and assist in antigen presentation. MPS own specialized mechanisms for recognition of different inflammatory stimuli. If the stimulus is microbe, they will evolve number of killing mechanisms. They evolve three highly efficient recognition and clearing mechanisms of immune complexes through phagocytosis. The presence of MPS in continuum of  inflammatory  responses  they indicated either sub-acute or chronic inflammatory state. MPS cells act as; second line defender of human and mammalian body, antigen processing, antigen presentation, cytokine production and recognition of inflammatory responses [1-3].

Cell Molecular Immunology

There are varieties of natural immune protein that have the ability to recognize and detect human infection. These proteins are either soluble or structural entities like; soluble lysosome, complement components or complement receptors. The recognition process is through pattern recognition on the surface of the microbes. Among these receptors are the Toll-like receptor, the TLRs which are proteins of collectin nature that have the ability to recognize certain molecular patterns on the microbial surface. Such recognition molecules are forming strong features of natural (innate) immunity. TLRs are considered as a part of normal immune physiology. In the structural sense TLRs are forming a family of trans-membrane proteins that belongs to a class of animal lectins known as collectin proteins. TLR family is composed of more than ten different receptors. Most of the human and mammalian body tissues express at least one type of TLR. Though all TLRs are expressed onto; macrophages, dendritic cells, mast cells and B cells. TLRs interplayed an array of immune functions like; microbial sensors, cell signaling activators, enhancers for the expression of both; inflammatory and immune response genes, cross- linking of pattern-recognition molecules on the surface of microbe with TLRs act as danger signal to increase the microbicidal activity of phagocytic macrophages and allow them to activate T cells. TLRs made an important link between innate and adaptive immune responses. On TLRs activation, macrophage co-stimulatory molecules will converts macrophage phagocytes into antigen presenting macrophages which able to activate T cells [4-7].

Immunology of MPS in Human Virus Infections

The invading mammalian and human viruses when gain foot- hold in their respective hosts. They are recognized via their surface pattern recognition molecules PRM by the surface TLRs of MPS in blood stream and tissue resident. PRM cross-linked TLRs then virus pinocytosed, or through macro-pinocytosis and/or receptor mediated endocytosis in to the cell interior, Nikitina et al. [2018] the cross-link lead to transform of pinocytosed cell into antigen presenting cells. The processed virus peptides conjugated with MHC molecule and migrate out on the surface of MPS. The antigen presenting MPS will either activate naïve T cell to be Th2 triggering naïve b lymphocytes to grow, proliferate and expand as an effector antibody producing and memory B cells. Or activate naïve T cells to Th1 cells triggering T cells to be effector CD8+ cytotoxic T cells, CD4 T cells and memory T cells. The burden of virus load can be eradicated by the action of cytotoxic T cells or neutralized by the antiviral antibody and/or cleared by the direct action of interferons. The presence of molecular mimicking viral epitope with host tissue cells may initiate through the action of autoantibodies or auto-reactive cells immune tissue injuries terminated by autoimmune diseases. Excessive cytokine production by T cells or MPS also lead to inflammatory and immune responses with consequences of immune tissue injuries. The possible occurrence of viral immunosuppressive epitopes will trigger a state of infectious immunosuppressive conditions [6-9].

Immunology of MPS in SARS-COV-2 Infections

The circulating and tissue resident MPS cells, the monocyte and tissue macrophages participate in all stages of SARS-COV-2 human infection. They contribute to: (i) innate immune reactions (ii) shaping adaptive immune reactions, (iii) comorbidity predisposing to clinical infections, (iv) virus resistance, (v) virus dissemination, (vi) the host factors that determine disease severity, (vii) induction of immune tissue injury, (viii) recovery and (ix) sequalae (Table 1) [10-13].

Table 1: Immune cell deviations in human COVID-19 lungs

Immuno-Inflammatory Responses

Severe SARS-COV-2 infection induce haemo-phagocytic syndrome due to the infiltration of pro-inflammatory monocytes, a rare condition expressed as an over excerbant inflammatory response due to development of hyper-cytokinemia together with depletion of the adaptive immune compartment which may explain the appearance of sepsis in many severe COVID-19 cases Gomez-Rail [23]. Macrophage activation syndrome MAS is a condition of systemic hyper- inflammation and often be noted in infection and malfunctioning. It is typified by marked up-regulated expression of pro-inflammatory cytokines. This sort of strong inflammation results in severe tissue injury. Macrophage within MAS state produce high amount of pro- inflammatory cytokine upon stimulation.  Inflammation  is  known to destruct the balance between coagulation and fibrionolysis [14]. The inflammatory cytokines TNFalpha and IL1 instruct macrophage and monocytes to produce tissue factor TF. TF activate coagulation while IL1 anIL6 increase the production of plasmalogin activation inhibitor. Hence, overproduction of inflammatory cytokines along with MAS also promotes intravascular coagulation Otsuka and Senio [15]. Dys-regulated inflammatory syndrome DIS. DIS is generated by mononuclear phagocytes (a rich source of pro-inflammatory cytokines) upon encounter of the virus within the tissue continuum via two stage activation mechanisms which is not specific to the initiating virus. This is relevant to the case of SARS-COV-2 virus infection were age and predisposing comorbidities enhances the risk of severe outcome due to DIS [11].

Human severe SARS-COV-2 pulmonary infection leads to inflammation and tissue destruction with a consequence of an immune mediated fibrosis which remains even in to convalescent phase. In a group of severe pulmonary infected patients with COVID-19. IA aided CT scan were used to score fibrosis via fibrosis index IF. Twelve patients with severe COVID-19 were investigated for IF, they were sub-grouped into IFlo and IF hi. Mononuclear cell were collected from those patients and investigated by single cell RNA sequencing, IF hi group have shown low mononuclear phagocytic cell, low IFN gene profiling as compared to that of IFlo subgroup   of patients. Mononuclear phagocyte could probe the prognosis of immune mediated ling fibrosis [16-21].

Monocyte Responses

In an in-vivo study setting SARS-COV-2 infection sensed by monocyte and macrophages, such sensation forms the inflammosomes that activate caspase I and gasdermin D leading to inflammatory cell death, the pyroptosis and release of potent inflammatory mediators. About 6% of blood monocyte of COVID-19 patients are infected with SARS-COV-2 virus. This virus infection of monocyte depends on the uptake of antibody-opsoinzed virus by FC gamma receptors. The internalized virus begins to replicate within the infected monocyte but infection is aborted and the infectious virus was not detected in the culture supernatant of the infected monocyte. Instead the infected monocyte undergoes pyroptosis mediated by activation of NLRP3 and AIM2 inflammosomes, caspase I and gasdermin D. In same culture settings, the addition of the COVID-19 vaccinee plasma does not promote AB dependent monocyte infection. Moreover tissue resident macrophages but not the infected epithelium and endothelium from lung autopsies from the deceased patients with COVID-19 have activated inflammosomes. The overall of findings suggest that ab-dependent SARS-COV-2 uptake by monocytes and macrophages triggers inflammatory cell death that abort the products of the infectious virus but cause systemic inflammation [14]. Though there was a report discounts the possibility of infection of both lymphocyte and monocytes [22,23].

Among the manifestation of SARS-COV-2 infection in man is the high systemic inflammation and immune dys-regulation. To obtains a mechanistic insight. An ex-vivo cell culture setting in which epithelial cells were co-cultured with monocytes and B cells. Epithelial cells were infected with SARS-COV-2 virus during the incubation period infected epithelial cells interacted with monocyte and B lymphocyte. Strong responses were induced both in monocyte and B cells with SARS-COV-2 inflammatory gene clusters which reproduce immune cell deviation. Similar to that deviation noted in the blood and lung myeloid cells from COVID-19 patients. Earliest infection of epithelial cells with SARS-COV-2 virus triggers inflammatory malformation of COVID-19 patients leading to raise of virus specific monocyte inflammatory phenotypes Leon et al. [24].

In a series of moderate COVID-19 patients, peripheral blood monocyte were investigated, the infection triggers inflammatory responses that stimulate an interferon stimulated gene driven phenotypes, cellular dysfunction epitomized by loss of HLADR receptor expression and induction of alarmin expression is documented in their features in severe cases. Pulmonary macrophages in COVID-19 were derived from infiltrating inflammatory monocytes are in a hyperactivated state resulting in determintal loop of pro- inflammatory cytokine release and recruitment of cytotoxic effector cells, thereby, exacerbating tissue damage in the site of infection [25].

Alveolar Macrophage Responses

In an experimental setting, two deceased severe COVID-19 patients were subjected within few hours to an anatomical and pathological study. Mucous plugs were found in all respiratory tracts, terminal bronchioles and pulmonary alveoli. Autopsy samples were processed and tissue samples were collected, sectioned and stained then examined. Real time PCR was performed to detect SARS- COV-2 Viral RNA. Flow cytometeric analysis was done to detect the direct binding of S protein and expression ofACE2 receptors on the macrophage surface. It was evident an extensive impairment of type I alveolar epithelial cells and atypical hyperplasia in type II alveolar epithelium with formation of halyn membrane, focal hemorrhage, exudation, pulmonary edema and consolidation. The mucous plug with fibrous exudates in alveoli together with alveolar macrophage dysfunction was the characteristic abnormalities. The SARS-COV-2 infection was detected in; alveolar epithelium, alveolar macrophages and hilum associated lymphoid tissue. SARS-COV-2 spike proteins interact with and bind ACE2 receptors. Infection of alveolar macrophages might derives cytokine storm [21].

SARS-COV-2 alveolitis were mapped in a human clinical setting in which broncho-alveolar lavage fluid samples were collected from within 48 of intubation from 86 severe COVID patients needing ventilation. In the  majority  of  these  patients  the  alveolar  space  is persistently enriched with alveolar macrophages and T cells without neutrophils. Single cell RNA sequencing was done to five  of the broncho-alveolar lavage fluids. Besides bulk and single cell transcriptomic profiling were suggesting that SARS-COV-2 infect alveolar macrophages, the infected alveolar macrophages in turn respond by recruiting T cells. These T cells release interferon gamma that triggers alveolar macrophages to secrete inflammatory cytokines and further promte T cell recruitments. Findings suggested that SARS- COV-2 causes slowly infecting, specifically-limited alveolitis in which aveolar macrophages incubating virus transcripts and T cells form positive feedback loop that derives progressive alveolar inflammation. Thus, SARS-COV-2 infected alveolar macrophages forms positive feedback loop with T cells in severe COVID-19 disease [20].

Tempts were made to investigate host responses at the level of lung tissue using single nucleus sequencing of 116000 nuclei from lungs of COVID-19 deceased in individuals and underwent rapid autopsies along with seven control individuals. Integrated analysis identified alterations in cellular compositions, transcriptional cell state and cell-cell interactions. The lungs from COVID patients were highly inflamed with dense infiltrates of aberrantly activated monocytes- derived macrophages and alveolar macrophages but had impaired T cell responses. Monocyte/macrophage derives interleukine 1B and epithelial cell derived IL6 were the unique features of COVID lung infection as compared to other viral pneumonias. Alveolar type II cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition to into alveolar type I cells resulting in imparted lung regeneration with expansion of pathologic fibroblasts accounting for the rapidly ensuing pulmonary fibrosis in COVID-19 [19,26].

