Chronic Viral Infection Compromises the Quality of Circulating Mucosal–Associated Invariant T Cells and Follicular T Helper Cells via Expression of Inhibitory Receptors

Background : Chronic viral infection results in impaired immune responses rendering viral persistence. Here, we compared the quality of T-cell responses among chronic hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV)-infected individuals by examining the levels of expression of selected immune activation and exhaustion molecules on circulating MAIT cells and Tfh cells. Methods : Cytokines were measured using a commercial Bio-plex Pro Human Cytokine Grp I Panel 17-plex kit (BioRad, Hercules, CA, USA). Inflammation was assessed by measuring an array of plasma cytokines, and phenotypic alterations in CD4 + T cells including circulating Tfh cells, CD8 + T cells, and TCR iV α 7.2 + MAIT cells in chronic HBV, HCV, and HIV-infected patients and healthy controls. The cells were characterized based on markers pertaining to immune activation (CD69, ICOS, and CD27) proliferation (Ki67), cytokine production (TNF-α , IFN-γ ) and exhaustion (PD-1). The cytokine levels and T cell phenotypes together with cell markers were correlated with surrogate markers of disease progression. Results : The activation marker CD69 was significantly increased in CD4 +hi T cells, while CD8 + MAIT cells producing IFN-γ were significantly increased in chronic HBV, HCV and HIV infections. Six cell phenotypes, viz., TNF-α + CD4 +lo T cells, CD69 + CD8 + T cells, CD69 + CD4 + MAIT cells, PD-1 + CD4 +hi T cells, PD-1 + CD8 + T cells, and Ki67 + CD4 + MAIT cells, were independently associated with decelerating the plasma viral load (PVL). TNF-α levels showed a positive correlation with increase in cytokine levels and decrease in PVL. Conclusion : Chronic viral infection negatively impacts the quality of peripheral MAIT cells and Tfh cells via differential expression of both activating and inhibitory receptors


Introduction
Chronic viral infections result in immune cell dysfunctions in the host [1], but often persist without inflicting any serious cell damage [2].Many chronic viruses, especially the hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus 1 (HIV-1) in humans are adept at circumventing the host's immune responses, primarily by imposing the expression of coinhibitory molecules to the advantage of the pathogen [3].Given the non-cytopathic nature of HBV and HCV, the immune system is attributed to hepatocellular damage as well as viral clearance [4].Inability to attain viral clearance and development of chronic HBV disease is suggestive of dysfunctional immune responses [5].The expansion of regulatory T cells (Tregs), high antigen loads, anti-inflammatory cytokines, and biosignatures of exhaustion are the likely indications of dysfunctional HBV-specific responses [6].Evasion of the host's immune surveillance augments active replication of chronic viruses.In addition, viral persistence also stems from clonal deletion of HBV-specific T cells and/or their functional insufficiency together with increased expression of signatures associated with immune activation, senescence, and exhaustion.
Chronic HCV infection leads to persistent upregulation of genes associated with innate immune activation leading to liver inflammation and consequently cirrhosis [7].Exhausted T cells in concert with TNF-α and IFN-γ, are capable of driving non-specific immune responses in order to prolong the infection [8].During HIV infection, it's clear that the virus dominates with the loss of immunological control over viral replication in treatment-naïve individuals.Exhausted virus-specific CD8 + T cells progressively lose their ability to clear cellular reservoirs of viruses [9], due to chronic immune activation that results in functional immune exhaustion [10].
Mucosal-associated invariant T (MAIT) cells are a unique subset of innate-like T cells that link the innate and the adaptive immune systems [11].Although MAIT cells comprise only ~5% of the peripheral T cell pool in adults, the cell population is enriched in up to ~40% in the liver [12].MAIT cells play a paramount role in innate host defense responses against bacterial and viral infections [13,14].Given the primary localization of MAIT cells is confined to the peripheral mucosal tissues, their functions are weakened (characterized by heightened expression of immune exhaustion markers, reduced cytokine production and cytotoxicity) in chronic viral infections, including HIV [15], HCV [16,17] and HIV-TB co-infections [18], the current research intends to generically and descriptively determine the quality of host immune responses in chronic HBV, HIV, and HCV infections.We investigated the role of immune activation and potentially compromised T-cell responses in the three different chronic viral infections by exploring conventional CD4 + and CD8 + T cells along with their counterpart follicular T helper cells (Tfh) and mucosal-associated invariant T cells (MAIT).

