Major T cell exhaustion pathways are up-regulated in HSV-specific CD8+ T cells from symptomatic HSV-1 patients

Blood-derived CD8+ T cells, specific to two HLA-A*0201-restricted HSV-1 epitopes selected from the membrane glycoprotein B (gB561–569), and tegument protein VP11/12 (VP11–12702–710), were sorted (Supplementary Fig. S2) by fluorescence-activated cell sorting (FACS) from: (1) HLA-A*0201-positive SYMP individuals that exhibit frequent and severe recurrent ocular herpetic disease (n = 2); and (2) HLA-A*0201-positive ASYMP individuals that never have any recurrent herpetic disease despite being seropositive (n = 2) (Fig. 1a). Significantly higher frequencies of CD8+ T cells specific to HLA-A*0201-restricted HSV-1 epitopes gB561–569 (3.5% vs. 1.9%, P = 0.04) and VP11–12702–710 (5.4% vs. 2.7%, P = 0.01) were detected in ASYMP individuals compared to SYMP individuals (Fig. 1b).

Figure 1

figure1

Differential gene expression in HSV-specific CD8+ T cells from HSV-1 infected symptomatic vs. asymptomatic individuals. (a) Experimental design and validation of differentially expressed genes in CD8+ T cells sharing the same HSV-1 epitope-specificities, from SYMP and ASYMP individuals. CD8+ T cells specific to HLA-A*0201-restricted HSV-1 gB561–567 and VP11/12702–710 epitopes were sorted from HLA-A*0201-positive SYMP and ASYMP individuals, using specific tetramers. Total RNA was extracted from each clone of epitope-specific CD8+ T cells, and whole transcriptome analysis was performed using bulk RNA sequencing to determine the levels of expression of 25,638 genes. (b) Frequencies of CD8+ T cells specific to HLA-A*0201-restricted HSV-1 gB561–567 and VP11/12702–710 epitopes detected by FACS in SYMP vs. ASYMP individuals. (c) Heatmap is showing 772 differentially expressed genes among SYMP and ASYMP individuals. (d) Heatmap showing statistically significant pathways that are affected in HSV-specific CD8+ T cells from SYMP vs. SYMP individuals. Parametric Gene Set Enrichment Analysis (PSGEA) method was applied based on data curated in Gene Ontology and KEGG. Pathway significance cut-off with a false discovery date (FDR) ≥ 0.2 was applied. (e) Bulk RNA heatmap comparing differentially expressed CAM pathway associated T cell co-stimulatory and T cell exhaustion genes in HSV-specific CD8+ T cells from SYMP vs. SYMP individuals.

Total RNA samples were isolated from sorted HSV-specific CD8+ T cells and subsequently processed for bulk RNA sequencing to screen the whole human transcriptome comprising 25,638 genes. Of this pool of genes, 20,126 genes were found with a minimum count per million (CPM) value ≥ 0.5. Our gene expression analysis revealed 772 genes to be statistically significant (FDR ≥ 0.1 and fold change ≥ 2) and differentially expressed among CD8+ T cells that share the same HSV-1 epitope-specificities, from SYMP vs. ASYMP groups (Fig. 1c). Of these genes, 583 genes were up-regulated, and 189 genes were down-regulated in HSV-specific CD8+ T cells from ASYMP individuals compared to SYMP patients (Supplementary Fig. S1a, upper panel). Correlation matrix and Pearson’s correlation coefficient confirmed a high degree of relatedness in the pattern of gene expression between SYMP and ASYMP individuals (Supplementary Fig. S1a, middle panel). The Volcano plot showed significant log2 fold changes and -log10 (FDR) of each of the differentially expressed genes in HSV-specific CD8+ T cells from ASYMP vs. SYMP patients (Supplementary Fig. S1a, lower panel). Further pathway enrichment analysis of the 772 differentially expressed genes between SYMP vs. ASYMP groups revealed a significant up-regulation of the cell adhesion molecule (CAM) pathway comprising major T cell exhaustion genes in HSV-specific CD8+ T cells from the SYMP group (P = 3.2e−04) (Fig. 1d). A bulk RNA sequencing specific heatmap confirmed the significant upregulation of CAM pathway-specific gene expression (i.e., CEACAM8, NRCAM, LAMA1, SELE, and NLGN3) and T cell exhaustion genes (i.e., PD-1, LAG-3, PSGL-1, CTLA-4, TIM3, and TIGIT) in HSV-specific CD8+ T cells from the SYMP patients compared to HSV-specific CD8+ T cells from the ASYMP individuals (Fig. 1e). These results indicate that an upregulation of CAM pathway associated genes along with major T cell exhaustion molecules in HSV-specific CD8+ T cells from SYMP patients is associated with frequent and severe recurrent ocular herpetic disease.