Dendritic Cells Responses

In a study on a series of convalescent COVID-19 patients peripheral blood mononuclear phagocyte cell were investigated in an in-vitro settings. Early infectious events of SARS-COV-2 with MPS has shown; impaired type I interferon responses, elevated inflammatory cytokine and chemokine levels. The virus even in absence of productive replication in the plasmocytoid DC mediate vigrous TLR7/TLR8 dependent production of both interferon type I and III and inflammatory cytokine as well as chemokine known to contribute to a state of hyper-cytokinemia’ Cytokine Storm”. Which were released from these DC in an ACE2 independent but Neuropilin-1 dependent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PDL-1 marker, a feature of type I IFN producing cells. In comparison hospitalized COVID-19 patients displayed low frequency of circulating pDC with inflammatory phenotype. Early interaction of SARS-COV-2 and immune cells  occurring  invitro and proved ex-vivo indicate the role of pDC-interferon axis regulate antiviral state in asymptomatic and severe COVID-19 patients. Such findings may indicate crucial and protective role of pDC/IFN I axis in COVID-19 patients [18].

In an in-vivo experimental settings tackling moderate to severe COVID-19 patients. These patients were subjected to high dimensional flow cytometery focusing on MPS cells. It was evident that there were redistribution of monocyte subsets towards intermediate monocyte and general decrease in circulating DCs was observed in response to infection. Severe disease coincided with the appearance of monocyte-myeloid-derived cell suppressor-like cells and high frequency of pre- DC2. Such MPS cell phenotypic alteration and their precursors were cell lineage specific and associated with either the general response to infection of COVID-19 severity. This included an interferon- imprint DCs observed in all patients and a decreased expression of co-inhibitory molecules CD200R in pDcs, DC2 and DC3 subsets in the severely sick patients. Such findings stands as a prove for the MPS dys-regulation associated with severe COVID-19 patients [17].

DCs recognize viral infections and trigger innate as well as adaptive immune responses. COVID-19 severity is highly influenced by the host immune responses and modulation of DCs generation and functions. After the establishment of SARS-COV-2 infection, DCs could play an important role in the immunopathology of the disease. In a series of 65 COVID-19 patients covering mild, moderate to severe infection forms were subjected to analysis of DC circulating populations. Results of such analysis has shown long lasting reduction in DC subpopulation with an expression of functionally impaired – HLADR+ cells lacking DC markers. A higher CD163+ CD14+ cells among DCs subpopulations correlate with systemic inflammation. Depletion and functional impairment of DCs beyond the acute phase play a role in inflammatory responses of COVID-19 patients [16].

Circuit

In the molecular immune sense circuit means a communication form between two immune cells living with in one tissue continuum. In which one cell activated by an inducer, infection will produce mediator, cytokine that activate the other immune cell to produce other mediator, cytokine which in turn affect the first immune cell to produce other mediator. In severe COVID-19 pneumonia infection of alveolar macrophage lead them to produce T cell chemo-attractants. These T cells produce interferon gamma to induce inflammatory cytokine release from the alveolar macrophages and further promote T cell activation. Thus Alveolar macrophages containing the virus and T cells form a positive feedback loop that derives persistent alveolar inflammation [20,25]

Six Points Severity Index

Since studies performed on severe COVID-19 patients were from different nations and different geographical regions across the globe. As well as the sampling and techniques tempted were somewhat different. Thus to hypothetical suggestions for a COVID-19 severity index, one should hold four assumptions as; (i) The sense of severity is of relative homogeneity (ii) Different MPS cells have equal opportunity to face local or systemic viral loads (iii) The function of the MPS cell functions are of similar state and (iv) The invented index components are of equal weights in the evaluation consideration;

1. Low counts of DCs subpopulations in the peripheral blood stream, impaired function and loss of surface.
2. DCs-interferon axis function stand as an index of severity and viral persistence.
3. Low count of broncho-alveolar macrophages and poor transition of type II to type I epithelial cells parallels with severity.
4. Alveolar Macrophage-alveolar T cell are set on parallel with severity.
5. Epithelial prior infection pave the way for alveolar macrophage and B cell infectious activation.
6. Deceased COVID-19 patients associated with alveolar macrophage infection consequences, the pyroptosis and sepsis.

Conclusions

During clinical human SARS-COV-2 pulmonary infection forms, mononuclear phagocyte system cells and epithelial cells are prone to this virus infection. The infection of APS cells is of antibody dependent type. On APS cell infection virus replication cause molecular alterations in; cellular composition, cellular transcription state, and cell-cell interactions. Secretory protein exports in these infected cells are amplified leading to the production of pro-inflammatory and inflammatory cytokine. At the whole cellular levels infected APS cells undergoes immune deviations like; lack of cell surface markers, appearance of intermediate phenotypes, suppressor phenotypes, inhibitory marker bearing phenotypes as well as reduction in numbers in the peripheral blood stream. Reduction in numbers of APS cells  as well as alveolar epithelial cell phase transition impairments were implicated with immune mediated pathological pulmonary fibrosis. Alveolar epithelial cell infection may triggers alveolar macrophage infection and B lymphocytes. Alveolar macrophage infection initiates bilateral positive feedback mechanisms with T lymphocytes. DC cells undergo long lasting reduction in numbers, functional impairments and lack of surface markers. DC functions together with interferons in an axis form leading to regulation of antiviral state in asymptomatic and severe cases. APS cell infections initiate haemo-macrophage syndrome, macrophage activation syndrome, and intravascular coagulation, implicated in immune mediated pulmonary fibrosis, pyroptosis, and terminated by sepsis.

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Abstract

Natural Killer T lymphocytes [NKT cells] share the characteristics of innate and adaptive immune cells. Though their immune cell identity is still a matter of debate among lymphocyte immunologists. Currently, NKT cells are of three subsets as; NKT type I, NKT type II and NKT like cells. NKT type I is quite filling this cell definition. Three immune phenomena are evident concerning NKT interplay with viral infected human host as; immune evasion, immuno-pathogenesis, and immune protection. Immuno-pathogenesis and immune protection appeared to be operable in COVID-19 disease. The present opinion paper was aimed at briefing the role interplayed by NKT cells in severe SARS-COV-2 human infections. Host response showed reduced circulating total NKT cells, but some NKT immune subtypes may express an increment shifts in this severe infection form. The biology of NKT cells in severe SARS-COV-2 infections own an array of immune features as; immune-metabolic dysfunction, mitochondrial dysfunction, marked expression of apoptotic and inhibitory receptor genes and ramified in to six immune subtypes.

Keywords

Evasion, Immune, Inhibitory, Metabolism, Pathogenesis, Protection, Receptors and SARS-COV-2

Introduction

Human lymphocytes are of several immune-types as; B, T, NK and NKT. The efforts of scientists concerning the immunology of NKT cells appeared to be little as compared to other lymphocyte immune-types. NKT as an immune-type and definition is a matter  of debates among lymphocyte immunologists. Recently, evident published efforts concerning NKT cells. They share characteristics of both innate and adaptive immune functions. NKT can be immune- pathogenic and/or immune-protective depending  on  the  intensity of the tissue micro-environmental stimuli during human microbial infections. Some human pathogenic viruses displayed an immune mediated evasion mechanism against NKT cells both in function  and in count limiting. The present opinion was aimed at briefing the interplay of NKT in human severe SARS-COV-2 infection [1-5].

NKT Cell Biology

Ontogeny

Lymphoid cell progenitor migrates from bone marrow to thymus. Arrival of these progenitors to the thymic micro-environment insults these cells to undergo positive and negative selection procedures. These selection procedures mediated by variety of cytokines cell surface molecule, signal transducers, transcription factors and other regulatory factors. A key step in NKT maturation is their acquisition of innate effector function mediated by pro-myelocytic leukemia zinc finger PLZF. Another key step is the acquisition of cytokine secretion ability in which an adoption of constitutional expression of cytokine gene transcripts. Such cytokine gene transcripts need intra-thymic signaling through GM-CSF in order to become competent cytokine secretors. VDJ recombination via stochastic events leads to generation of invariant TCR. Acquisition of NKT phenotype appear to be driven by invariant TCR CD1d. Positive selection of NKT needs interaction of invariant TCR on immature NKT expressing both CD4 and CD8 with CD1d expressed on cortical thymocyte themselves. Both alpha galactosyl Cer NKT agnosts and DCs overexpressing CD1d played critical role in NKT thymic negative selection [4].

Identity

NKT cells are defined as lymphocyte expressing CD3+CD56+ surface receptors [6,7]. Lipid antigen reactive CD1d restricted  T cells most of which do not express CD56 [8]. Koay et al. [9] were formulating important NKT defining criteria as; (i) CD56+ T cells do not equate NKT cells, (ii) CD56+ T cells are heterogeneous T cells but not NKT cells and (iii) The use of CD56 T cells in predicating COVID-19 outcomes needs more validation. Khan and Khan [3] mentioned three subsets of NKT of which only NKTtype I with Koay et al. [9] defining criteria as in Table 1 as well as six subtypes, Table 2.

Table 1: NKT CELL subsets*

Table 2: NKT subtypes*

NKT Cell Molecular Biology

NKT cells  recognize  lipid  stimulating  antigens  through  CDR3 alpha and CDR3 loops. These complementary determining regions are the hyper-variable regions of TCR that complement an antigen shape. Crystallographic and biophysical analysis of alpha galactosylceramide (alpha GalCer) recognition by human CD-1d resistant TCR that utilize a V alpha 3-1-J alpha 18 re-arranged and displays more restored specificity of alpha linked glycolipid than iNKT TCRs. TCR alpha and CDR2 alpha loops have frequent divergence. This TCR employs convergence recognition strategy to engage CD-1d (Alpha Galcer) with binding affinity approximate 2um almost identical to that of an iNKT [10,11]. The hydrophilic groups of the lipoidal antigen contribute relatively little to CD-1d groove and the top of the alpha helcies are involved in lipid antigen presentation which suggest a conventional mode of presentation and recognition. NKT differentiation has unique management for their differentiation which is highly lympha-toxin dependent [12].

NKT Cellular Evasion

Pathogenic human viruses adopt number of strategies for evasion of both innate and adaptive arms of immune responses in human host. HIV weakens the immune system functions by depleting the numbers of CD4 T cells [13]. HIV-1 reduces the expression ofCD-1d molecules by increasing internalization and retains them in the trans- Golgi network. The down regulation in cell surface CD-1d is caused by interaction with in the continuum of intra-cytoplasmic tyrosine with HIV-1 NEF protein, leading to an early NKT depletion in HIV-1 infected individuals. West-Nile virus interferes with the interaction of DCs with NKT cells with net result of pro-inflammatory cytokine secretion [3].