Ethics Approval
The cross-sectional case-control study was carried out in accordance with the guidelines of the International

Subjects and Analytical Parameters
HBV-infected individuals with plasma HBsAg and anti-HBc positivity (n = 13), HCV-infected individuals as determined by anti-HCV (n = 8), HIV-infected individuals (as per the criteria of the National AIDS Control Organization (NACO), India) (n = 7), and healthy controls (HCs) (n = 10) were recruited into the cross-sectional study.The HIV-infected patients were on ART as per NACO recommendations (at least for over five years).Peripheral blood was obtained from all the participants by a trained phlebotomist.HCs were identified as individuals free from HBV, HCV, HIV, Mycobacterium tuberculosis infections as well as HBV/HIV co-infections.

Plasma Viral Load
The Pathodetect™ (Mylab Discovery, Pune, India) quantitative Real-Time PCR was used to quantify the viral loads of HBV and HCV using an in vitro nucleic acid amplification assay on a QuantStudio 5 real-time PCR (Applied Biosystems, ThermoFisher Scientific, MA, USA).We determined HIV-1 viral load using the Abbott Real-time HIV-1 assay (Abbott, Abbot Park, IL, USA), via an in vitro reverse transcription-polymerase chain reaction assay with the sensitivity 40 copies/mL volume of the sample.

Peripheral Blood Mononuclear Cells
Ten milliliters of peripheral blood were collected by venipuncture, and stored in lithium heparin BD Vacutainer (BD Biosciences, Franklin Lakes, NJ, USA) tubes at room temperature.PBMCs were extracted using a commercial Sepmate™ (Stemcell Technologies, Vancouver, Canada) by density gradient centrifugation.Cell viability was determined by 0.4% Trypan blue vital staining.Purified PBMCs were suspended in a Bambanker™ serum-free cell freezing medium (Nippon Genetics Europe GmbH, Duren, Germany) for storage at -80 °C, not more than 3 months before use in the experiments.PBMCs were thawed in a water bath at 37 °C before use in the experiments.

Flow Cytometry Multi-Parametric Immunophenotyping
All antibodies were purchased from BD Pharmin-gen™ (BD Biosciences, San Jose, CA, USA) unless otherwise specified.

Intracellular Cytokine Staining
Mononuclear cells were incubated with PMA (50 ng/mL) and ionomycin (500 ng/mL) or cultured in RPMI containing 10% FBS (R10) alone.Samples were incubated at a concentration of 10 µg/mL, and Golgi Plug (brefeldin A) and Golgi Stop (monensin) were included at 10 µg/mL.Samples were incubated for overnight at 37 °C in 5% CO 2 and then permeabilized using Fix & Perm reagents (BD Bioscience) and stained intracellularly with BV421conjugated anti-IFN-γ and APC-conjugated anti-TNF-α.At the end of stimulation, cells were washed once with FACS wash (PBS containing 2% [vol/vol] FBS and 0.25% of sodium azide) and surface stained with anti-CD3, anti-TCR7.2(3C10), anti-Ki67 (B56) anti-CD8 (SK1), anti-CD4 (OKT4).Cell stain at room temperature for 30 minutes.Cells were then fixed with Cytofix/Cytoperm (BD Pharmingen) for 20 minutes at 4 °C and washed with Perm wash (BD Pharmingen).Cells were then incubated for 30 minutes at 4 °C with antibodies specific to IFN-γ and TNFα, washed once with Perm wash and once with FACS wash, and resuspended in PBS containing 1% formalin.Cells were acquired on a BD FACS Canto II Immunocytometric system.FlowJo for Windows, Ver.10.0.8 (FlowJo LLC, Ashland, OR, USA) was used to perform the analysis.At least 100,000 events were acquired for each sample.

