Chronic viral infection compromises the quality of circulating mucosal-invariant T cells and follicular T helper cells via expression of both activating and inhibitory receptors

Chronic viral infection results in impaired immune responses rendering viral persistence. Here, we investigated the role of immune activation and compared the quality of T-cell responses in chronic HBV, HCV, and HIV infections. 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 peripheral CD4+ T cells including circulating Tfh cells, CD8+ T cells, and TCR iVα7.2+ MAIT cells in chronic HBV, HCV, and HIV-infected patients and healthy controls. The cells were characterized based 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. The activation marker CD69 was significantly increased in CD4+ hi T cells, while CD8+ MAIT cells expressing 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, 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. Chronic viral infection negatively impacts the quality of peripheral MAIT cells and TFH cells via expression of both activating and inhibitory receptors.


Introduction
Chronic viral infections results in immune cell dysfunctions in the host [1], but often persist without in icting any serious cell damage [2]. Many chronic viruses, especially the hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunode ciency virus 1 (HIV-1) in humans are adept at circumventing the host's immune responses, primarily by imposing the expression of co-inhibitory 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-in ammatory cytokines, and biosignatures of exhaustion are the likely indications of dysfunctional HBV-speci c responses [6]. Evasion of the host's immune surveillance augments active replication of chronic viruses.
Besides, viral persistence also stems from clonal deletion of HBV-speci c T cells and/or their functional insu ciency 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 in ammation and consequently cirrhosis [7]. Exhausted T cells in concert with TNF-α and IFN-γ, are capable of driving non-speci c 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-speci c 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]. MAIT cells appear to undergo functional impairment during chronic infections. 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 study was carried out in accordance with the guidelines of the International Conference on Harmonization Guidelines and the Declaration of Helsinki. 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 crosssectional study. Peripheral blood was obtained from all the participants by a trained phlebotomist. HCs were identi ed as individuals free from HBV, HCV, HIV and Mycobacterium tuberculosis 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 ampli cation assay on a QuantStudio 5 realtime PCR (Applied Biosystems, ThermoFisher Scienti c, MA, USA).
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. Puri ed PBMCs were suspended in a Bambanker™ serum-free cell freezing medium (Nippon Genetics Europe GmbH, Duren, Germany) for storage at -80°C. PBMCs were thawed in a water bath at 37°C before use in the experiments.
Cells were then incubated for 30 minutes at 4°C with antibodies speci c to IFN-γ and TNF-α, 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 samples.

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 PVL were assessed using the linear regression model. * P < 0.05, ** <0.01, *** <0.001, and **** <0.0001 were used to determine signi cance. 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% (n=7) HBV-infected individuals, 87% (n=7) HCV-infected individuals, and 47.5% (n=3) HIV-infected individuals were diagnosed with signs of liver injury, while 46% (n=6) of the HBV-infected, 13% (n=1) of the HCV-infected, and 52.5% (n=4) 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 ow cytometry gating strategy to delineate the CD4 +hi , CD4 +lo and CD8 + T cells is depicted in Figure   1A. The CD8 + T cell levels were signi cantly higher for HIV ( Figure 1B) as expected. There was negligible alteration in CD69, ICOS, or PD-1 (CD279)-expressing T cells between the different groups ( Figure 1C-D).
CD69 was signi cantly 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 signi cantly higher in CD4 +hi T cells in HBV (p<0.05), HCV-infected (p<0.01) and HIVinfected (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 signi cantly higher in all infected groups whereas the levels of ICOS + CD4 +lo T cells were only signi cantly higher in chronic HBV (p<0.01) infection. CD69 + CD8 + T cell levels were signi cantly increased in HBV (p<0.05) and HCV-infected individuals (p<0.01). Furthermore, all groups had signi cantly higher levels of PD-1 + CD8 + T cells. (Figure 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 (Figure 2A). Total TCR iVα7.2 + MAIT cells were signi cantly lower in all the infected groups (p<0.01) compared with HCs. The CD4 + MAIT cell levels were signi cantly 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 ( Figure 2B). The circulating Tfh cell levels in HIV-infected individuals were signi cantly higher (p<0.001) in HIV infected group, but comparable in the HBV and HCV-infected groups ( Figure 2C). The CD69 + CD4 + MAIT cell levels were signi cantly increased in the HIV and HBV groups (<0.05) but not in the HCV-infected group. PD-1 + CD4 + MAIT cell levels were signi cantly elevated in all the infected groups ( Figure 2D). The ICOS + CD8 + MAIT cells were signi cantly decreased for HCV and HBV groups, and the PD-1 + CD8 + T cell levels were highly signi cant in HIVinfected individuals with p<0.001 ( Figure 2D). Spearman correlation of CD4 + TCR iVα7.2, CD8 + TCR iVα7.2 with CD69, ICOS, and PD-1 is presented in Figure 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 during chronic HIV infection the Tfh cells become activated (see Figure F-G).