Due to the ethical and practical limitations in obtaining human TG samples from HSV-1-infected, SYMP and ASYMP individuals, the remainder of this study utilized our established HLA-A*0201 transgenic rabbit (HLA Tg rabbits) model of ocular herpes, which develops spontaneous virus reactivation, virus shedding in tears, and symptomatic recurrent ocular herpetic disease, as occurs in humans.

Blockade of PD-1 and LAG-3 immune checkpoint reduces recurrent ocular herpes infection and disease in latently infected symptomatic HLA transgenic rabbits

Since up-regulation of genes for the PD-1 and LAG-3 exhaustion pathways in HSV-specific CD8+ T cells is associated with symptomatic herpes in humans, it was of interest to determine whether the blockade of PD-1 and LAG-3 immune checkpoint pathways would reduce virus reactivation and ease symptomatic recurrent ocular herpes.

As illustrated in Fig. 2a, HLA Tg rabbits (n = 20) were infected with 2 × 105 pfu of HSV-1 (McKrae Strain). Half of the rabbits (Group-1; n = 10) were treated with a combination of blocking α-PD-1 and α-LAG-3 mAbs, injected intravenously (i.v.) on days -3, -5, -7 before infection and then on days 3, 5, and 7 post-infection (p.i.) at 200 μg/dose. The other half of the rabbits (Group-2; n = 10) received saline injections (untreated controls). Eye swabs were collected daily for 5 days p.i., and recurrent ocular herpetic disease was monitored 15 days p.i. Rabbits were subsequently segregated into (1) asymptomatic (ASYMP) rabbits, with no apparent recurrent ocular herpetic disease, and (2) symptomatic (SYMP) rabbits with higher rates and severe recurrent ocular herpetic disease. The detailed characteristics of the SYMP and ASYMP HLA Tg rabbits used are described in “Methods”. The α-PD-1/α-LAG-3 mAbs-treated group had the most ASYMP animals, with significantly less virus titers in the eyes (P < 0.04, Fig. 2b) and no apparent recurrent corneal herpetic disease (Fig. 2c). In contrast, the untreated group had the most SYMP animals, which showed a significantly higher level of virus replication in eyes associated with severe recurrent ocular herpetic disease (P < 0.04, Fig. 2b,c).

Figure 2

figure2

Effect of immune checkpoint-blockade on recurrent ocular herpes infection and disease in HSV-1 infected HLA Tg rabbits. (a) Schematic representation of HSV-1 ocular infection, α-PD-1/α-LAG-3 mAbs treatment, immunological, virological, and disease analyses in HLA Tg rabbits ocularly infected with HSV-1. HLA-Tg rabbits from Group-1 were ocularly infected with HSV-1 and treated with α-PD-1/α-LAG-3 mAbs whereas HLA-Tg rabbits placed in Group-2 were also ocularly infected with HSV-1 but not treated with α-PD-1/α-LAG-3 mAbs. Based on the severity and frequency of recurrent ocular herpetic disease, the rabbits were scored between 0 and 4 and subsequently categorized into SYMP and ASYMP groups. (b) HSV-1 viral titers in the eyes of α-PD-1/α-LAG-3 treated and α-PD-1/α-LAG-3 untreated HLA Tg rabbits. Infectious virus particles were quantified from eye swabs of α-PD-1/α-LAG-3 treated and α-PD-1/α-LAG-3 untreated HLA Tg rabbits. (c) Representative images showing recurrent ocular herpetic disease in HSV-1 infected SYMP HLA Tg rabbits. (d) Trigeminal ganglia were extracted from HSV-1 infected non-treated HLA Tg rabbits on day 15 post-infection. The effect of α-PD-1 or α-LAG-3 mAbs treatment on virus reactivation, ex vivo from TG explants, was assessed along with that of isotype mAb control.