NKT Cellular Immunobiology

T lymphocytes are of many subsets among which the NKT cells that have common surface markers and functional characteristics with both conventional T lymphocyte and natural killer cells. Major NKT cells express semi-invariant T cell receptor TCR that reacts with glycolipid antigens presented by major histocompatibility complex class I related protein CD-1d on the surface of antigen presenting cells APC. Both infectious and inflammatory conditions do activate NKT to be rapidly producing immune-modulatory cytokines. NKT may influence the function and the activation state of other immune cells [4]. The NKT semi-invariant alpha Beta TCRs recognize mammalian glycol-sphigo-lipid and microbial alpha glycuranylceramides found on the cell wall LPS of gram negative bacteria. This dipartite recognition of the auto and microbial ligands underlies innate-like antimicrobial functions mediated by CD40L induction, massive helper TH1, TH2 cytokines and release of chemokines. NKT and DCs performed a sort of reciprocal activation NKT can regulate a range of immunopathologic condition through unknown mechanisms. NKT legends holds a position between innate and adaptive immunity serving as a model system for structural biology of glycolipid trafficking and recognition [14]. NKT lymphocyte tissue distribution is unusual, they are found in large numbers in liver and lymph nodes but to a lesser extent La Jolla Institute of Immunology [15]. NKT served as an important regulator of the immune responses [16].

NKT Immune Functions

NKT cells are able to substantial cross-talk with other innate and adaptive immune cells. NKT when activated with alpha-GalCer, the activation will lead to rapid cytokine production of both Th1 and TH2 cytokines and chemokines though other NKT subsets when activated produce IL17 cytokine. The mode of cytokine activation of NKT cell starts rapid early in activation events, then late in the activation process the production ceased. NKT can mediate immune protection against a wide range of pathogens including bacteria viruses, fungi and parasites. In an experimental laboratory animal settings NKT mediate immune protection against low dose pathogenic challenge. Though in high dose challenge can induce hyper-cytokinemia terminated by sepsis [4].

NKT in Virus Disease

NKT Cells have anti-viral potentials against hepatitis B virus [17], herpes simplex virus-1, lymphocytic chorio-meningitis virus and influenza A virus [18]. They constitute an important arm of the innate immune responses against pathogenic viruses and can regulate adaptive immune responses through modulation of the antigen presenting cells. NKT exerts direct cytolytic effects and retards viral replication [3].

NKT in SARS-COV-2 Infections

A show case analysis of four series of severe SARS-COV-2 infection in different parts of the world is performed by four different research teams. The analysis covered number of patients, age/sex, clinical samples, and nature of the cellular investigations. At most single cell RNA sequencing, transcriptomic analysis and flow cytometery. The tracked immune cell types were NKT, NK, and T cells. As the SARS- COV-2 infection progressed NKT cells reduced with an apparent immune-metabolic dys-regulation, cellular dysfunction as well as mitochondrial dys-regulation (Table 3) [7,10,19,20]. Among these four studies Yang et al. [10] presented a novel detailed investigation on the role of NKT in SARS-COV-2 severe infection and be briefed in the following paragraph.

Table 3: NKT interplay in severe and mild sars-cov-2 human infections

In a clinical setting in to which COVID-19 series of patients and controls were subjected to single cell RNA sequencing in order to determine lymphocyte and mononuclear cell profiles. The number  of patients and subjects were; mild 24, moderate, severe 36, critical, died 13, mild recovered 79, severe recovered 50.  Fifty  patients were tested by single cell RNA sequencing. There was evident that decrease in percentages of lymphocyte in patients is associated with severity. The lymphocyte profiles in PBMC were found that CD8+ T, MAITs, gamma delta T cell, Mono DCs and pDCs decrease significantly as disease progress. While the percentages of plasma   B CD94+, monocytes and platelets increased significantly. The NKT cell percentages in severe COVID-19 decreased significantly as the disease progressed and in convalescent. TM3 expression in NKT cells of 202 COVID patients and controls were grouped into six cell subtypes. Increased Tm3 expression in NKT associated with NKT depletion in severe SARS-COV-2 infections [10]. In the followings  a deduction of features of NKT in COVID-19 from Yang et al. [10] study;

• High levels of expression of CD147CD26
• High expression of Tim3 promotes NKT depletion and dysfunction.
• Decrease in circulating NKT counts as the disease
• High expression of apoptotic and mitochondrial
• Tim3NKT has capacity to secret IFNgamma, IL4 and
• Expression of co-stimulatory inhibitory receptors PD-L, CTL4 and

Conclusions

Lymphocyte profile studies of SARS-COV-2 infection forms have shown variable picture of increase in one immune-type and decrease in others. Generally speaking, lymphogenesis increased as the disease progressed. T, NK, and NKT were affected to a variable degree. Total NKT count in circulation decreased as the disease progressed. Though there are some subtypes of NKT cells increased as the disease progressed.

References

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Abstract

TH17 cells displayed multiple immune functions in viral human infections including SARS-COV-2. The objective of the present opinion paper was to deduce the actual contribution of these cells in various infection phases of SARS-COV-2 in man. The deduction tempts to: (i) map the immune-typing of TH17 cells in: severe, critical, deceased and Vaccinee via show case analysis and (ii) suggest the pathogenic mechanism of TH17 cells in this disease. The show case analysis of five research papers  published  between  2020  and  2022  indicated  that:  TH17  cells  are  of  two  main  subsets,  the  nonpathogenic and the pathogenic was with, marked plasticity, pleomorphism and instability. On the onset of the clinical infection through hospital admission the patient peripheral blood has shown twice TH17 counts than in normal controls. In uncontrolled progressed COVID-19, the TH17 cell count drop in peripheral blood and enriched in lungs with marked elevation in counts and clonal expansion therein in severe critical and deceased cases. Critical cases on recovery showed TH17 cell counts restoration to normal. Peripheral blood Th17 cells inhibit Teg while in lung both of TH17 and T reg counts were elevated. TH17 cell recruit neutrophils during the infection progress and interacts with various subsets of macrophages and DCs with an outcome of hypercytokinemia and tissue pathology. Based on these facts, the opinion suggested: (i) TH17 as predictor of severity, critical and deceased as well as (ii) the possibleTH17 cells pathogenic mechanisms operable in cases of SARS-COV-2 human disease.

Keywords

Cell, Clonal, COVID-19, Expansion, Pathogenic, TH17

Introduction

TH17 cells are heterogeneous distinct lineage of CD4+ T cells. They are differentiated from naïve T cells through the action of cytokine micro-environmental stimuli. TH17 are basically of two subsets the nonpathogenic and the pathogenic [1]. These helper cells take part in extra-cellular bacterial infections, yeast infections, viral infections including SARS-COV-2 and auto-immune diseases. TH17 performed dual immune functions: the immune-pathogenic and to lesser extent the immune-protective [1-6]. The objective of the present opinion paper was to: (i) map the role of TH17 through the show case analysis of five immune-typing studies of TH17 cells and (ii) suggest the pathogenic mechanisms of these cells in COVID-19, during the period of 2020 till 2022.

Show Case Analysis Approaches

To assess the current holdings of the scientific workers in immunology of SARS-COV-2 infections in man, a sum of 150 current published works through the period of 2020 till 2022 were allocated. These efforts were analyzed so far concerning the CD4+ T cell subsets in COVID-19. Among which ten were concern the role of TH17 in this disease. Of the ten, one was proving TH17 role indirectly from cytokine profiles, five adopted flow cytometery, single cell mRNA sequencing and immunoinformatic approaches to the immune cells recovered from peripheral blood and broncho-alveolar leavage. The rest four review articles were already depending on flow cytometery proving that TH17 cells in association with severe COVID-19 disease and considered as supplementary to this work. The adopted five research works (Table 1) were the raw materials for the show case analysis to deduce the role of TH17 in various phases of human COVID-19 disease [1-12].

Table 1: The show case analysis test research articles

TH17 Cells

Basic TH17 Cell Biology

TH17 cells are distinct lineage of CD4+ T cells that differentiated from naïve T cells, secret the cytokines IL17 A and IL17f and express the lineage specific transcription factor RORC. Both of TH17 and TH17/Th1 clones showed selective expression of IL23R and CCR6 in addition to RORC. Th17 help B cells, express low cytotoxicity and low susceptibility to action of autologous T reg. and critical in clearance of extracellular microbial pathogens [14]. They are of two subsets pathogenic and nonpathogenic [1] (Table 2). These helper cells are pleomorphic, instable and exhibit a sort of plasticity. The TH17/TH1 subset can revert to Th1 cells. Hence some workers denote them as heterogenic helper cells [1,14].

Table 2: Th17 cell subset characteristics

TH17 Cell Differentiation

T helper lymphocytes featured by the expression of CD4+ T cells surface markers are the central cell subset of adaptive immunity. CD4 T cells can recognize proteins of microbial pathogens by their unique surface TCR. TCR can shape antigens and organize against them. Both of the TCR-antigen recognition and the signal of TCR engagement integrated stimuli initiate sort of transcriptional changes that guide naïve T cells towards a specialized function. These stimuli include cytokine, soluble mediators or bacterial products in the microenvironment. This differentiation process needs the regulatory interplay of specific intracellular signal transducer and activator STAT protein in the process. STAT eventually induces the dominant transcription factor TF. TF represent the master lineage specific factor. TF controls the transcriptional program of the cell covering: cytokine production and chemokine receptor expression that mediate trafficking to various organs: this network helps each T cell subsets to exert their specific functions in response to antigens available in the assigned tissue. The T cell TF is a T-Box protein in TH1, GATA b binding protein in TH2, and retinoic acid related orphan receptor gamma-t RORCg-t in TH17 and fork head box3 in T regs [16-19]. Any insult of what so ever nature to this differentiation mechanisms lead to dys-regulation mechanism in various steps of the T cell growth, maturation and response to challenge. Such dysregulation can contribute to pathological responses just as in case of immune mediated diseases. For TH1 and TH17 cells and allergenic responses for TH2. The fate decision of the naïve T cells is largely affected by the cytokine surrounding environment. The Th17 differentiation process encoded by the expression of two effector genes: IL17a and IL17f together with multiple player processes are involved in the different stages of differentiation. The STAT, RORC-g-t axis, RORA, Ahr IPF4 and BATF markers set the initial chromatin accessibility that allows the transcriptional programs. Among which the RO RCg-t is determinative for the expression of IL17a and IL17f genes.

Two different cytokine cocktails lead to two different TH17 subsets. TGFB and IL6 induce nonpathogenic TH17 cells characterized by the co-expression of IL10. While IL6 and IL23 but not TGFB lead  to differentiation of the pathogenic TH17 cells. Both of the subsets would express RORCg-t but the pathogenic subset of TH17 cells are more plastic, polymorphic and have tendency for transition towards TH1 cells. For any naïve T cell differentiation, the concentration and the gradient of TGFB is crucial, high concentration induces T regs associated genes. While restraining T bet and TH1 genes possibly inhibit TH17 pathogenic responses. There is a developmental overlap between TH17, Th1 and T regs. Such overlap might be caused by the complex cytokine dynamics [1,2,20-22].

TH17 Cell and Cellular Interactions

TH17 cells are known to inhibit T reg. responses in peripheral blood of COVID-19 patients [7]. They can induce the neutrophil  and epithelial responses provided by the presence of environmental microbial insults [2]. Within the continuum of COVID-19 pneumonic lungs, TH17 cells interact with pro-inflammatory, pro-fibrotic macrophages, DCs and pDCs [9].