Signi cant 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 , CD8 + T cells and compared them with HCs results indicates that there is a signi cant 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 signi cant in HBV and HIV-infected individuals (p<0.0001). Ki67 + CD4 +hi was signi cantly higher in HIV-infected individuals (p<0.0001). CD4 +lo TNF-α was signi cant in HCV-infected individuals (p<0.001). CD4 +lo IFN-γ and Ki67was highly signi cant in HIV (p<0.0001) infection. CD8 + IFN-γ and Ki67 had signi cance (p<0.0001) in HBV and HCV-infected subjects, respectively ( Figure 3A and 3B).
Next, we also looked at the proliferating potential of T cells in the same assay, which indicates 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. They displayed signi cance (p<0.05) and (p<0.0001) in HIV-and HBV-infected individuals, respectively. The proliferating MAIT cells, Ki67 + CD4 + MAIT and Ki67 + CD8 + MAIT cells, showed more signi cance in HIV and HBVinfected individuals ( Figure 3C) compared to the HCV-infected group. Spearman correlation analysis and the signi cance of various cytokines in chronic HBV, HCV and HIV-infected individuals are shown in Figure 3D.

Similar expression pro le 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 pro les with total CD4 + , CD8 + T cells along with Tfh, and MAIT cells being the principal cells showing elevated activation and cytokine levels ( Figure 4A and 4B). The cells that elevated >2-fold for each chronic infection were identi ed and displayed in Venn diagrams after the cytokine fold change was graded by descending order (Figures 4C and 4D). The analysis revealed that among all the immune cells, seven viz., CD4 +lo TNF-α, MAIT CD4 + TNF-α, MAIT CD4 + IFN-γ, MAIT CD8 + IFN-γ,CD4 +lo IFN-γ, CD4 +hi IFN-γ, CD8 + IFN-γ were common among patients chronically-infected 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. The bar chart describes the %fold change of each cell subset (see Figure 4C-D and Supplementary Figure 1). The activation, exhaustion and functional markers in association with viral load of T-cell subsets are presented in Table 2.

Discussion
Virus-speci c T cells express multiple inhibitory receptors during chronic infections eventually impairing T cell functions. Studies in murine [11] simian [12] 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 synthesis and/or Fas-FasL interactions to render in viral elimination [13]. Chronic viral infections frequently result in decreased CD8 + T cell functions as compared to the potent effector T cells activated during acute infections [14]. Exhausted T cells in chronic infections can be classi ed into terminally-exhausted CD8 + T cells, and cells with a preserved ability to proliferate [15]. Exhausted T cells along with TNF-α, and IFN-γ induce non-speci c immune responses aiding in retaining 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 [16] during viral infections. In chronic HBV and HIV infections plasma IL-5 and IL-7, respectively may play a signi cant role in viral suppression [17]. MAIT cells have been shown to act against several viral agents, particularly HBV, HCV, HIV, dengue, and in uenza viruses [18,19]. Although available ndings portray their signi cance 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 ndings 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 [20]. 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 [21]. Similarly, here we observed that the levels of CD69-expressing CD8 + T cells and MAIT CD4 + cells were associated with a decrease in PVL indicating the likely importance of CD69 in viral control. The major limitation of our study is that HBV and HCV are hepatotropic viruses, whereas HIV is lympho-tropic 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 a nity maturation [22]. 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 pro les have seldom been studied in head-tohead 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.

Conclusions
In summary, our ndings suggest a possible role of phenotypic markers as biological indicators of disease progression in chronic HBV, HCV, and HIV infections with their close association with disease severity, immune responses, and viral persistence. 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.

Declarations
Author contributions EMS, VV, AM, PK, PB and ML designed the study. AM coordinated patient recruitment and sample collections. JV, RA, SR and AM developed the laboratory works and co-ordinated the development of the study. YKY and EMS per-formed data analysis. YKY, HYT, and YZ did the biostatistical analysis. JV, EMS, YKY, VV and MS co-wrote the manuscript. All authors critically reviewed the article, have accepted responsibility for its entire contents and approved its submission.  Tables   Table 1 Clinico-demographic characteristics of study participants Characteristics Total      Figure 1 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 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  A) Gating strategies of intercellular cytokines in conventional and unconventional T cells. Total CD3 + cells were gated against TCR iVα7.2 (MAIT cells). From MAIT cells CD8 + and CD4 + T cell populations were gated whereas CD3 + was gated as CD4 +hi , CD4 +lo , and CD8 + T cells. The functional markers of different gates were determined: CD4 + TCR iVα7.2, CD8 + TCR iVα7.2, CD4 + , and CD8 + cells B)