It was next determined whether the observed reduction of virus replication in the eyes of ASYMP HLA Tg rabbits treated with PD-1 and LAG-3 immune checkpoint blockade was associated with a reduction in virus reactivation locally in the TG, the site of latent HSV-1 infection/reactivation cycles. The viral load during ex-vivo TG reactivation is often determined using RT-PCR as a more sensitive alternative to plaque assay34,36,37. The excised latently infected TG from the same rabbit was digested and equally distributed in control, and the mAb treated wells, so the possibility of variation in DNA copy numbers due to variation in the level of latency does not arise. The amount of reactivated virus detected ex vivo in treated and untreated HSV-1 infected TG explants was determined daily for 8 days post-treatment. As shown in Fig. 2d, the HSV-1 infected TG explants incubated with α-PD-1, or α-LAG-3 mAbs showed a significant decrease of the reactivated virus on day 8 following the blockade (P < 0.05). The blockade of the LAG-3 pathway (P = 0.04) appeared slightly better in reducing virus reactivation from TG compared to blockade of the PD-1 pathway (P < 0.05).

Altogether, these results demonstrate that blockade of PD-1 and/or LAG-3 immune checkpoints reduced recurrent ocular herpes infection and disease in vivo in latently infected HLA Tg rabbits. The observed reduction of virus replication and recurrent ocular herpetic disease in PD-1 and LAG-3 treated HLA Tg rabbits was associated with a significant reduction in virus reactivation locally in the HSV-1 infected TG.

Since the HLA Tg rabbit model develops human-like CD8+ T cell responses to HLA-A*0201 restricted epitopes3,21,38, it offers the possibility to determine the phenotype, function, and transcriptome of TG-resident HLA-A*0201 restricted HSV-1 epitopes-specific CD8+ T cells associated with symptomatic vs. asymptomatic recurrent ocular herpes.

Single-cell RNA sequencing revealed CD8+ T cells and monocytes as the most significant CD45+ leukocytes infiltrating trigeminal ganglia of “protected” asymptomatic HLA Tg rabbits

TG-derived CD45+ leukocytes were sorted by fluorescence-activated cell sorting (FACS) from (1) SYMP HLA Tg rabbits (n = 4); and (2) ASYMP HLA Tg rabbits (n = 4), as illustrated in Figs. 2a and 3a and subsequently processed for single-cell RNA sequencing (scRNASeq) using the 10 × Genomics platform. Eight cell populations were observed among the sorted CD45+ leukocytes from TG of ASYMP and SYMP groups: CD4+ T cells, CD8+ T cells, NK cells, B cells, macrophages, monocytes, granulocytes, and dendritic cells (Fig. 3b).

Figure 3

figure3

Single-cell RNA sequencing of trigeminal ganglia-resident CD45+ leukocytes from HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits. (a) Illustration of the experimental design and validation of differentially expressed genes in CD45+ leukocytes sorted on day 15 p.i. from the trigeminal ganglia (TG) of SYMP and ASYMP HLA Tg rabbits. (b) Heatmap expression of the most significant 140 differentially expressed genes among eight different clusters detected in TG-resident CD45+ leukocytes from HSV-1 infected SYMP and ASYMP HLA Tg rabbits (top two heatmap panels). Each cluster represents an individual immune cell population, determined on the basis of specific molecular markers: CD8+ T cells (CD8A), CD4+ T cells (CD4), NK cells (NKG7), B cells (CD19), macrophages (CD68), monocytes (CD14), granulocytes (FUT4) and dendritic cells (CD1c). The t-SNE dimensionality reduction, applied to single-cell RNA sequencing data revealed eight distinct clusters of immune cell populations among CD45+ leukocytes for the TG of HSV-1 infected ASYMP HLA Tg rabbits (middle panels). The total number of differentially expressed genes within each immune cell clusters (nCount) (lower panels). (c) Average frequencies of different immune cell populations detected within TG-resident CD45+ leukocytes of SYMP and ASYMP HLA Tg rabbits. (d) Volcano plot illustrates the total copy number reads observed for all the genes within one single cell (nFeature).