TH17 Cell Immune Functions

TH17 cells performed dual immune function in immune- pathogenesis of viral infections and/or immune protection [6]. They are involved in neutrophil and epithelial cell immune response to extracellular microbes and the initiation of autoimmune diseases [2]. Th17 cell may interplay with the pathogenicity of allergy, asthma and human inflammatory diseases [14].

TH17 Cells in Viral Infections

TH17/IL17 hinders and limits viral infections via several mechanisms as: Enhancing  TH1  responses,  promoting  cytotoxic  T cell activity, modulating antiviral B cell responses and inducing protective inflammatory responses. They may limit the viral induced organ pathology, inhibiting inflammation and mediating protective immune responses. Th17 cells/IL17 cytokine may promote viral infection through different mechanisms as: Antagonizing antiviral TH1, T regs and CTL responses, enhancing survival of infected cells, promoting viral intracellular replication, take part in evolution of tissue pathology and fibrosis [6].

TH17 Cells in Various Phases of SARS-COV-2 Human Infection

The TH17 cells were confirmed both in peripheral blood and lung compartments of various phases of COVID-19, Tables 3-6. Early acute infection and on admission to hospital, TH17 cells were of twice count than that of asymptomatic and controls. T regs were reduced in count and function [1]. On progress under un-controlled conditions, severe state, TH17 counts were reduced in circulation. Both TH17 cells and T regs were increased with clonal expansion in lung compartment in severe COVID-19. In critical or deceased cases both TH17 and T regs were amplified in counts and expansion. But on recovery from severe or critical states TH17 and T reg counts were restored to normal. In line with Th17 count elevation these cases there were reduction in CD4+ T cells, Cd8+ T cells both in circulation and lung compartment. TH17 suppress the T regs and triggers neutrophils causing recruitment to the affected tissue compartment and interacts with each of pro-inflammatory, and pro-fibrotic macrophages, DCs, pDCs, and monocytes. Such intercellular interactions may terminated by an overt inflammatory cytokine production leading to a state of hyper-cytolinemia, the cytokine storm [9]. Th17 cell clone expansions in lung compartment were higher than that in circulation. Lung resident TH17 cell clones can be either merely resident or of mixed resident and migratory forms from circulation. Other T cell subsets were showing various degrees of clonal expansion [9].

Table 3: Circulatory TH17 cells in severe and vaccine of COVID-19 as evident in the three show case analyzed groups

Table 4: Circulatory and pulmonary Th17 cells severe, critical, deceased and vaccine of COVID-19 as evident in the two show case analyzed groups

Table 5: Circulatory Th17 cells severe, critical, critical deceased and vaccine COVID-19 as evident in the five show case analyzed groups

Table 6: Pulmonary existed TH17 cells in various forms of COVID-19 as evident in two show case analyzed groups in comparison to gut Th17 in Chron’s disease

TH17 Cell Suggested Pathogenic Mechanisms

Since TH17 cells expressed low cytotoxicity, though to be a pathogenic helper cell it should express other supportive means to make it able to perform its pathogenic influences. Hence, this opinion paper tempted to hypothesize theoretical suggested mechanisms operable in induction of immune tissue injuries in the lung compartments. They can be coined as follows:

• TH17 cells when interacts with pro-inflammatory macrophages and inflammatory macrophages, they will induce excessive inflammatory cytokines forming cytokine storm mediating tissue pathology consequences of COVID-19 [9].
• Th17 cells recruits neutrophils to lung compartment whereby the affected tissue niche, therein neutrophils produce excessive inflammatory cytokines and reactive O2 intermediates mediating immune tissue injury [9].
• On inhibition of T reg by TH17 cells, they lend the cellular microenvironment allowance of up regulation of auto-reactive T cells to initiate autoimmune pathologic tissue injury [7,10].
• The TH17 cell interaction with pro-fibrotic macrophages may initiate lung tissue fibrotic lesions, the known consequences of COVID-19 pneumonia [9].
• TH17/Th1 cells are known to be: plastic, pleomorphic and instable they my undergoes transition to TH1 cells producing IFNG cytokines and other inflammatory cytokines leading to hyper inflammation in lung paranechyma the sign of COVID-19 pneumonic lungs [15].

Conclusions

TH17 cells associated with the pathogenesis of COVID-19. Circulatory TH17 subset is distinct from lung tissue resident TH17 cell subset. The tissue resident TH17 cells are expanded as tissue specific subset, as mixed clones of migratory and tissue resident TH17 cell clones. On clinical onset of the disease they were of twice count level than controls and inhibit T regs. But in progression during uncontrolled affection, Th17 cell and T reg cells gaining higher counts and marked clonal expansion therein lung compartments as compared to peripheral blood both in severe and critical cases. Though they both reduced to variable degrees in circulation, on recovery of severe and critical cases TH17/T reg ratios restored to normal.

References

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Abstract

The objective of the present opinion paper was aimed to present an at glance review of B cell immunology in COVID-19. B cell system is important both in human health and disease. B cells in health interplayed a role in organization of lymphoid tissue and regulation of lympho-angiogenesis. The antigen presenting, antibody producing and cytokine producing subsets of B cells are crucial for the activities of human immune system both in health and disease. The memory B cell functions and  counts  are  acting  as  a  valid  probe  for  vaccine  efficacy,  vaccine  breakthrough  infections,  severe  infection  form and hypoxic severe infection form of COVID-19. B cell depletion serves as indicator for septic COVID-19 disease conditions.

Keywords

B cell, Breakthrough, COVID-19, Deletion, Hypoxia, Vaccine

Introduction

Bone marrow plays a niche for the leukocyte primordium and    a site where they differentiated. This primordium is the pluripotent stem cell that serves as the mono-chotomus origin of the immune cells. Stem cells are differentiated through the action cytokine into the tri-chotomus progenitor cell lines. The myeloid progenitor cell which forms the origin of granulocytes, the lympho-myeloid progenitor cell, the origin of mononuclear cell system and the lymphoid cell progenitor which constitute the origin of lymphocytes. Macrophages, T cells and B cells are forming the functional back bone of the immune system. B cell system displayed an array of immune functions in human health and disease. The present opinion paper concerned with the role played by B cell system in COVID-19 immunity [1].

B Cell Immunology

B cell can have two main subsets B1 and B2 [2]. Though, functionally they were subset as systemic and mucosal B cells. B cell may display; antibody production,  cytokine  production  [3]  and antigen presentation [4,5]. This story is ensemble under the umbrella of B cell system. B cell descends from the lymphoid cell progenitor of the pluri-potent haemo-poietic stem cell. In general lymphocytes were considered from the morphological point of view as more heterogeneous than mono and poly-nuclear leukocytes. The ratio of nucleus to cytoplasm is large. They are devoid from Golgi apparatus and from endoplasmic reticulum. Their mobility is rather slower than other leukocytes and has amoebic movement fashion.   B cells bear surface Ig that acts as a recognition molecules and IgFc fragment receptor. B cells are devoid from thymus antigens. They have transmembrane cluster of differentiation; CD19, CD20, CD22, CDR1, CDR2, CD5, CD35, BCR and B7 (Table 1) [6].

Table 1: Developmental B cell phases and their receptors

Maturation of B cell from the B lymphocyte progenitor may establish in bone marrow or it may migrate as an immature B lymphocyte to spleen or to other peripheral lymph nodes and maturate there. The onto-genic maturation steps starts as; progenitor the primordium, early Pro B, Bro B, pre B to immature B cell then to mature B cells. Mature B lymphocytes when stimulated by an antigen it well grow, differentiate and expand the transform to an effector plasma cells producing either antibodies  and/or  cytokines  IL10  and IL12. As well memory B cells, Memory B cells are elected from those cells that undergoes heavy chain translocation and V fragment amplifying mutation which lead to the production of high affinity cells producing Ig G, Ig A, IgE, their BCR have high affinity for antigen binding [7-9].

B Cell Molecular Immunology

The molecular genetic system of B cells contains an array of gene sets that encodes for the various immune functions like those encoding genes for mitotic cell cycle, Ig gene superfamily, cytokine production genes, antigen presenting genes and the apoptosis encoding genes. The ontogeny of the different cell differentiation phases based on molecular genetic mechanisms like; gene re- arrangement, gene exclusion, alternative splicing and  apoptotic  gene expression. B cell on antigenic epitope stimulation, the epitope triggers naïve B cells to be activated for growth, proliferation, expansion and change to effector plasma cell and memory B cells.

Early in the Immune response time curve B cell produce IgM then class switched to other isotype. IL4, IL10, TGFB and IFN gamma promotes class switching [7]. When antigen primed B cell acts as   an antigen presenting cell, it well take up and process antigen then present it onto their surface with MHCII molecules to be presented to T helper cell. It appear that there was a shift from antibody production to APC function. Primed B cells that are transformed    to plasma cells are devoid from either IgM or IgD. Memory B cells once reactivated will perform class switching of Ig iso-types. BCR are composed of two identical Ig heavy and two identical light chains together with Ig alpha and Ig beta chains that transmit signals to   the cell interior when legends are bound to BCR. Each chain has amino and carboxy termini. Domains are found on the amino  termini of each chain. The epitope specificity of Ig molecule of BCR is determined before they are facing the antigen. The number of the possible BCR-epitope binding specificities high exceeds that number of genes forming human genome. This present a paradox. Such paradox can be solved through molecular re-arrangement, splicing and /or gene exclusion. The Kappa light chain encoding genes are mapped onto chromosome 2. While, those encoding lambda light chains are mapped onto chromosome 22. The gene cluster encoding is located onto chromosome 14. The potential antigen binding segment combinations are greater than 26 million. The heavy and light chain gene segments for both variable and constant regions are rearranged, transcribed into RNA and translated into single heavy or light chain polypeptide. The restriction of VLCL, and VHCH expression to a single member of each of the involved chromosome pairs is termed as allele exclusion. However, the collective combination of all of B cell means that both maternal and paternal of allele genes are expressed within any particular individual. There has been a guess that each B cell has the ability to produce a range of individual antigen specific receptor capable for binding as many as ten different epitopes. Significant part of this BCR epitope diversity are attributed to chromosomal DNA rearrangement. BCR represent a case of natural construct of a set of gene clusters for both of the heavy and light chains in which each set of gene clusters construct RNA transcript that express as a single polypeptide. Then these polypeptides undergo post-translational assembly. A single B cell synthesizes Ig of one single specificity at a time. This is because of its nature of combination of VL and VH regions at the effect of allelic exclusion. Un-stimulated B cell synthesizes and display monomeric IgM and IgD on their surfaces. Upon stimulation B cell may change their iso-type but not the epitope specificity of the Ig they produce. This process is known as iso-type switching. Iso-type switching influences the ultimate nature of humoral immune responses. Memory B cell producing IgM can undergo further DNA re-arrangement to change the Ig iso-type they produce [7-11].

Antigen Presenting B Cell

B lymphocytes including B1 may act as antigen presenting cell. When they do so B cells use specialized MHCII complex antigen presentation pathway to process BCR bound and internalized  protein antigens and present them to CD4 T cells. Such processes  are performed through several efficient molecular mechanisms in a stepwise manner as; (i) antigen capture and uptake, (ii) intersection of the internalized antigen-BCR complexes with MHCII complexes,(iii) generation and regulation of MHCII-peptide complexes, (iv) exo- cytotic transport for presentation of MHCII-peptide complexes at the surface of B cells and (v) activation of CD4 T cells. Collectively, these molecular mechanisms affect both of the fate of lymphocyte and shape the immune response [4,5,12].