Statistical Analyses
We examined the percentages and expression of biomarkers on distinct subsets of T cells, MAIT and Tfh cells between the four study groups.For multiple group comparisons, categorical variables were examined using the Chi-square test of Fisher's Exact Test, while continuous variables were tested using non-parametric Kruskal-Wallis Test.If the p values were <0.05, four-way comparisons were made between the four patient groups using the Mann-Whitney Test.The Spearman's Rank correlation was used to compare the correlation between two continuous variables.The association between the surface markers, and functional markers and plasma viral load (PVL) were assessed using the linear regression model.* p < 0.05, * * <0.01, * * * <0.001, and * * * * <0.0001 were used to determine significance.GraphPad Prism Ver.6.0 software (GraphPad, La Jolla, CA, USA) was used to perform all the analyses.

Clinico-Demographic Characteristics of Participants
The four groups, non-randomized study design consisted of 38 individuals.Thirteen subjects with chronic HBV infection who tested positive for HBsAg, anti-HBc as well as HBV DNA: Group 1 (G1), eight subjects with HCV RNA positive and anti-HCV positive; G2, seven subjects with HIV RNA positive, and 10 healthy controls (HC) (G4).The samples were collected between September and October of 2021.As per the analytical parameters, 54% HBV-infected individuals, 87% HCV-infected individuals, and 47.5% HIV-infected individuals were diagnosed with signs of liver injury, while 46% of the HBV-infected, 13% of the HCV-infected, and 52.5% of the HIV-infected participants were chronically infected without any underlying clinical or biochemical signs of liver injury (Table 1).

Activated T Cells with High PD-1 Expression was Observed in Chronic HBV, HCV and HIV-Infected Subjects
The flow cytometry gating strategy to delineate the CD4 +hi , CD4 +lo and CD8 + T cells is depicted in Fig. 1A.The CD8 + T cell levels were significantly higher for HIV (Fig. 1B) as expected.There was negligible alteration in CD69, ICOS, or PD-1 (CD279)-expressing T cells between the different groups (Fig. 1C,D).CD69 was significantly increased on CD4 +hi T cells in chronic HBV, HCV and HIV in comparison with HCs (p < 0.05, p < 0.01, and p < 0.05, respectively).The expression of co-stimulatory (ICOS) and co-inhibitory (PD-1) markers were significantly higher in CD4 +hi T cells in HBV (p < 0.05), HCV-infected (p < 0.01) and HIV-infected (p < 0.01) individuals.Likewise, CD69 + CD4 +lo T cell levels (p < 0.01) PD-1 + CD4 +lo T cell levels and (p < 0.001) were significantly higher in all infected groups whereas the levels of ICOS + CD4 +lo T cells were only significantly higher in chronic HBV (p < 0.01) infection.CD69 + CD8 + T cell levels were significantly increased in HBV (p < 0.05) and HCV-infected individuals (p < 0.01).Furthermore, all groups had significantly higher levels of PD-1 + CD8 + T cells (Fig. 1C,D).Together, these data highlight that markers of T-cell activation coupled with exhaustion were increasingly expressed on T cells in chronic HBV, HCV and HIV infections.

Circulating MAIT and Tfh Cells Represented Activated Phenotypes Coupled with Higher PD-1 Expression in Chronic HBV, HCV, and HIV-Infected Subjects
Next, we set out to study the MAIT and Tfh cells across the different study groups with a pre-determined gating strategy (Fig. 2A).Total TCR iVα7.2 + MAIT cells were significantly lower in all the infected groups (p < 0.01) compared with HCs.The CD4 + MAIT cell levels were significantly increased in HCV and HBV groups (p < 0.01) but not in the HIV-infected group compared to HCs.The level of CD8 + MAIT cells (p < 0.01) as compared to HCs were higher in all the groups (Fig. 2B).The circulating Tfh cell levels in HIV-infected individuals were significantly higher (p < 0.001) in HIV infected group, but comparable in the HBV and HCV-infected groups (Fig. 2C).The CD69 + CD4 + MAIT cell levels were significantly increased in the HIV and HBV groups (<0.05) but not in the HCV-infected group.PD-1 + CD4 + MAIT cell levels were significantly elevated in all the infected groups (Fig. 2D).The ICOS + CD8 + MAIT cells were significantly decreased for HCV and HBV groups, and the PD-1 + CD8 + T cell levels were highly significant in HIV-infected individuals with p < 0.001 (Fig. 2D).Spearman correlation of CD4 + TCR iVα7.2,CD8 + TCR iVα7.2 with CD69, ICOS, and PD-1 is presented in Fig. 2E.The functional markers for Tfh viz., CD27, ICOS and PD-1 were comparable between HBV, HCV and HCs.Nonetheless, PD-1 was highly elevated in the HIV-infected group suggesting that Tfh cells become activated (see Fig. 2F).

