Phenotypic and Functional Signatures of Herpes Simplex Virus–Specific Effector Memory CD73+CD45RAhighCCR7lowCD8+ TEMRA and CD73+CD45RAlowCCR7lowCD8+ TEM Cells Are Associated with Asymptomatic Ocular Herpes

Ruchi Srivastava, Pierre-Grégoire Coulon, Soumyabrata Roy, Sravya Chilukuri, Sumit Garg and Lbachir BenMohamed
J Immunol October 15, 2018, 201 (8) 2315-2330; DOI: https://doi.org/10.4049/jimmunol.1800725

Abstract

HSV type 1 (HSV-1)–specific CD8+ T cells protect from herpes infection and disease. However, the nature of protective CD8+ T cells in HSV-1 seropositive healthy asymptomatic (ASYMP) individuals (with no history of clinical herpes disease) remains to be determined. In this study, we compared the phenotype and function of HSV-specific CD8+ T cells from HLA-A*02:01–positive ASYMP and symptomatic (SYMP) individuals (with a documented history of numerous episodes of recurrent ocular herpetic disease). We report that although SYMP and ASYMP individuals have similar frequencies of HSV-specific CD8+ T cells, the “naturally” protected ASYMP individuals have a significantly higher proportion of multifunctional HSV-specific effector memory CD8+ T cells (CD73+CD45RAhighCCR7lowCD8+ effector memory RA (TEMRA) and CD73+CD45RAlowCCR7lowCD8+ effector memory (TEM) as compared with SYMP individuals. Similar to humans, HSV-1–infected ASYMP B6 mice had frequent multifunctional HSV-specific CD73+CD8+ T cells in the cornea, as compared with SYMP mice. Moreover, in contrast to wild type B6, CD73−/− deficient mice infected ocularly with HSV-1 developed more recurrent corneal herpetic infection and disease. This was associated with less functional CD8+ T cells in the cornea and trigeminal ganglia, the sites of acute and latent infection. The phenotypic and functional characteristics of HSV-specific circulating and in situ CD73+CD8+ T cells, demonstrated in both ASYMP humans and mice, suggest a positive role for effector memory CD8+ T cells expressing the CD73 costimulatory molecule in the protection against ocular herpes infection and disease. These findings are important for the development of safe and effective T cell–based herpes immunotherapy.

Introduction

Herpes simplex virus type 1 (HSV-1) infection is widespread in human populations (1–5). A staggering 3.72 billion individuals worldwide currently carry the virus that causes a wide range of mild to life-threatening diseases (1–7). Complications range from mild, such as cold sores and genital lesions, to more serious complications, including permanent brain damage from encephalitis in adults and neonates and blinding corneal inflammation (5, 8). HSV infections are prevalent and permanent, as the virus establishes latency in the neurons of sensory ganglia after a primary infection (9–12). Although the virus reactivates from latency and is shed multiple times each year in body fluids (i.e., tears, saliva, nasal and vaginal secretions), most reactivations are subclinical due to an efficient immune-mediated containment of the infection and disease (2, 3, 13, 14). Thus, most infected individuals are asymptomatic (ASYMP) and do not present any apparent recurrent herpetic disease (e.g., cold sores, genital or ocular herpetic disease). However, a small proportion of individuals experience endless recurrences of herpetic disease, usually multiple times a year, often necessitating continuous antiviral therapy (i.e., Acyclovir and derivatives) (15, 16). In those symptomatic (SYMP) individuals, HSV-1 frequently reactivates from latency, reinfects the eyes, and may trigger recurrent and severe corneal herpetic disease, a leading cause of infectious corneal blindness in the industrialized world (17–19). In the United States, up to 450,000 individuals have a history of recurrent herpetic stromal keratitis (HSK), a T cell–mediated immunopathological lesion of the cornea (17–19). Ergo, a better understanding of the immune mechanisms that protect from HSV-1 is highly desirable for the development of more efficacious vaccines and immunotherapies to reduce herpes infection and disease.

Despite recent progress, a clear understanding of the molecular and cellular basis of memory T cells in herpes simplex infection is still lacking. In animal models of herpes infection and disease, HSV-specific memory CD8+ T cells play a critical role in aborting attempts of virus reactivation from latency and in reduction of herpetic disease (1, 7, 13, 20–22). However, herpetic corneal disease is also associated with HSV-specific CD8+ T cell responses (23, 24). Although HSV glycoprotein B (gB) and glycoprotein D (gD) are major targets of CD8+ T cells in seropositive ASYMP individuals (14, 25), they only produced a transient protective immunity in vaccine clinical trials (19, 26, 27). In B6 mice, an immunodominant CD8+ T cell epitope, gB498–505, achieved at least partial protection against herpes infection and disease (15, 19, 28, 29). Considering the wealth of data addressing the phenotype and function of HSV-1 gB498–505 epitope-specific CD8+ T cells in B6 mice (2, 3, 8, 13, 30), it is surprising how only a few reports characterizing the phenotype and function of “protective” CD8+ T cells, specific to human epitopes (instead of mouse epitopes) that are developed from HSV-seropositive healthy ASYMP individuals who appear to have acquired a “natural” protection from recurrent herpetic disease (1, 31), actually exist. This information is necessary for the successful design of effective T cell–based immunotherapeutic strategies.