Antibody Producing B Cells

A naïve B cell subset, on priming with SARS-COV-2 antigenic epitope(s) in continuum with in-vivo, ex-vivo and in-vitro settings will be activated through; cell growth, proliferation, expansion and transition into plasma cell producing antibodies specific for spike, receptor binding domain and other epitopes. In a study performed   in 2020 workers isolate 61 SARS-COV-2 neutralizing monoclonal antibodies from five hospitalized severe COVID-19 infection. Of which 19 were potent neutralizing to authentic SARS-COV-2 in-vitro [13,14].

Cytokine Producing B Cells

B cell performed an array of immune functions both in human and mammals independent on that of antibody formation. Like; tolerance, tumor rejection, immune response suppression autoimmunity, organization of lymphoid tissue and regulate lymphoangiogenesis. Broadly speaking it can be bio-typed into antibody producing and cytokine producing. Cytokine producing B cells are ramified into an effector and regulator B cells. The cytokine profiles of the effector are somewhat different from regulator B cells. The ontogenic origin of antibody producing B cells is different from that of cytokine producing B cells (Table 2) [3].

Table 2: Immune Functions of B cells

Proposed COVID-19 Immunity

It seems to be that the nature of human immunity; during SARS- COV-2 infections, recovery and post-infection is somewhat different from other human viral infections. Collective scientific task has been made by STAT scientists [15] proposed four scenarios for immunity to SARS-COV-2 infections. These are; sterilizing, functional, waning and lost. Other idea tempted to put-down a collective scenario that mix up more than one of the mentioned scenarios. Sterilizing; The immune system of the host is armed to a foe and able to fend it off before infection take a hold. Functional; The immune system produce specific antibody responses but the antibodies wan on time post-infection but many cellular responses are not waning. Under this scenario, people whose immune system have been primed to recognize and fit the virus in continuum with either infection or vaccination could contract it again in the future but with either mild or symptomless. Waning; In this scenario people who have been infected or vaccinated would lose their protection overtime. But even if immunity wans, reinfection would be less severe and be a variation to the functional immunity. Lost; In this scenario people who have been infected would lose all of their immunity against the virus within some time frame. A re- infection after that point would be like that of first infection. Mixed Scenario; The overall picture of human immunity to SARS-COV-2 infection will be mixed. Some people will have sterilizing but most will fit about functional or waning immunity. Neither sterilizing nor lost immunity gains support by the scientific immunity [15].

Evidence Based COVID-19 Immunity

Collective local experience from the in-practice COVID-19 patient some of them recover with use of convalescent sera others not. Most patient produce specific antibodies but few fail to raise antibodies though they clinically infected [unpublished data, early in the waves of the pandemic]. The to date information in this context indicated that both humoral and cellular immunity can be lasting up to eight months post infection [14,16-19]. Effective effector B, memory B, effector T and memory T cells are involved in the protection against COVID illness. Breakthrough infection during post-vaccination periods could function exactly the same as a booster immunization. Though this may be a theoretical concept rather than practical one [20].

Memory B Cell Probe COVID-19 Vaccine Efficacy

In an experiment two groups of human subjects were assigned as; SARS-COV-2 naïve 33, and SARS-COV-2 recovered 11. Antibody and antigen specific B cells were mapped overtime. SARS-COV-2 naïve group were found to be requiring both vaccine doses for antibody response. Likewise memory B cells tracing have shown full length spike protein and spike receptor binding domain were detectable at second mRNA vaccine shot. While in SARS-COV-2 recovered group antibody and B cell responses were significantly boosted after first vaccine dose. Second dose neither increase antibody nor B cells. First vaccine dose strongly correlated with the pre-existing memory B cells in recovered group. COVID mRNA vaccine priming to individuals have shown distinct response based on prior SARS-COV-2 exposure. Memory B cells could be used as a probe for vaccine efficacy both in naïve and recovered COVID-19 individuals [21].

Memory B Cells Breakthrough Infection

In an experimental setting in which two group of patients. One vaccine breakthrough infection 55 patients and the second vaccinated close contacts who did not contract infection 88 subjects. Vaccine breakthrough infection was sharing lower memory B cell frequency but high antibody in plasma cell and those produced by memory B cells. Inflammatory cytokines, the IL1B and TNF were lowered in vaccine breakthrough infection than that infection of similar settings. Hence, lower memory B cells are correlated with vaccine breakthrough infections of COVID-19 [22].

Memory B Cells and Severity

In a retrospective study that was planned as 208 laboratory confirmed COVID-19 patients in which severity was checked on a scale graded from 0-10 severity score. Two parameters were adopted for evaluation of severity; the memory B cell count and the serum immunoglobulin levels. The age range for those patients  35-63  years with median of 50 years of which 88 were females (42%). The survivors were 191:208(91.82%) and diseased were 17:208(8.18%). The severity was ranged from6-8 with a median of 8 in deceased and 0-2 with median of 1 in survivors. Significant low levels of total B, naïve B, switched memory B, and serum immunoglobulin levels; IgA, IgG1, IgG2 in deceased than in survivors. Negative significant correlation between serum Igs and memory B cells was evident. The prognosis of COVID-19 disease is associated with B cell subsets and serum immunoglobulin levels [23].

B Cell and Hypoxia

In an experimental setting including COVID-19 patients with variable severity and hypoxia and genetically modified VHL deficient mice kept in hypoxia exposed mice. Blood was collected  from  those patients for detailed B cell phenotypes in peripheral blood lymphocytes using flow cytometery. Single cell transcription and whole blood sequence analysis were done to evaluate the impact of hypoxia on patients B cells and same was done onto the genetically modified hypoxia kept mice. There was an early and perminant defects in B cell subsets in moderate to severe COVID-19 patients including marginal zone-like, memory and transitional B cells. Similar findings were noted on genetically modified hypoxia kept mice. To this end hypoxia may contribute to pronounce and persistent defects of B cell pathology observed both in COVID hypoxic patients and hypoxia kept mice [24].

B Cell and Sepsis

In a clinical settings of an abscess forming super-infection viral COVID-19 pneumonia. Bone marrow and splenic B cell count have shown severe B cell loss in bone marrow or spleen in 64% of the of the patients. This was reflected by peripheral blood lymphopenia. B cell loss was associated with higher pulmonary SARS-COV-2 burden and only with marginal decrease in T cell counts. Study suggests the presence of sepsis related immunodeficiency in sever COVID-19 pneumonia with super-infection [25].

B Cell in COVID-19 with Seasonal Coronavirus

In this clinical setting tempts were made to evaluate the role of the pre-existing seasonal endemic coronavirus B cell on the development of sars cov-2 specific IgG responses. The tried parameters were; kinetics, breadth, magnitude and levels of cross-reactivity of IgG antibodies against SARS-COV-2  and  heterologous  corona  virus  at the clonal levels in patients with mild or severe COVID-19 and controls. Assessment were made onto antibody reactivity to nucleo- capsed and spike antigens and correlate this with IgG responses to SARS-COV-2 neutralization. Patients with COVID mounted a mostly type specific SARS-COV-2 responses. IgG clones directed against seasonal coronavirus were boosted in patients with severe covd-19. these boosted clones did not neutralize SARS-COV-2. Such findings indicate a boost of poorly protective COVID-19 specific antibodies in patients with COVID-19 that correlate with disease severity, reveals “Original antigenic sin” [26].

Relaxing B Cell Tolerance

Five groups of individuals were the test and controls. First COVID-19 convaluscent, second mild COVID-19, third severe COVID-19 fourth COVID-19 vaccinated and fifth control. Blood were collected from these groups. Mononuclear cells were separated from the test and control blood samples. Flow cytometery was performed on these preparations. Anergic B cells were found in high frequency in severe COVID-19 patients as compared to mild cases. They were in activated state displaying reduced inhibitory receptor expression and restored BCR signaling indicative of breach of anergy during viral infection, supported by increment shift in autoantibody levels. This together with phenotypic and functional alterations significantly correlated with hyper-inflammation in severe SARS-COV-2 infection. Hence, B –ND undergoes relaxation in their peripheral tolerance in severe COVID-19 [27].

Memory B Cell Pulmonary Subsets

Memory B cells are immune cells produced primarily in the lymph node and spleen. They persist for long time there in these regions and retain the memory of the infectious agents. If the subject body facing with same agent in future. These cells immediately mobilized and rapidly reactive immune system for effective protection. Recently workers have been documented the presence of memory B cells in lungs of laboratory animal models. Whereby, the laboratory animal model infected with the virus as influenza or COVID-19. Ten weeks later the virus eliminated from the body of infected animals. Memory B cells were tracked in lungs of the infected animals using flurescent markers followed by single cell transcriptomic analysis. Such techniques enable localizing memory B cells in lung and their gene expression profile cell by cell. Results have shown groups of memory B cells in the bronchial respiratory mucosa in two subpopulations of different gene expression profile and functions. The Bona fide and Bystandard subsets. The bona fide subset showed high affinity to virus and trigger their appearance immediately on entry and infection. While the bystandard is not directly recognize the virus but bind to the receptor of the immune complexes formed by the antibodies produced by bona fide cells. Both of the subsets exhibit synergistic function as a two tiers system [28].

Conclusions

B cell immunology of COVID-19 is in the forefront and current scientific mode. Memory B cell was found pathognomic with an array of human COVID-19 pathological changes. The changes cover both subnormal count of the B cell subsets and cell defects in different COVID-19 disease forms.

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Abstract

The ontogeny, the basic and molecular biology and the subsets of human lymphocytes were briefed. Functional, anergic, exhausted, and senescent lymphocyte subsets were detected in severe SARS-COV-2 human infection forms. The aim of the present opinion paper tempted to reveal the comparative biology of the regulatory lymphocyte subsets RLS both in health and disease. RLS, are regulatory natural killer cells NK reg., regulatory B cell, B reg. and regulatory T cell, T reg. to visualize their roles in severe SARS-COV-2 human infections. RLS of NK, B and T cells were found almost associated with dampening the immune mediated tissue injuries following COVID-19 illness. Though it was not clear whether these regulatory lymphocytes     did their dampening role in simultaneous, sequential, synergistic and/or antagonistic manner. Show case analysis was made for Nk reg and T reg in COVID-19 cases. This opinion paper suggest a clinical experimental setting in which a group of severely ill COVID-19 patients and normal controls will be blood collected and their lymphocyte separated then subjected to regulatory lymphocyte [NK reg, B reg. and T reg.], flow cytometry, single cell RNA sequencing and interactome studies in order to visualize the way they perform their immunologic roles and the possible interplay between them.

Keywords

Anergy, COVID-19, Exhausted, Lymphocyte, Regulatory, Senescent

Introduction

Regulatory Cells, regulatory immune cells, and regulatory lymphocytes are terms denoted to immunosuppressive lymphocytes RLS. RLS functioned both in the innate and adaptive immune responses and their functions mainly in down regulation of immune over-reaction and/or immune unwanted reactions that served as cellular bases for anergy, tolerance and suppression. Major regulatory subsets mapped both in human health and disease are: NK reg., B reg., and T reg. [1-15]. The present opinion tempts to reveal the actual roles played by regulatory lymphocytes in COVID-19 in a comparative attitude and show case analysis.