Significant Elevation in Cytokine-Producing T Cells during Chronic Viral Infection
Next, we compared the cytokine-producing ability of T cells with respect to CD4 +hi , CD4 +lo and CD8 + T cells.Our results indicates that there was a significant increase in the cytokine + CD4 + T cells and CD8 + T cells during chronic viral infection when compared to HCs.The comparison analysis of CD4 +hi IFN-γ was significant in HBV and HIV-infected individuals (p < 0.0001).Ki67 + CD4 +hi was significantly higher in HIV-infected individuals (p < 0.0001).CD4 +lo TNF-α was significant in HCV-infected individuals (p < 0.001).CD4 +lo IFN-γ and Ki67 was highly significant in HIV (p < 0.0001) infection.CD8 + IFN-γ and Ki67 had significance (p < 0.0001) in HBV and HCVinfected subjects, respectively (Fig. 3A,B).
Next, we also looked at the proliferating potential of T cells, which indicated that Ki67 + T cells were higher in chronic viral infections as compared to HCs.Then, we examined the production of cytokines by the CD4 + and CD8 + MAIT cells.Both CD4 + and CD8 + MAIT cells expressed higher levels of TNF-α, and IFN-γ, together with higher proliferating ability as compared to HCs.The proliferating MAIT cells, Ki67 + CD4 + MAIT and Ki67 + CD8 + MAIT cells, had higher significance in HIV and HBV-infected individuals (Fig. 3C) compared to the HCV-infected group.PBMCs using height and area of forward scatter, then singlet gates were utilized to remove doublet populations.This was followed by lymphocyte gating using forward and side scatters areas.This was followed by a total CD3 + cell gate against TCR iVα7.2.From CD3 + cells, total CD8 + cells CD4 +hi , and CD4 +lo were gated out.From this CD8 + , CD4 +hi , and CD4 +lo we determined CD69 + , PD-1 and ICOS.(B) The results of these gates are three T cell populations: CD8 + , CD4 +hi , and, CD4 +lo .Comparison of the levels of, CD4 +lo , CD4 +hi and CD8 + among patients chronically infected with HBV, HCV, HIV, and HCs.(C) CD69, PD-1 and ICOS expression was determined by using a CD69, PD-1, and ICOS mean fluorescence intensity (MFI), respectively, which was used as a negative control for CD69, PD-1, and ICOS staining and allowed for accurate gating on the positive populations only.
Spearman correlation analysis and the significance of various cytokines in chronic HBV, HCV and HIV-infected individuals are shown in Fig. 3D.

Similar Expression Profile of Markers Across Individuals with Chronic HBV, HCV and HIV Infections
In this study, we observed that the infected groups shared a similar activation and cytokine profiles with total CD4 + , CD8 + T cells along with Tfh, and MAIT cells displaying elevated activation and cytokine levels (Fig. 4A,B).The cells that showed >2-fold for each chronic infection were identified and displayed in Venn diagrams after the cytokine fold change was graded by descending order (Fig. 4C,D).The analysis revealed that among all the immune cells, seven altered phenotypic profiles viz., CD4 +lo TNF-α, MAIT CD4 + TNF-α, MAIT CD4 + IFNγ, MAIT CD8 + IFN-γ, CD4 +lo IFN-γ, CD4 +hi IFN-γ, CD8 + IFN-γ was common among patients chronicallyinfected with HBV, HCV, and HIV.Ki67 + MAIT CD4 + and CXCR5 CD4 +lo was common among those chronically infected with HCV and HIV.CD4 + MAIT was common among chronic HBV and HCV-infected individuals.(Fig. 4C,D and Supplementary Fig. 1).The activation, exhaustion and functional markers in association with viral load of T-cell subsets are presented in Table 2.