CD8+ T cells (defined by CD8A gene) and monocytes (defined by CD14 gene) represented the most frequent CD45+ leukocyte populations in the TG of “protected” ASYMP HLA Tg rabbits compared to TG of “non-protected” SYMP HLA Tg rabbits. We detected a total of 198 (26.7%) CD8+ T cells per TG in ASYMP HLA Tg rabbits, while only 116 (15.6%) CD8+ T cells were found in the TG of SYMP HLA Tg rabbits (Fig. 3c). After CD8+ T cells, the next most significant cell population detected in the TG of ASYMP HLA Tg rabbits were the monocytes at a frequency of 20.4% of the total cell population in comparison to only 4.3% among the SYMP HLA Tg rabbits (Fig. 3c). A relatively higher mRNA copy number was also found in the TG of ASYMP HLA Tg rabbits (Fig. 3d).

These results indicate that: (1) anti-PD-1 and anti-LAG-3 blockade induced compartmental remodeling of TG-infiltrating immune cells of HSV-infected HLA Tg rabbits; and (2) expansion of CD8+ T cells and monocytes in the TG of HSV-1 infected asymptomatic HLA Tg rabbits is associated with reduced virus reactivation and less recurrent ocular herpetic disease.

Up-regulation of major T cell exhaustion pathways confirmed by bulk RNA sequencing in TG-resident HSV-specific CD8+ T cells from symptomatic HLA Tg rabbits

TG-derived CD8+ T cells, specific to three HLA-A*0201-restricted HSV-1 epitopes selected from the glycoprotein B (gB561–569), the glycoprotein D (gD53–61), and the tegument protein VP11/12 (VP11–12702–710), were enriched by fluorescence-activated cell sorting (FACS) from: (1) SYMP HLA Tg rabbits (n = 2); and (2) ASYMP HLA Tg rabbits (n = 2), as illustrated in Fig. 4a. Higher frequencies of CD8+ T cells specific to HLA-A*0201-restricted HSV-1 epitopes gB561–569 (6.5% vs. 3.3%, P = 0.01), VP11–12702–710 (9.6% vs. 7.8%, P = 0.03), and gD53–61 (7.4% vs. 5.1%, P = 0.01) were detected in HSV-1 infected and protected ASYMP HLA Tg rabbits compared to non-protected SYMP HLA Tg rabbits (Fig. 4b).

Figure 4

figure4

Differential gene expression in HSV-specific CD8+ T cells from trigeminal ganglia of HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits. (a) Experimental design and validation of differentially expressed genes in CD8+ T cells sharing the same HSV-1 epitope-specificities, from SYMP and ASYMP HLA Tg rabbits. CD8+ T cells specific to HLA-A*0201-restricted HSV-1 gB561–567, VP11/12702–710, and gD53–61 epitopes were sorted from TG of HLA-A*0201-positive SYMP and ASYMP HLA Tg rabbits, using specific tetramers. Total RNA was extracted from each clone of epitope-specific CD8+ T cells, and whole transcriptome analysis was performed using bulk RNA sequencing to determine the levels of expression of 23,669 rabbit genes (OryCun2.0 (GCA_000003625.1). (b) Frequencies of CD8+ T cells specific to HLA-A*0201-restricted HSV-1 gB561–567, VP11/12702–710, and gD53–61 epitopes detected by FACS in TG of HLA-Tg rabbits. (c) The heatmap is showing the most significant 2,879 differentially expressed genes among SYMP and ASYMP HLA Tg rabbits. Genes with minimum count per million (CPM) ≥ 0.5 were used for obtaining the transformed counts data for clustering using regularized log (rlog). (d) Bulk RNA heatmap shows the pathways that are different among ASYMP and SYMP HLA Tg rabbits. Genes differentially expressed in both single-cell RNA sequencing and bulk RNA sequencing were considered for pathway analyses. Parametric gene set enrichment analysis (PSGEA) method based on data curated in Gene Ontology and KEGG was applied. Pathway significance cut-off with a false discovery date (FDR) ≥ 0.2 was applied.