Ontogeny

The primordial mother cell, the heamopoietic stem cells of all   of immune cells was found in fetal liver in the pregnant woman.    As gestation proceeds it will migrate to fetal bone marrow and remained there in postnatal life and adulthood life. In bone marrow the haemopoietic stem cells differentiated  into  three  progenitor  cell types; the myeloid, the lymphoid and the lympho-myeloid. The lymphoid cell descend from the lymphoid progenitor cells through the action of haemopoietic cytokines. Lymphoid cells within the continuum of bone marrow microenvironments either stay therein and differentiated in to naïve B cells or trafficked and migrate to thymus. In thymic tissue niche naïve cells undergoes thymic negative and positive selection through thymic proteins and cytokines then mature to be T cells [15]. In two groups of infants, first healthy while the second was premature and stressed. Cord blood lymphocytes were separated from both groups and their phenotypes compared. The phenotyping process was done by fluorometric analysis. This analysis had shown that early third trimester cord blood lymphocytes were as 80-85% fetal T cells belongs to T4+ inducer population and 10% as T8+ suppressor/ cytokine subsets. As gestation proceeds, the T4 and T8 ratio shifts towards adults value together with increase in expression of mature antigen T12. The naïve B cells differentiated into antigens of B1, B2 and B 4 were not changed during gestation of normal healthy infants. Antenatal stress which threatens fetal survival leads to the appearance of phenotypically less mature B cells in circulation for both of the lineages than expected for the gestational age. Cells expressing very early B cell markers B2, B4 increase and exceed the numbers of the more mature B1+ cells in the cord blood most notably due to hypoxic stress during antenatal condition [1]. The fetal blood from fetuses in the second trimester of gestation were collected, lymphocyte separated and analyzed by monoclonal antibody two color immune-fluorescence technique as well as flow cytometry. Lymphocyte surface markers were evaluated. The study indicate that cells of B, T and NK lineages as well as precursor cells can be detected in fetal blood at 18 to 20 weeks of gestation. During this stage of development variable proportions of T & B cells express surface molecules such as CD 1, CD10, CD38, CD45RA indicative of precursor or naïve state; on the other hand the CD57 molecules is not detectable on membrane of NK and T cells and the RO isoform of CD45 leukocyte antigens is synthesized at low percentages of T cells. Such findings suggest that the observed phenotypic peculiarities of lymphoid cells might be due to the inductive easiness of tolerance that occur in early ontogenic stages of the immune system [2]. T, B and NK lymphocytes and their respective phenotypic subsets originate from bone marrow stem cells and their progenitor lineages. Lymphoid cell that migrate to the thymus receive signals through notch commit to the T cell lineage. The lineage development in human beings is critically dependent on IL7 for T cell, IL 4 for B cell and IL15 for NK cells. The specificity and diversity of lymphocytes are gained during the process of generation TCR in T cells and BCR in B cells. The T and B cell repertoire is determined by random variable V, diversity  D and joining J somatic gene segments that recombine with an imprecise addition of nucleotides at the segment connection. This recombination is performed by an enzyme complex known as VDJ recombinase that contains the recombination activation gene RAG. RAG proteins expressed onto B & T cells. Successful recombination is determined by the expression of a functional antigen receptor which allows survival and durability of development [3].

Molecular Biology of Lymphocytes

In an experimental setting for a human lymphocyte cell line that was irradiated with 12C ion beams at; 0, 0.1, 0.5 and 2.0 Gy. The transcriptional profiles were evaluated by human gene expression microarray method at 24 hr. post-irradiation. In accordance with microarray assays, there are 1113 genes were up regulated and 833 genes were down regulated in human lymphocyte irradiated with 0.1 Gy 12C ion beams compared to the control group. 1095 genes were up regulated and 1220 down regulated in cells irradiated with 0.5 Gy 12C ion beams and 1055 genes were up regulated and 1356 genes were down regulated with 2.0 Gy. A total sum of 504 genes were differentially expressed in all irradiated groups of which 88 were up regulated and 416 were down regulated. Most of these altered genes were related to; cell cycle, apoptosis, signal transduction, DNA transcription, repair and replication. Thus, the differentially expressed genes at 24 hr post- irradiation increased as irradiation dose increased, up regulated genes gradually decreased and down regulated genes increased. 12C ion beams irradiation could express a number of genes in dose dependent manner which might initiate failure of multiple biological functions of the cell [4].

Lymphocyte Functional Phenotypes and Subsets

Lymphocytes are white blood cells that forms parts of the systemic and mucosal immune system with uniform appearance and varied functions. Bone marrow derived cells, the B cells are involved in the innate immune function of antigen presenting cells, adaptive antibody and IL10 responses. T cells functions in; cell mediated immune response, TH1, TH2, Th17 cytokine production as well as delayed type IV hypersensitivity reactions. TH17 take part in the immune cross- road functions both for innate and adaptive responses. Regulatory T cells the T regs. are commited in down regulation of over-immune and/or unwanted immune reactions. Natural killer cell the NK interplay direct cell mediated cytotoxicity of virus and tumor cells. NK includes innate and adaptive phenotypes [LaRosa and Orange 2008, National human genome institute 2022 [3,5].

Regulatory Lymphocyte Subsets

The B, T and NK lymphocytes are expressing regulatory phenotypes as; B reg., T reg. and NK reg. All of which are involved in down regulation of immune over and/or  unwanted  reaction  noted through the cellular events of immune responses. Such down regulation processes almost mediated by various cytokines through; signal transduction, cell-cell communication and cross-talk [3,7,12].

Regulatory Natural Killer Cells

Cellular Immuno-Biology

A large lymphocyte with finely granular cytoplasm commit in antiviral and anti-cancerous cells. These cells are known as Natural Killer Cells NK cell that belongs to innate immunity with an adaptive immune potentials. From the structural point of view these cells are devoid from both TCR an BCR. But they have; cytotoxicity receptor family (NKp30, NKp44, NKp16, NKp80), type C lectin domain containing receptors (NKG2D), the CD2 superfamily receptor and IgG receptor (CD16). The legend of some of these receptors up regulated by stress and infection like vial haemagglutinins for NKp46, Cd46 for CD2 and IgG for CD16 [LaRosa and Orange [3]. NK cells have distinct subsets with desperate function, location and developmental origin. Peripheral blood NK cells can be of two functional subsets are known based on expression of CD56 and CD16. the first subset isCD56 dim CD 16+ form 90% of the total blood NK effective in killing target cells and secret low cytokines. While, the second is CD56 bright CD16- constitute 10% of the total blood NK but are enriched in secondary lymphoid tissues. The effector functions of the NK are cytotoxicity and cytokine production. These functions are separate in NK subpopulations. The bright and the dim subsets were differing in; (i) the expression of inhibitory and activating receptors (ii) adhesion molecules and (iii) chemockine receptors (Leunemann et al.) [6]. The subsets, Table 1 and immune regulatory subsets of NK cells are; Bright [Gross et al. [7] and CD73 + [8].

Table 1: Natural Killer Cell subsets*

*Adapted from Lunemann et al. [6]

NK Immune Functions

NK have an array of immune functions as; Controlling T cell responses and maintaining homeostasis [Gross et al. [7], Orchestrate immune responses, linking innate and adaptive immune responses and regulating, T, B and DC [9].

Regulation by Natural Killer Cell, A Mechanistic View

NK cells performed their immune regulatory function using either of the following mechanisms; (i) direct contact cytotoxicity of myeloid cells, (ii) polarization of TH1, shaping and dampening through cell mediated cytotoxicity and (iii) antibody dependent blocked of Qa 1 NKG2A interaction resulted in potent NK dependent elimination of T cells [7,12].

Regulatory NK in Human Diseases

The immune regulatory actions of NK cells are operable in a number of human diseases; CD72 NK mediated pathology in tumor micro-environment [8], autoimmune and inflammatory diseases [6] autoimmune liver diseases [Jian and Wang [9] and multiple seclerosis [6,7].

NK in COVID-19

SARS-COV-2 clearance rate, antibody response and disease progression in COVID-19 correlate with NK pathophysiological status and NK dysfunction is linked to disease susceptibility. Thus NK may act as a key element in the switch events from effective to harmful immune responses in COVID-19 illness. NK depletion and dysfunction correlated with severity and anti-fibrotic activity [10]. There were significant reduction in number and function of NK attributed to their exhaustion [11].

Regulatory NK in COVID-19

A bidirectional of cell-cell cross-talk of NK with, neutrophil, DC, and monocyte/ macrophages have shown elimination of neutrophil and DC in a separate experimental settings [12]. High NK count, low T cell CD8+ and regulatory CD56 bright CD16- dim in severe COVID-19 patients [13] as shown in the show case analysis with a notable NK subsets of NK reg. in COVID-19 disease (Table 2).

Table 2: Show case Analysis of natural killer cells in COVID-19

Regulatory B Lymphocyte

Cell Immunobiology

B lymphocytes are bone marrow derived lymphocytes. They developed in bone marrow and matured in the peripheral lymphoid tissues starting with pro B then Pre B, immature B and mature B. The immune potentials of B lymphocytes are multiple. As antibody producing, antigen presenting and cytokine producing as well as immune regulating [3,17].

The Immune Functions of B lymphocytes

Naive B lymphocytes on activation through stimulatory and co- stimulatory signals by either of; antigen, mitogen and/or cytokine they may perform one or more of the following immune functions; (i) antibody production, (ii) antigen presentation, (iii) cytokine production, (iv) immune regulatory and (v) toleragenesis [17,18].

Regulatory B lymphocytes [B reg.]

Regulatory B lymphocyte express immunosuppressive functions via diverse mechanisms. Of which B reg. modulate immune responses through secretion of cytokines, IL10, IL35, and TGFB.   Or by direct cell-cell contact dependent mechanisms. B reg. is important to human welfare both in health and disease. In disease, however, they are associated with abnormalities both in numbers  and functions. B reg. accounts for 0.5% of human peripheral blood lymphocytes. And expressed a variety of protective and pathologic. They take part in the tissue transplantation tolerance and involved in creating immune prevealiged sites for the uterine environment and neonatal life [17-19].

Regulatory B Lymphocytes Subsets

Human regulatory B lymphocyte is heterogeneous in term of number, origin and localities as shown in the following (Table 3).

Table 3: The Subsets of Human B reg.*

*Adapted from Menon et al. [17]

Mechanisms of Action of B reg

The operable mechanisms by which naïve B lymphocytes becomes B reg. are; (i) change in CD markers and surface immunoglobulins, (i) stimulation by IL10, (iii) stimulation by IL35 and TGFB, (iv) direct cell-cell contact dependent which might be attributed to CD 80 CD86 PD-L 1 CD40L and CDd 1 d, (v) granezyme dependent cell killing, (vi) anergic or tolerizing mechnisms and TLR-BCR engagement mechanisms [17,19].

B reg. in Human health and Disease

B reg. take part in; pathogenesis, protection and or promotion of human disease as in; cancer of lung, infections, malaria, allergic air way inflammation and transplantation Diabetes and pregnancy [17,19].