Discussion
Virus-specific T cells express multiple inhibitory receptors during chronic infections eventually impairing T cell functions.Evidence suggests that exhausted virusspecific CD8 + T cells eventually lose their ability to clear intracellular viral reserves [9] associated with the onset of chronic immune activation [10].Studies in murine [19] simian [20] and humans have shown that blockade of the inhibitory molecules restores immune functions in vitro as well as in vivo [9].CD4 + T cells provide help to other effector cells, especially CD8 + T cells to aid in their activation and cytokine production (TNF-α and IFN-γ), and contact-dependent cytotoxicity via perforin/granzyme syn-thesis and/or Fas-FasL interactions to render viral elimination [21].Chronic viral infections frequently result in decreased CD8 + T cell functions as compared to the potent effector T cells activated during acute infections [22].Exhausted T cells in chronic infections can be classified into terminally-exhausted CD8 + T cells, and cells with a preserved ability to proliferate [23].Exhausted T cells along with TNF-α, and IFN-γ induce non-specific immune responses aiding in controlling the infection [9].
Recent evidence suggest that MAIT cells are key to immune surveillance, especially in chronic viral infections.MAIT cells aid in host defense in an antigen-independent manner, as they respond to a variety of cytokines such as IL-12 and IL-18 [24] during viral infections.In chronic HBV and HIV infections plasma IL-5 and IL-7, respectively may play a significant role in viral suppression [25].MAIT cells have been shown to act against several viral agents, particularly HBV, HCV, HIV, dengue, and influenza viruses [26,27].Although available findings portray their significance in chronic HBV, HCV and HIV infections, very few compared their role in cross-sectional studies conducted across the infections from same region.In addition, we have also explored into the activation and functional status of circulating Tfh cells across the different study groups.
In the current study, we observed the activation and proliferation of MAIT cells with higher expression of CD69 and Ki67, respectively.Similar findings have also been observed during chronic HCV infection, where they exhibit an activated phenotype with higher levels of the activation markers CD69, HLA-DR, and CD38 [28].Importantly, here we found that the co-stimulatory potential of MAIT cells were elevated in the three chronic viral infections, as these cells expressed higher levels of ICOS as compared to HCs.The correlation between CD69 levels and polyfunctional TCR iVα7.2 + CD4 + T cells points to the critical role of CD69 against chronic HBV infection [29].Similarly, here we observed that the levels of CD69expressing CD8 + T cells and MAIT CD4 + cells were associated with a decrease in PVL indicating the likely importance of CD69 in viral control.Further, chronicallyinfected individuals were in a hyper-activated state; a stage that precedes immune exhaustion because the later refers to a phase that occurs sequentially only next to immune activation (i.e., sequentially, immune activation will be followed by immune exhaustion, culminating in immune senescence) [30].The major limitation of our study is that HBV and HCV are hepatotropic viruses, whereas HIV is lymphotropic macrophage-tropic, and these distinct virus tropisms should have had an impact on the phenotypes of various T cell subsets during the chronic phase, which have not been contemplated in the current work.
Experiments conducted thus far on CD4 + T cells during chronic infections have focused on Th1, and only recently investigators have started looking at Tfh cells, in particular the circulating Tfh cells.Tfh, a diverse group of CD4 + T cell subsets are essential for complete B cell responses that include germinal center (GC) reactions, isotype-switching, and affinity maturation [31].Data available from all the chronic infections that we have studied herein have shown evidence on the induction of Tfh cells.However, the circulating Tfh activation, proliferation and their cytokine profiles have seldom been studied in headto-head comparisons.Similar to the MAIT cells reported herein, we also observed increased activation of Tfh cells along with higher proliferation (Ki67), and co-stimulation with higher levels of ICOS expression.In all the chronic infections studied herein, we have shown evidence for higher levels of Tfh cells compared to HCs.