Total RNA was isolated from sorted CD8+ T cells and subsequently processed for bulk RNA sequencing to screen the whole rabbit transcriptome. Our gene expression analysis revealed 12,689 genes among CD8+ T cells, with the same HSV-1 epitope-specificities, from ASYMP vs. SYMP HLA Tg rabbits with a minimum count per million (CPM) value ≥ 0.5. Out of these genes, 2,879 genes were found to be statistically significant and differentially expressed between ASYMP and SYMP HSV-specific CD8+ T cells using stringent criteria (FDR-adjusted P < 0.05, fold change ≥ 2, paired moderated two-tailed t test) (Fig. 4c). Further, among the 2,879 genes, we found 1,605 genes were up-regulated, while 1,274 genes were down-regulated in HSV-specific CD8+ T cells from TG of ASYMP HLA Tg rabbits (Supplementary Fig. S1b, upper panel). Correlation matrix and Pearson’s coefficient confirmed a high degree of relatedness amongst the SYMP and ASYMP HLA Tg rabbit groups (Supplementary Fig. S1b, middle panel). The Volcano plot showed significant log2 fold changes and − log10 (P value) of each of the differentially expressed genes in CD8+ T cells from SYMP vs. CD8+ T cells, that shared the same HSV-1 epitopes, from SYMP HLA Tg rabbits (Supplementary Fig. S1b, lower panels).

The pathway enrichment analysis carried out on the basis of differentially expressed genes among scRNA-Seq, and bulk RNA-Seq demonstrated downregulation of prominent T cell exhaustion molecules present in the cell adhesion molecules (CAMs) pathway (P = 0.04) (Accession no: ocu04514) among the ASYMP group (Fig. 4d). The CAMs pathway is comprised of PD-1-, LAG-3-, TIM3-, TIGIT-, CTLA4-, PSGL-1 genes. In contrast, the T cell activation pathway (P = 0.03) (Accession no: GO: 0042110), the chemokine-chemokine receptor signaling pathway (P = 0.04) (Accession no: ocu04062), and the cytokine-cytokine receptor interaction (P = 0.04) (Accession no: ocu04060) were all up-regulated in TG-resident HSV-specific CD8+ T cells from ASYMP HLA Tg rabbits (Fig. 4d).

These results indicate that similar to SYMP patients above, in SYMP HLA Tg rabbits, there was an up-regulation of T cell exhaustion pathways in HSV-specific CD8+ T cells associated with frequent and severe recurrent ocular herpetic disease.

High frequencies of HSV-specific memory CD8+ TEM and CD8+ TRM cell subsets, with upregulated T-cell activation pathways, detected in TG of asymptomatic HLA Tg rabbits

We next compared the frequencies of the three major subsets of memory CD8+ T cells that share the same HSV-1 epitope-specificities and are present in the TG of ASYMP vs. SYMP HLA Tg rabbits. As illustrated in Fig. 2a above, HLA Tg rabbits (n = 8) were first infected with 2 × 105 pfu of HSV-1 (McKrae Strain).

As shown in Fig. 5a, several genes associated with T-cell activation pathway were upregulated in HSV-specific CD8+ T cells from TG of “protected” ASYMP HLA Tg rabbits, as compared to HSV-specific CD8+ T cells from TG of “non-protected” SYMP HLA Tg rabbits (P < 0.05). Prominent genes that were found to be significantly upregulated among ASYMP group include CD69 (P = 0.03, logFC = 2.72), CD62L (P = 0.004, logFC = 6.48), CD44 (P = 1.52E−06, logFC = 2.79), CD107 (P = 7.04E–05, logFC = 2.14), and IFN-γ (P = 0.01, logFC = 2.03) (Fig. 5a; Supplementary Table S1).

Figure 5

figure5

Activation and exhaustion genes differentially expressed in trigeminal ganglia-resident HSV-specific CD8+ T cells from HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits. (a) Expression of T cell activation genes (CD69, CD62L, CD44, CD107, and IFN-γ) detected by single-cell RNA sequencing from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (top panels). Expression of T cell exhaustion genes (PD-1, LAG-3, CTLA4, ICOS, and BLIMP1) detected by single-cell RNA sequencing from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (lower panels). (b) Average frequencies of specific genes representing memory CD8+ TCM, CD8+ TEM, and CD8+ TRM cell subsets from TG of SYMP vs. ASYMP HLA Tg rabbits (left panel). Average frequencies of CD8+ TRM cells expressing various exhaustion genes in HSV-1 infected TG of in SYMP vs. ASYMP HLA Tg rabbits (top right panel). Average frequencies of functional CD107a/b+IFN-γ +CD8+ TRM cells in HSV-1 infected TG of in SYMP vs. ASYMP HLA Tg rabbits (lower right panel). (c) Bulk RNA sequencing showing expression of T-cell activation (left panel) and T-cell exhaustion genes (right panel) in HSV-specific TRM cells from SYMP vs. ASYMP HLA Tg rabbits. (d) Frequencies of memory CD8+ TCM, CD8+ TEM, and CD8+ TRM cell subsets detected by FACS in HSV-1 infected TG of SYMP vs. ASYMP HLA Tg rabbits. (e) Fluorescence microscopy images showing infiltration of CD8+ T cells in HSV-1 infected TG from SYMP vs. ASYMP HLA Tg rabbits. TG sections were co-stained using DAPI and mAb specific to rabbit CD8+ T cells (magnification, × 20). Blue, DAPI: DNA, green: CD8+ T cells.