B reg. in Viral Infections

In human hepatitis B disease immature transition CD19+ CD24hi CD38hi and CD19+Il10+ Breg. involved in regulation of the antigen specific CD8+ Tcells. While, incaseofacquiredimmunedeficiency HIV disease, activated immature transtionalCD19+CD24hiCD38hi Breg. reduces the frequency of HIV infected individuals through suppression of CTL function which cause viral persistence. In Deng fever disease, CD19+CD24hi CD38hi reduces the severity of the disease. Neonatal CD5hiCD10-CD 1chiCD45RACd23loCd24hiC38loIgDloIgMlo Breg. produce IL10 that dampen the beneficial cytokine production by TH1 cells and contribute to severe disease [18].

Breg in COVID-19 Disease

In severe SARS-COV-2 infections the precursors of human transitional CD19hi CD38hi B reg. were reduced [19]. In a clinical setting in which two men vaccinee were enrolled in immune cell flow cytometery, it was evident that Breg was increased post first shot but reduced post to the second COVID-19 vaccine dosage [20].

Regulatory T lymphocytes

Cell Immunobiology

The CD4+ T lymphocytes may be differentiated into lymphocytes that have the ability to suppress other immune cells including T cell responses. These differentiated cells are known as regulatory T cells. Their development from naïve lymphocytes can either be thymic or extra-thymic. The thymic developed are natural T regs and stably expressFoxp3 transcription factor and is eligible for its immune suppressive function. The natural T regs are IL2 dependent and Foxp3 transcription factor producer. The surface markers of natural T regs are CD3+CD4+Cd25+ Foxp3+. While, the extra thymic developed T regs are of inducible nature. The surface markers of the inducible type 1 T regs are CD3+CD4+ [3,21].

Human Tregs Subsets

Three main subsets of T regs are known till date. One of which  is natural and the other two were of inducible nature. The inducible subsets are either TGFB or IL10 induced subsets (Table 4).

Table 4: Human Regulatory Lymphocyte Subsets*

*LaRosa and Orange [3]

T regs Immune Functions

T regs expresses an array of immune functions including; negative regulation of T cell responses, sustaining immune tolerance, reserve immune homeostasis, taking part in establishment of lymphocyte anergic state [21].

T regs in Human Diseases

In human host microenvironment T regs. may express either protective or pathologic functions depending on the nature of the surrounding micro-environmental factors. Loss of function mutation in the Foxp3 gene lead to; inflammatory autoimmune disease, immune dysregulation, poly-endocrino-pathy, enteropathy and or graft versus host reaction GVHR. Such mutation may aggraviate human virus infections [21].

Tregs in COVID-19

In early stage of SARS-COV-2 infection, activated Tregs. Populations potently suppress the recruitment of immune cells such as Th1 a CD8+CTLs leading to reducing of the immune responses. In mild infection form, T regs becomes increased in numbers and could attenuate the inflammatory responses and quench the cytokine storm, this event could promotes recovery of the patients. When the infection form proceeds towards severity, Treg depletion can enhance the activation of pro-inflammatory immune cells and production of pro-inflammatory cytokine that leads to hyper-cytokinemia and lung injury [21]. The exact role played by T reg. in COVID-19 seemed   to be controversial among different workers all over the world. In a study were 57 mild, severe and recovered patients, peripheral blood mononuclear cell were subjected to study Tregs CD25+ foxp3 has shown increase in proportion of these cell [22]. Other working group use to study 109 mild, severe and recovered patients T regs they were found Cd4+CD25+ CD127+ got increased in both proportion and numbers of Tregs [23]. No change in CD4+CD25+Foxp3+ Tregs have been found in a group of 17 moderate,27 severe and 8 control [24].

Show Case Analysis

A show case analysis was made for two published works; one concerning SARS-COV-2 infected patients in Japan and the other done on two COVID-19 vaccinee men. The investigation in both    of the infected Table A and vaccinee Table B, tempted to analyze peripheral blood lymphocytes using flow cytometery and immune informatic approach of lymphocytes including T regs. Researchers reached results of different subsets of Tregs (Table 5) [20,25].

Table 5: Show case analysis of T reg subsets in COVID-19 infected and COVID-19 vaccinee

*Sondergraad et al. [25];**Gupta et al. [20]

Conclusion

In comparative Biologic sense regulatory lymphocytes as ordered in accordance with their chronological order of discovery are; T reg., Breg. and NK reg. They are at most immunosuppressive lymphocytes. Each of which  may  include  typical  or  conventional  and  atypical  or unconventional phenotypes. The main conventional regulatory lymphocytes are; Three T regs, six B regs and two Nk regs. The current newly reported regulatory lymphocytes are; Eight T regs, one Bregs and one NK regs. Hence the total number of these regulatory subsets are; Eleven T regs, seven B regs and three NK regs. These regulatory lymphocytes are of suggested basic sharing in common immune features as: (i) Natural or induced, (ii) initiated by activation and action mechanisms, (iii) mobilized by chemkines and activated by  cytokines, (iv) own sets of surface inhibitory and/or stimulatory functions, (v) on function they may associated with age sex or severity of the disease, (vi) the consequences of their immune function in disease either promote protection or promote disease and vii- the mechanistic path can be of use as chemotherapeutic target. The regulatory lymphocytes shared essential roles in pathogenesis and immunology of COVID-19. Though therein presence of gaps in understanding of their exact interplay. The role of T regs in COVID-19 to date it is still in debate. Leem et al. [16] have done a landmark  study  of natural killer cell including NKregs in COVID-19 patients. Maucourant et al. [14] have been performing landmark investigation on NK immunotypes as related to severity, Bergantini et al. [13] did immunological signature of T cell and NK cells on hospitalized COVID-19 patients. They conclude that higher frequencies of NK and NKreg. Corresponded to lower frequencies of CD3+ and CD4+TCM in severe cases and B cells were not mapped. High frequencies of senescent and exhausted NK, and memory CD4+, CD8+ T cells associated with severe infection forms. B cells compartment were not mapped [Srivastava et al. [26]. Regulatory NK and T regs as well as B cells were investigated through mass flow cytometery with an emphasis on cell networking were tempted [25] performed profiling study to lymphocytes for NK, B and T cells including regulatory lymphocyte subsets have been reported in two vaccine men [Gupta et al. [20]. Hence full lymphocyte profiling using flow cytometery, single cell RNA sequencing and interactome studies including regulatory subsets as well as the deduced network in between lymphocyte and other immune cells to visualize the way they interacts between them and with other cells in mild, moderate, severe and deceased COVID-19 patients still need to uncovered. Thus it is being suggested.

References

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Abstract

MAIT cells are innate like T cells served as; bacterial and fungal sensors through TCR dependent mechanism and viral sensors via cytokine TCR independent mechanism. These mechanisms are along with an overlapping tissue repair mechanisms. The objective of the present opinion paper was to deduce the immune functions and immune features of MAIT cells in; Homeostasis, SARS-COV-2 infection and SARS-COV-2 vaccination as appeared in the current 2020 up to 2022 publications. Single cell transcriptomics, mass transcriptomics, flow cytometery, unsupervised analysis and full immune      cell landscapes were the major approaches followed by lymphocyte immunologists. During homeostasis and activation, early antigen specific MAIT        cells activation with MRI legand5-OP-RU and non-specific TCR stimulation, it has been found an array of phenotypes as; homeostatic, effector, helper,  tissue infiltrating, regulatory and exhausted phenotypes. While, in prolonged stimulation, proliferative, cytotoxic, immune modulating and exhausted phenotypes were identified. In SARS-COV-2 human infection, MAIT cells may be reduced in circulation and enriched in the airways, dys-regulated, or activated then migrate to lungs in the pneumonic COVID-19 pathotype. In vaccinee, however, MAIT cells in mRNA COVID-19 vaccinee was found to be positively correlated with magnitude of humoral and cellular response to vaccine in normal healthy vaccinee but their cytotoxic function post activation        is negatively correlated with humoral and cellular immune response to vaccine. MAIT cells were found to be helpful mucosal cellular immune-adjuvant in influenza nasal vaccination strategy. Thus MAIT cell immune functions may be summed up as;  microbial  immune  sensors,  immune-pathogenic, immune modulating, mucosal cellular adjuvant to viral protein antigen and intrinsic systemic cellular adjuvant in COVID-19 vaccinee. The evolution mechanisms for MAIT functional phenotypes were suggested.

Keywords

Adjuvant, Activation, cell, cytotoxicity, mucosal, MAIT, TCR dependent, TCR independent, vaccine.

Introduction

MAIT cells are innate-like of recirculating T cells. They can function as bacterial and fungal sensors through TCR dependent mechanism and as viral sensors via cytokine activation mechanisms. These mechanisms are overlapping with their ability to repair tissues. MAIT cells are also found in association with autoimmune, immune mediated and cancer diseases. They are enriched in human; liver, lung, gut with an evident variable existence in peripheral blood of normal healthy individuals. In human viral infections they are of reduced frequencies in blood and enriched in local tissues. The situation      in human SARS-COV-2 infection so far concerning the MAIT distribution seems to be not far from that of other viral infections [1- 8]. The aim of the present opinion paper was to deduce the immune functions and immune features of MAIT cells in in; normal human homeostasis, SARS-COV-2 infected and SARS-COV-2 vaccinee.

Investigative Approaches

Google mapping the MAIT published contributions all over the world paved the author to the years 2020 up to 2022. The publication covers the areas of biology, molecular biology, pathogenesis in microbial, autoimmune, immune mediated and cancer besides their role in tissue repair. The aiming of the present opinion paper was focused on their role in homeostasis, COVID-19 and COVID-19 vaccinee. Table 1 lists the investigative approaches ensembled in current MAIT-COVID-19 research.

Table 1: Molecular and Immune investigation approaches in COVID-19, homeostasis and COVID-19 vaccinee

MAIT Cell Immunobiology

Ontogeny

The human MAIT cells expressing V alpha 7-CD161hi T cells are generated during gestation and likely share a common prenatal development program. Within cord blood niche the total MAIT cells, V alpha 7+ CD161hi. T cells are forming the minority recognizing MR1; 5Op-RU display a TRAV/TRBV repertoire very similar to adult MAIT cells. During the few weeks of postnatal life only MR1:5-OP-RU reactive to V alpha 7.2+CD161hi T cells aquire memory phenotype Only these cells expand to form adult MAIT pool diluting out other V alpha.7-2+ CD161hi. And V alpha 7-2-CD161hi. Population in a process requiring at least6 years to reach adult level [1].