Although memory CD8+ T cell subpopulations are heterogeneous in terms of phenotype, function, and anatomical distribution, they can generally be divided into two major subsets: effector memory (TEM) cells and central memory CD8+ T (TCM) cells (31, 32). We recently reported two distinct phenotypic and functional patterns of protective and nonprotective HSV-1 gB-specific CD8+ T cells that are associated with ASYMP versus SYMP ocular herpes, respectively. Whereas a significantly higher proportion of HSV-1 gB-specific CD8+ TEM cells were detected in ASYMP individuals, a significantly higher proportion of HSV-1 gB-specific CD8+ TCM cells were detected in SYMP patients. The mechanisms by which HSV-specific CD8+ TCM and TEM cells play different roles in herpes infection and disease remain to be fully determined.

The CD73 receptor is both a coactivator molecule of T cells and an immunosuppressive ectoenzyme through adenosine production (33, 34). However, the precise role of the CD73 costimulatory molecule and its involvement in CD8+ T cell function during HSV infection has not been reported. In this report, we hypothesized that 1) CD73 may have a role in the modulation of T cell responses to herpes infection and disease, and 2) ASYMP individuals develop more protective HSV-specific CD73+CD8+ TEM cells, whereas SYMP patients develop more nonprotective (or possibly pathogenic) HSV-specific CD73+CD8+ TCM cells, compared with SYMP individuals (35, 36). In this article, we report for the first time, to our knowledge, that “naturally” protected ASYMP individuals have a significantly higher proportion of multifunctional HSV-1 VP11/12220–228 epitope-specific (CD73+CD45RAhighCCR7lowCD8+ effector memory RA [TEMRA] and CD73+CD45RAlowCCR7lowCD8+ TEM cells) when compared with SYMP individuals who present with repetitive recurrent herpetic disease. Moreover, ASYMP wild type (WT) B6 mice, which are ocularly infected with HSV-1 but did not develop corneal disease, have more HSV-specific CD73+CD8+ T cells in the cornea and trigeminal ganglia (TG), compared with SYMP mice that did develop corneal disease. Similar to ASYMP humans, the HSV-specific effector CD73+CD8+ T cells from ASYMP mice are multifunctional. In contrast to WT B6 mice, CD73−/− deficient mice develop significantly more herpes infection and disease. These findings draw attention to a role of circulating and in situ effector memory CD73+CD8+ T cells in protection against herpes infection and disease, and this should unequivocally be considered in the development of a safe and effective T cell–based herpes immunotherapy.

Materials and Methods

Human study population

All clinical investigations in this study were conducted according to the Declaration of Helsinki. All subjects were enrolled at the University of California, Irvine, under approved Institutional Review Board–approved protocols (no. 2003-3111 and no. 2009-6963). Written informed consent was received from all participants prior to inclusion in the study.

During the last 15 y (i.e., January 2003 to January 2018), we have screened 875 individuals for HSV-1 and HSV-2 seropositivity. Five hundred seventy-four were white, 301 were nonwhite (African, Asian, Hispanic, and other), 446 were female, and 429 were male. Among this sample, a cohort of 306 immunocompetent individuals, ranging from 21 to 67 y old (median 39), were seropositive for HSV-1 and seronegative for HSV-2. All patients were negative for HIV and hepatitis B virus, with no history of immunodeficiency. Seven hundred ninety-two patients were HSV-1, HSV-2, or HSV-1/HSV-2 seropositive; among them, 698 patients were healthy and defined as ASYMP. These patients have never had any herpes disease (ocular, genital, or dermal) based on self-reporting and clinical examination. Even a single episode of any herpetic disease would exclude the individual from this group. The remaining 94 patients were defined as HSV-seropositive SYMP individuals who suffered from frequent and severe recurrent genital, ocular, and/or orofacial lesions. Signs of recurrent disease in SYMP patients were defined as herpetic lid lesions, herpetic conjunctivitis, dendritic or geographic keratitis, stromal keratitis, and iritis consistent with HSK, with one or more episodes per year for the past 2 y. However, at the time of blood collection, SYMP patients had no recurrent disease (other than corneal scarring) and had no recurrences during the past 30 d. They had no ocular disease other than HSK, no history of recurrent genital herpes, and were HSV-1 seropositive and HSV-2 seronegative. Because the spectrum of recurrent ocular herpetic disease is wide, our emphasis was mainly focused on the number of recurrent episodes and not on the severity of the recurrent disease. No attempt was made to assign specific T cell epitopes to the severity of recurrent lesions. Patients were also excluded if they 1) had an active ocular (or elsewhere) herpetic lesion, or had one within the past 30 d, 2) were seropositive for HSV-2, 3) were pregnant or breastfeeding, or 4) were on Acyclovir and other related antiviral drugs or any other immunosuppressive drugs at the time of blood draw. Among this large cohort of SYMP and ASYMP individuals, 29 patients were enrolled in this study (Table I). SYMP and ASYMP groups were matched for age, gender, serological status, and race. We also collected and tested blood samples from 10 healthy control individuals who were seronegative for both HSV-1 and HSV-2 and had no history of ocular herpes, genital lesions, or orofacial herpes disease.