Conclusion
In summary, our findings suggest a likely association of compromised immune responses in chronic HBV, HCV, and HIV infections although not conclusive of the role of the markers studied herein.The upregulation of CD69 may aid in regulating immune response by determining the patterns of cytokine and chemokine release as well as the activation of lymphocytes during chronic infections.Enhanced TNF-α levels during chronic infection likely imply their protective role in viral elimination possibly via recruiting T cells.

Abbreviations
Conference on Harmonization Guidelines and the Declaration of Helsinki.The study protocols were reviewed by the Institutional Ethical Committee (IEC) of the Government Medical College, Theni, for necessary approval for the conduct of the research (Ref.No. 2544/ME1/18 and Ref. No. 1515/MEIII/21).Prior approval was also obtained from the Tamil Nadu State AIDS Control Society, Chennai, India (TANSACS Approval-00529/TANSACS/M&E/2019 NACO-T-11020/08/2020_NACO) for the conduct of work.Institutional Biosafety Committee (IBSC) approval was secured (Ref.No.: CUTN/SLS/1st IBSC/2020/04).All the human subjects were adults and written consents were duly obtained from all the participants.

Fig. 1 .
Fig. 1.Immune activation and exhaustion markers associated with CD4 + T cells and CD8 + T cells.(A) Gating strategy for CD69, ICOS and PD-1 expression on peripheral CD4 +hi , CD4 lo , and CD8 + T cell populations.Lymphocytes were gated from whole human (D) Expression (MFI) of CD69, ICOS, and PD-1 in CD4 +hi , CD4 lo and CD8 + T cells among patients chronically-infected with HBV, HCV, HIV and HCs.The level of expression of each marker was reflected by the color scale of the heatmap.The cells were compared across the four groups by the Kruskal-Wallis test.Post-hoc Mann-Whitney U tests were subsequently performed only for those biomarkers with a Kruskal-Wallis test p value of <0.05.p < 0.05 are considered significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.n.s.represents 'not significant'.

Fig. 4 .
Fig. 4. Immune cell profiling in chronic HBV-, HCV-, and HIV-infected individuals.(A,B) The fold change in immune cells with identified intracellular and extracellular markers in individuals chronically infected with HBV, HCV and HIV normalized against HCs.(C) Bar plot depicting mean 2-fold change among identified markers in specific immune cell type.(D) Venn diagram showing immune cells that are upregulated >2-fold.The Venn diagram identified a profile of seven immune cell populations that are commonly expressed among chronically HBV-, HCV-, and HIV-infected individuals.(v) %CD4 hi ; (w) %MAIT CD4 + ; (x) %CD4 lo TNF-α + , %MAIT CD4 +

Fig. 5 .
Fig. 5. Predictors of plasma viral load.(A,B) Network analysis of the six predictors of plasma viral load.(A) depicts the complexity of the interactions between the six predictors.(B) Spearman correlation between the six predictors of plasma viral load.(C) The six markers were subjected to multivariate linear regression analysis to determine the markers that independently predict the plasma viral load.(D) Expression of TNF-α and their association with plasma cytokines.(E) Plasma level of cytokines associated with plasma viral load.Variables with p values < 0.05 were considered independent predictors in their respective models.*, **, **** represent p < 0.05, <0.01, and <0.0001, respectively.

Table 2 . Activation, exhaustion and functional markers associated with plasma viral load.Table 2 .
Continued.Univariate analyses by linear regression were performed to identify potential predictors of PVL.Given the small sample size, the linear regression modeling for T-cells subsets, activation, exhaustion and functional markers were performed separately.Variables with p < 0.05 were considered as candidates.The candidate predictors were then included in a multivariate model, and variables with p values were <0.05 were considered as independent predictors in their respective models.*, **, ***, **** represent p < 0.05, <0.01, <0.001 and <0.0001, respectively.†, the Ki67 expression of these subpopulations were in co-linearity with % MAIT CD4 Ki67.For all the Ki67+ subpopulation, only % MAIT CD4 Ki67 was included in multivariate analysis.Coef, coefficient; CI, Confidence interval.