In the cell cluster representing 198 CD8+ T cells; 86 cells (43.4%) expressed CD69 gene, 28 cells (14.1%) expressed CD62L gene, 54 cells (27.2%) expressed CD44 gene, and 8 cells (4.5%) expressed CD107 gene (Fig. 5b). Notably, the IFN-γ and CD107 genes were expressed in 22.09% (P = 0.005) and 53.4% (P = 0.02) of CD69+CD8+ TRM cells in TG of ASYMP HLA Tg rabbits (Fig. 5b, left lower panel). In contrast, only ~ 20% of CD69+CD8+ TRM cells from TG of “non-protected” SYMP HLA Tg rabbits expressed genes for the PD-1 and LAG-3 exhaustion pathway compared to less than 5% of CD69+CD8+ TRM cells from TG of “protected” SYMP HLA Tg rabbits that expressed PD-1 and LAG-3 exhaustion genes (P ≤ 0.01) (Fig. 5b, left upper panel).

The CAMs pathway-specific genes were significantly down-regulated in TG-resident CD103+CD69+CD8+ TRM cells from ASYMP HLA Tg rabbits compared to TG-resident CD103+CD69+CD8+ TRM cells, with same epitopes-specificities, from SYMP HLA Tg rabbits. Major T cell exhaustion molecules like PD-1 (P = 0.005, logFC = − 2.02), LAG-3 (P = 0.04, logFC = − 4.15), CTLA4 (P = 0.02, logFC = − 1.78), were found to be downregulated among ASYMP HLA Tg rabbits in the CD8+ TRM cells as evidenced from the single cell RNA sequencing results (Fig. 5a; Supplementary Table S2). Whereas, Inducible T-cell Co-Stimulator (ICOS) gene (P = 0.04, logFC = 4.03), and PRDM1 (BLIMP1) (P = 0.01, logFC = 3.1) known to be associated with T cell activation and differentiation were observed with a higher expression in the TG resident TRM cells among ASYMP HLA Tg rabbits (Fig. 5a; Supplementary Table S2).

PD-1 and LAG-3 genes were expressed in 21.6% and 18.9% of TG-resident CD103+CD69+CD8+ TRM cells in the SYMP HLA Tg rabbits compared to only 1.16% and 2.3% of TG-resident CD103+CD69+CD8+ TRM cells in the ASYMP HLA Tg rabbits (Fig. 5b).

When differential gene expression analysis was carried out between ASYMP vs. SYMP groups using bulk RNA sequencing, TIGIT (P = 0.05, logFC = − 2.70), CTLA4 (P = 0.01, logFC = − 1.04), PD-1 (P = 1.01E−06, logFC = − 5.58) genes were found to be significantly down-regulated along with other T cell exhaustion molecules (Fig. 5c, right panel; Supplementary Table S2). The bulk RNA sequencing-based on differential gene expression (DGE) analysis performed on TG-resident memory CD103+CD69+CD8+ TRM cells, with the same HSV-1 epitope-specificities, confirmed a heightened expression level of several genes associated with the T cell activation pathway in “protected” ASYMP HLA Tg rabbits (Fig. 5c). In contrast, significantly higher expression levels of several genes associated with the T cell exhaustion were confirmed by bulk RNA sequencing in “non-protected” SYMP HLA Tg rabbits (Fig. 5c). Importantly, the single-cell RNA sequencing-based transcriptome of CD45+ leukocytes clearly showed increased frequencies of memory CD8+ TRM and CD8+ TEM cell subsets in the TG of “protected” ASYMP HLA Tg rabbits.