HSV-specific serotyping through ELISA

The sera collected from random donors were tested for anti-HSV Abs. ELISA was performed on sterile 96-well flat-bottom microplates coated with the HSV-1 Ag in coating buffer overnight at 4°C. The next day, plates were washed with PBS–1% Tween 20 five times. Nonspecific binding was blocked by incubating them with a 5% solution of skimmed milk in PBS (200 μl/well) at 4°C for 1 h at room temperature (RT). The microplates were washed three times with PBS–Tween and incubated with various sera at 37°C for 2 h. Following five washes, biotinylated rabbit anti-human IgG, diluted 1:20,000 with PBS–1% Tween 20, was used as the secondary Ab and incubated at 37°C for 2 h. After five washes, streptavidin was added at a 1:5000 dilution and incubated for 30 min at RT. After five additional washes, the color was developed by adding 100 μl of TMB substrate. The mixture was incubated for 5–15 min at RT in the absence of light. The reaction was terminated by adding 1 M of H2SO4. The absorbance was measured at 450 nm.

HLA-A2 typing

The HLA-A2 status was confirmed by PBMC staining with 2 μl of anti–HLA-A2 mAb (clone BB7.2) (BD Pharmingen, Franklin Lakes, NJ), at 4°C for 30 min. The cells were washed and analyzed by flow cytometry using an LSR II (Becton Dickinson, Franklin Lakes, NJ). The acquired data were analyzed with FlowJo software (BD Biosciences, San Jose, CA).

Tetramer/VP11/12 peptide staining

Fresh PBMCs were analyzed for the frequency of CD8+ T cells recognizing the VP11/12 peptide/tetramer complexes, as we previously described (9–12). The cells were incubated with VP11/12 peptide/tetramer complex for 30–45 min at 37°C. The cell preparations were then washed with FACS buffer and stained with FITC-conjugated anti-human CD8 mAb (BD Pharmingen). The cells were washed and fixed with 1% paraformaldehyde in PBS and subsequently acquired on a BD LSRII. Data were analyzed using FlowJo version 9.5.6 (Tree Star, Ashland, OR).

CD107 cytotoxicity assay

To detect VP11/12-specific cytolytic CD8+ T cells in PBMC, spleen, TG, and cornea cells, intracellular CD107a/b cytotoxicity assay was performed as described by Betts et al. (37, 38) with a few modifications. Briefly, 1 × 106 PBMCs from patients, in addition to spleen cells, DLN cells, and TG cells from HSV-infected mice, were transferred into 96-well V-bottom plates in R10 medium and stimulated with VP11/12 peptide (10 μg/ml) in the presence of anti–CD107a-FITC and CD107b-FITC (BD Pharmingen) and BD GolgiStop (10 μg/ml) for 5–6 h at 37°C. PHA (10 μg/ml) (Sigma-Aldrich, St. Louis, MO) and no peptide were used as positive and negative controls, respectively. At the end of the incubation period, the cells were harvested into separate tubes and washed once with FACS buffer and then stained with PE-conjugated anti-human CD8 Ab for 30 min. Cells were then fixed, permeabilized, and stained with additional Abs against IFN-γ and TNF-α using Fixation/Permeabilization and Perm/Wash solution (BD Pharmingen).