MAIT Cell and Molecular Biology

MAIT cell are forming one subset of T cells with an evident unique characteristics. They function both in innate and adaptive immune responses. MAIT can perform their biologic functions both through TCR dependent and TCR independent manners. Their basic TCR structure composed of TCR alpha chain V alpha33 associated with limited TCR Beta chain repertoire and restricted by the non – polymorphic MHC class related MRI molecule. MAIT cells are innate- like immune cells produce wide range of cytokines. Resting cells are devoid from garnzyme and porins and no-cytotoxic. On activation by either microbial riboflavin through TCR or through non-TCR way by cytokines, they own granzyme and porins and becomes cytotoxic. Such activated MAIT cells allowed to migrate to the inflamed areas and functions therein. So can act as first line defenders against microbial infections. They also contribute in autoimmune and immune mediated diseases. MAIT cells harbor mucosal surfaces of lung, gut and in liver. Variable MAIT cell counts were found in peripheral blood of normal human blood doners. The surface markers of MAIT cells are; C type lectin CD161, integrin and chemokine receptors. The fine structure of the MAIT cellular system composed of g ranzyme type B, TCR alpa and TCR beta but the beta is very limited [5,7,8]. MAIT cell TCR can fine-tune MRI recognition through antigen dependent manner, by which MAIT cell recognition is modulated [4]. MAIT cell are ramified into a number of subsets so far concerned in COVID-19 (Table 2). Comparative view to MAIT and conventional T cells are depicted in Table 3.

Table 2: MAIT cell subsets in COVID-19 and in normal healthy donors

Table 3: Comparative View to MAIT cells And Conventional T cells*

*Based on to Vorkas et al. [10]

To be a functional MAIT cell phenotype there may be a number of suggested sequential steps and several influencing factors in effect leading to the rise of functional MAIT cell phenotype as;

• Primitive lymphoid cell progenitor in bone marrow migrates and homed into thymus.
• Therein thymus homed lymphoid progenitor cells undergoes positive and negative selection processes through the action of thymic factors and cytokines and maturate to naïve MAIT cells.
• Naïve MAIT matured cells undergoes further developmental events mediated by cytokines finalized by evolving of effector MAIT These effector cells leave the thymus.
• On leaving thymus effector MAIT cells migrate through circulation to peripheral tissues and homed therein. By this, two forms of MAIT cells are merging circulatory and tissue resident forms.
• Tissue micro-environmental stimuli are in action within the localized tissue niche.
• Genetic, epigenetic and metabolic reprograming happened.
• Evolving and acquisition of new surface and intracellular receptors or markers.
• Cytokine induction or cell-cell cross-talk are in action
• New functional MAIT phenotype emerged.

Immune Recognition by MAIT Cells

Two immune recognition mechanisms are known to date in MAIT cells. First is the invariant TCR dependent in which MAIT recognize microbial conserved microbial riboflavin derivatives antigens. The process terminated by MAIT cell activation. The second mechanism is TCR independent in which cell mediators the cytokines interacts with their surface receptors on MAIT cells, signal transduced in to the cell interior leading to cell activation. The activated MAIT cell in both mechanisms MAIT cells transformed from resting to activate cells form with acquisition of granzym B and porins. Activated MAIT cells performed immune and non-immune functions. The non- immune functions expressed as affected tissue repair, and the immune functions spans in innate and adaptive immune response arms. The activation by TCR dependent may renders MAIT cells cytotoxic killing virus infected cells. Activation by cytokine may induce MAIT cells to produce IL17A with pathological consequences in the affected tissue lungs [3].

MAIT Cell Immune Functions

MAIT cells of human and mice are programed in the thymus to seed and reside in barriers tissues. Therein local cues possibly modulate the MAIT transcription program so that MAIT cells isolated from different organs express distinct gene sets [15]. Hence MAIT cells seems to display specific properties according to the organ from which they recovered, suggesting that their function is related to the tissue they recovered from [16]. MAIT cells express both antimicrobial and wound healing molecules. So that they are well aquinted to contribute both of these overlapping phases of immune responses to infections according to lymphokine milieu and legend availability [2]. The assembly of granzym B and porins within the basic biology structure of MAIT cells renders them capable for direct infected cell cytotoxicity. MAIT cells hold the position of microbial sensor via microbial metabolite and first line defenders within the inflamed tissue niche against the tissue invasive microbial infections. In human pulmonary tissue microenvironment MAIT cells produce IL17A cytokine that implicated in viral infection including SARS- COV-2. They may contribute to immune protection against some virus human infection like influenza virus [7,8].

Homeostasis

In an experimental setting, peripheral blood was collected for   30 blood healthy donors from whom PBMC were separated by cell separation media Ficoll prep. Washed PBMC were subjected to an early and prolonged activation with 5-OP-RU or anti CD3/CD28 in presence of IL2 or Il2/TGFB and examined by single cell transcriptomics and flow cytometery. It was evident that CD4+MAIT cells are associated with expression of co-stimulatory receptors IL2 signaling and memory markers, whereas CD8+ MAIT cells are defined by granzym modulated cytotoxicity and type IFN signaling. CD4-CD8-expression on MAIT cells may define distinct functional subpopulation. The landscape   of MAIT cell clusters during homeostasis and early activation have shown that MAIT cell may respond earlier than conventional T cells. CD4+ and CD8+ MAIT cell subsets are transcriptionally distinct and can adopt similar program to innate lymphocyte and conventional    T cells. MAIT cells up-regulate Foxp3 prolonged activated MAIT cell expand homeostatic subpopulation and inducing; proliferative, cytotoxic, regulatory and exhausted phenotypes [10].

MAIT Cells and Human Diseases

MAIT cells decreased in blood stream of tuberculus patients but appeared to be accumulated in lungs suggesting that they are recruited to the infected lung tissue contributing to tissue defects. Migration  of blood MAIT cells to the infected tissue as activated and expanded clones in gut of typhoid patients. MAIT cells could be involved in autoimmune and immune mediated diseases. After activation MAIT cells could act doubly on targets of other immune cells in immune mediated and autoimmune diseases as reduced in blood, increase in the affected tissues and are in altered and dys-regulated forms [5].

MAIT Cells in Viral Infections

At resting state in human being MAIT cells are characterized by lack of granzyme B and low perforin expression. These two key proteins are required for cytotoxic activity. While once MAIT cells activated they can rapidly induce granzyme B and granzyme K. Pateint with human influenza A virus infection has shown significantly increased MAIT cell counts with an evident cytotoxic function as compared   to healthy controls. Influenza infection is capable of inducing MAIT cells to up-regulate antiviral IFN-g and cytolytic granzyme B in a TCR independent pathway, requiring IL18 and potentially other mediators from accessory cells include monocyte and macrophages clinical significant of these findings is a matter of debate [8].

Measles McV initially infects macrophages or conjunctival epithelial cells then migrate to regional lymph nodes where it infects lymphocytes and spread systematically through the lympho-reticular system and generation of viremia of secondary nature. McV utilize CD46-nectin 4 and CD150 to enter host cells. While nectin 4 is used as a receptor on epithelial cell, CD150 is used to infect immune cells leading to apoptosis of CD15 memory cells, resulting in immune amnesia. Human PBMC MAIT cells were highly expressing CD150. The rapid virus induced apoptosis in infected immune cells is likely share in induction of immunosuppression associated with measles virus disease [8,17,18].

MAIT in SARS-COV-2 Infections

In 24 moderate and severe SARS-COV-2 cases, It was found  that total and CD4, CD8 and double negative MAIT cell counts in circulation is reduced but with marked activation state. Meanwhile MAIT cells were enriched in the airways, on recovery MAIT cell counts restored to normal [12]. MAIT cell frequencies in 208 COVID-19 patients were investigated for MAIT cell profiles. They were of reduced counts in peripheral blood with appearance of; active, cytokine producing and CD4CD8 MAIT cell phenotypes. The activated MAIT cells were migrating to lungs and enriched therein in the affected pulmonary tissues [14]. In other study, 13 moderate and severe COVID-19 patients PBMC were mapped for MAIT cell profiles, they were decreased in circulation and activated, their activation initiate chemotaxis, apoptosis and found involved in the virus immune responses and possibly engaged in immune tissue damage [11]. MAIT cell reduction and functionally impaired in peripheral blood of; 23 mild, 22 severe, 6 asymptomatic patients and 44 were controls. Together with appearance of IFN stimulated gene up-regulated HLA-DRlo-monocytes and enrichment of suppressive macrophages [9]. Kim et al. [12], have been studied 50 severe and 50 normal control subjects, they were showing that MAIT cell reduction in number   but activated in circulation and they were inversely correlated with disease severity and mortality. The activated MAIT cells migrate to lung wherein stimulate macrophage. MAIT cells contribute to the worsening of inflammation in severe pneumonic lungs (Table 4).

Table 4: Role of MAIT cells in SARS-COV-2 human pneumonia*

*The infection of MAIT cells by SARS-COV-2 virus is rather unclear till date

MAIT Cells in SARS-COV-2 Vaccinee

At the days seven and 21 post mRNA COVID-19 vaccination of; 42, 42, and 24 normal, immune compromised patients. From whom peripheral blood were sampled. PBMC were separated and mapped for MAIT cells. The MAIT cell frequencies remained stable overall the normal Subject vaccinee, immune compromised vaccinee. There were a positive correlation between the size of MAIT cell compartment and the vaccine induced adaptive immune response to srs-cov-2 spike protein. The pre-vaccinee and post-vaccinee levels of MAIT cells correlated positively with magnitude of SARS-COV-2 spike specific antibodies and CD4 T cells in both vaccinee groups. In normal healthy donors the levels of MAIT cell activation is negatively correlated with the spike antigen specific immune responses. Hence, MAIT  cell compartment is involved in the early stages of primary adaptive immune to vaccine and may be they are important in the vaccine induced immunity [19].

The protective immune response to respiratory pathogens including influenza virus are initiated by mucosal immune system. The development of safe and effective mucosal vaccine has greatly been impaired due to lack of a properiat mucosal adjuvant. MAIT cells when co-administered with influenza A haeagglutinin protein induced protective immunity in mice and the resultant antibodies were of predominant IgA type. The heamagglutinin immune primed mice were found on influenza A live virus challenge immune protected. MAIT cell in the study act as a cellular adjuvant. Their adjuvanicity was mediated by CD40L dependent activation of DCs and subsequent priming of CD4+ follicular T cells. Hence MAIT cells acts as cellular mucosal adjuvant in a mucosal vaccine strategy [20]. The comparative role of MAIT cells in SARS-COV-2 infection and vaccination was depicted in Table 5.

Table 5: Comparative view to the role of MAIT cells in SARS-COV-2 infection and in vaccination*

*Based on Boulus [19], Pankurest et al. [20], Shi et al. [11].

Conclusions

MAIT cells are functioning in; Homeostasis, infection, vaccination, tissue repair, autoimmune diseases, immune mediated diseases and cancer. In SARS-COV-2 infections, MAIT displayed number of immune functions as; Chemo-attractant, initiation of antigen processing, antigen presentation in antigen presenting cells as well as activation of lymphocytes. During the adaptive immune responses to SARS-COV-2 virus infection it acts early in the adaptive immune response as an intrinsic cellular adjuvant to the immune cells involved in the immune response. While in vaccination with influenza haemagglutinins it was found that MAIT cell activate DCs through DC40L and prime follicular CD4+ helper T cells by this it may acts as mucosal cellular vaccine adjuvants. MAIT cells may adopt; pyroptic, apoptotic, active tissue infiltrating and exhausted phenotypes. During SARS-COV-2 severe infection, MAIT cell count in circulation dropped down but resume active phenotype then migrates to air ways then to lungs therein they expand and worsen the patients state. On recovery, however, MAIT cells restore their normal distribution both in circulation and mucosal tissues. The evolution of MAIT functional phenotype was suggested.

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