Moreover, these genomic results were supported by FACS-based immunophenotyping, which confirmed higher frequencies of the memory CD8+ TRM cell subset (ASYMP = 69.2% vs SYMP = 43.6%, P = 0.02) and memory CD8+ TEM cell subset expressing adhesion molecules, CD69 (ASYMP = 18.3% vs SYMP = 5.1%, P = 0.02) and CD103 (ASYMP = 23.2% vs SYMP = 6.5%; P = 0.01) in the TG of “protected” ASYMP HLA Tg rabbits (Fig. 5d). More-so, an increased infiltration of CD8+ T cells in the TG of ASYMP rabbits was visualized by immunostaining (Fig. 5e).

Interestingly, Fig. 5d shows upregulation of CD62L protein expression at the surface of the HSV-specific CD8+ T-cells from SYMP compared to ASYMP individuals, whereas the single-cell and bulk RNAseq data panels on Fig. 5a–c show downregulation of the CD62L mRNA expression in SYMP subjects. While the protein expression for the other markers (CD69, CD44, CD103) follows their mRNA expression, this does not seem to be the case with CD62L. This may be due to post-transcriptional/translational modification/regulation. Thus, it appears that the expression of CD62L on the surface on the T-cell (at the protein level) is regulated not only at the transcriptional level but also at a post-transcriptional level. In previous reports, it has been shown that the loss of CD62L by the activated T-cells can be regulated by the cleavage of CD62L from the cell membrane at K283–S284 by a disintegrin and metalloprotease ADAM17 in a process called CD62L shedding39,40,41,42.

Heightened expression of genes for T cell attracting chemokines/receptors and T cell maintaining cytokines/receptors detected in TG-resident CD8+ TRM cells from asymptomatic HLA Tg rabbits

The higher frequencies of HSV-specific CD8+ TRM cell subset observed in the TG of “protected” ASYMP HLA Tg rabbits prompted us to further compare in CD8+ T cells TG from SYMP vs. ASYMP HLA Tg rabbits the differential gene expression profile of genes for cytokines, chemokines and their receptors that are involved in T cell homing and T cell maintaining.

Using both the single cell and bulk RNA sequencing methods, we detected a significant up-regulation of several genes for T cell attracting chemokines and chemokine receptors in TG-resident HSV-specific CD103+CD69+CD8+ TRM cells from ASYMP HLA Tg rabbits. The genes found to be significantly upregulated among ASYMP HLA Tg rabbits represented CC-, CXC-, and CX3C-subfamilies of chemokines including CCL5 (P = 4.13E−26, logFC = 3.29), CCL14 (P = 0.05, logFC = 3.02), CCR7 (P = 7.52E−05, logFC = 5.98), and CXCR3 (P = 0.02, logFC = 2.63). (Fig. 6a,c, left panel; Supplementary Table S3). CCL5, CCR7, and CXCR3 genes were expressed in 6.9%, 16.2%, and 32.5% of CD8+ TRM cells in the ASYMP group compared to 2.7%, 5.4%, and 10.8% of CD8+ TRM cells, with the same epitope specificities, in the SYMP group (Fig. 6b, upper panel).

Figure 6

figure6

Genes of cytokines/chemokines and receptors differentially expressed in trigeminal ganglia-resident HSV-specific CD8+ T cells from HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits. (a) Expression of genes of chemokines and chemokine receptors detected by single-cell RNA sequencing from TG-resident HSV-specific CD8+ T cells from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (top panels). Expression of genes of cytokines and cytokine receptors detected by single-cell RNA sequencing from TG-resident HSV-specific CD8+ T cells from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (lower panels). (b) Average frequencies of CD8+ TRM cells expressing genes of cytokines/chemokines and receptors in TG of HSV-1 infected SYMP vs. ASYMP HLA Tg rabbits. (c) Bulk RNA sequencing showing expression of genes of chemokines/chemokine receptors (left panel) and genes of cytokines/cytokine receptors (right panel) in HSV-specific TRM cells from SYMP vs. ASYMP HLA Tg rabbits. (d) Representative (left panels) and average (right panels) frequencies of CCR3+CD8+ TRM cells in TG of HSV-1 infected SYMP vs. ASYMP HLA Tg rabbits. (e) Fluorescence microscopy images showing infiltration of HSV-1 infected TG from SYMP vs. ASYMP HLA Tg rabbits by CD8+ T cells expressing T cell-attracting chemokines and receptors (i.e., CXCR3, CXCL9, CXCL10, and CXCL11) (magnification, × 20). Blue: DAPI (DNA stain); red: CXCR3 cells.

Single cell and bulk RNA sequencing results (Fig. 6a,c, right panel) also indicate that several genes associated with the cytokine–cytokine receptor interaction pathway that are known for maintaining T cells within tissues were up-regulated among the ASYMP group of HLA Tg rabbits: IL-17R (P = 0.008, logFC = 2.13), IL-15R (P = 0.01, logFC = 2.51), IL6ST (P = 0.04, logFC = 6.69), IL7R (P = 4.13E−07, logFC = 4.35), CD137 (P = 2.17E−12, logFC = 2.35) to be upregulated in the CD8+ TRM cells among the ASYMP group of HLA Tg rabbits (Fig. 6a; Supplementary Table S4). The expression of these cytokines/cytokine receptors were significantly increased among the CD8+ TRM cell subset in ASYMP HLA Tg rabbits. The expression of genes IL-17R, IL-15R, IL6ST, IL7R, and CD137 (TNFRSF9) was in 22.09% (P = 0.004), 15.1% (P = 0.02), 30.2% (P = 0.002), 38.3% (P = 0.01) and 19.7% (P = 0.003) of CD8+ TRM cells, respectively (Fig. 6b).

Furthermore, we confirmed higher frequencies of CXCR3+CD8+ TRM cells in TG of ASYMP HLA Tg rabbits compared to TG of SYMP HLA Tg rabbits (ASYMP = 24.5%, SYMP = 7.3%; P = 0.03) by FACS (Fig. 6d). Using differential gene expression analysis between the ASYMP vs. SYMP groups, the genes for T cell attracting-chemokines CXCL9 (P = 0.003, logFC = 3.70), CXCL10 (P = 0.001, logFC = 4.32), and CXCL11 (P = 0.01, logFC = 2.84) genes were also found to be significantly up-regulated in TG-resident CD8+ TRM cells from “protected” ASYMP HLA Tg rabbits compared to TG-resident CD8+ TRM cells from “non-protected” SYMP HLA Tg rabbits (Fig. 6c). Similarly, increased infiltration of CXCR3+CD8+ TRM cells in the TG of ASYMP rabbits was visualized by immunostaining (Fig. 6e).

Altogether, these results suggest that a heightened activation of T cell attracting chemokines/receptors and T cell keeping cytokines/receptors pathways may lead to increased infiltration/retention of protective antiviral CXCR3+CD8+ TRM cells observed in the TG of “protected” ASYMP HLA Tg rabbits, as illustrated in Fig. 7.

Figure 7

figure7

TG-resident HSV-specific memory CD8+ TRM cells downregulate the T cell exhaustion associated pathway and confer protection from ocular herpes in HSV-1 infected asymptomatic humans and HLA transgenic rabbits. (1) Upon exposure to stressors, the HSV-1 enters into the cornea and travels through neurons to Trigeminal ganglia. (2) Following primary HSV-1 infection, the vast majority (up to 95%) of antiviral effector CD8+ T cells die, leaving behind only about 5% of CD8+ T cells destined to differentiate into a heterogeneous pool of memory CD8+ T cells. (3) The effector memory (TEM) and tissue-resident memory (TRM) CD8+ T-cell subsets are found mainly in the HSV-infected but “naturally protected” asymptomatic subjects, whereas the lymphoid organ-resident central memory (TCM) CD8+ T cell subsets are mainly present in non-protected Symptomatic subjects. (4) Reduced viral reactivation was observed among asymptomatic subjects possessing a higher frequency of CD8+ TRM cells resulting in a less severe herpes disease. (5) The findings study suggests that by blocking immune checkpoints, there is a reduced expression of T cell exhaustion molecules (PD-1, LAG-3, PSGL-1, CTLA-4, TIM3, and TIGIT) and T cell exhaustion associated Cell Adhesion Molecule pathway and increased retention of CD8+ TRM cell population in asymptomatic subjects. This memory CD8+ T cell population mediates recall responses and halts attempts of virus reactivation in the infected TG, thus accelerating viral clearance. More-so, reduced expression of T cell exhaustion pathway also gives rise to higher expression of genes associated with T cell function (CD107, IFN-γ), T cell homing (CXCR3, CCR7), and T-cell keeping (IL7R, IL15R). This helps in reducing the ocular herpes infection and recurrent herpetic disease.

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