Human PBMCs isolation

Individuals (negative for HIV and hepatitis B virus, and with or without any HSV infection history) were recruited at the University of California, Irvine, Institute for Clinical and Translational Science. Between 40 and 100 ml of blood was drawn into yellow-top Vacutainer Tubes (Becton Dickinson). The serum was isolated and stored at −80°C for the detection of anti–HSV-1 and HSV-2 Abs, as we have previously described (16). PBMCs were isolated by gradient centrifugation using leukocyte separation medium (Corning Life Sciences, Tewksbury, MA). The cells were then washed in PBS and resuspended in complete culture medium consisting of RPMI 1640 and 10% FBS (Gemini Bio-Products, Woodland, CA) supplemented with 1× penicillin/streptomycin/l-glutamine, 1× sodium pyruvate, 1× nonessential amino acids, and 50 μM of 2-ME (Life Technologies, Rockville, MD). Freshly isolated PBMCs were also cryo-preserved in 90% FCS and 10% DMSO in liquid nitrogen for future testing.

Human T cells flow cytometry assays

The following anti-human Abs were used for the flow cytometry assays: CD3 (clone SK7) PE-Cy7, CD8 (clone SK1) allophycocyanin-Cy7, CD73 (AD2) PE-Cy7, PD-1 (clone EH12.1) A647, CD45RA (L48) FITC, CCR7 (clone 3D12) PE-Cy7, IFN-γ (clone B27) Alexa Fluor 647, TNF-α (MAb11) (BD Biosciences), CD107b (clone H4B4) FITC (BioLegend). For surface stain, mAbs against cell markers were added to a total of 1 × 106 cells in 1× PBS containing 1% FBS and 0.1% sodium azide (FACS buffer) for 45 min at 4°C. After washing with FACS buffer, cells were then permeabilized for 20 min on ice using the Cytofix/Cytoperm Kit (BD Biosciences) and then washed twice with Perm/Wash Buffer (BD Biosciences). Intracellular cytokine mAbs were then added to the cells and incubated for 45 min on ice in the dark. Cells were washed again with Perm/Wash and FACS Buffer and fixed in PBS containing 2% paraformaldehyde (Sigma-Aldrich). For each sample, 100,000 total events were acquired on the BD LSR II. Ab capture beads (BD Biosciences) were used as individual compensation tubes for each fluorophore in the experiment. To define positive and negative populations, we employed fluorescence minus controls for each fluorophore used in this study when initially developing staining protocols. In addition, we further optimized gating by examining known negative cell populations for background expression levels. The gating strategy was similar to that used in our previous work (2). Briefly, we gated single cells, dump cells, viable cells (AquaBlue), lymphocytes, CD3+ cells, and CD8+ cells before finally gating human epitope-specific CD8+ T cells using HSV-specific tetramers. Data analysis was performed using FlowJo version 9.9.4 (Tree Star). Statistical analyses were done using GraphPad Prism version 5 (La Jolla, CA).

The intracellular assay to detect IFN-γ, TNF-α, and CD107a/b in response to in vitro peptide stimulations was performed as described (39–41) with a few modifications. On the day of the assay, 1 × 106 PBMCs were stimulated in vitro with peptide (10 μg/ml per peptide) at 37°C for an additional 6 h in a 96-well plate with BD GolgiStop (BD Biosciences) and 10 μl of CD107a FITC and CD107b FITC. PHA (5 μg/ml) (Sigma-Aldrich) and no peptides were used as positive and negative controls, respectively. At the end of the incubation period, the cells were transferred to 96-well round-bottom plate and washed twice with FACS buffer, then stained with PE-conjugated anti-human CD8 for 45 min at 4°C. Intracellular staining for the detection of IFN-γ and CD107a/b was performed as outlined above. The cells were washed again and fixed, then 100,000 total events were acquired on the BD LSR II, and data analysis was performed using FlowJo version 9.9.4 (Tree Star).

Peptide synthesis

HLA-A*0201 binding peptide VP11/12220–228 and HLA-2b binding peptide gB498–505 were synthesized by Magenex (San Diego, CA) on a 9050 Pep Synthesizer instrument using solid-phase peptide synthesis and standard 9-fluorenylmethoxy carbonyl technology (Applied Biosystems, Foster City, CA). Stock solutions were made at 1 mg/ml in PBS. gB498–505 and VP11/12220–228 peptides were aliquoted and were stored at −20°C until assayed.

Virus production

HSV-1 (strain McKrae) was grown and titrated on rabbit skin cells, as we have previously described (3).

Mice

Colonies of WT B6 and CD73 knockout mice (42) were bred and maintained at University of California Irvine, Laboratory Animal Resources, under specific pathogen–free conditions. All studies were conducted in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care and according to Institutional Animal Care and Use Committee–approved animal protocols (IACUC no. 2002-2372).

Ocular infection of mice with HSV-1

Two groups of age-matched female CD73−/− deficient and WT B6 mice were infected with 2 × 105 PFU of strain McKrae as eye drops, without corneal scarification. Control mice were inoculated using mock samples of the virus. Following ocular infection, mice were monitored for ocular herpes infection and disease.

Monitoring of ocular herpes infection and disease

Animals were examined for signs of ocular disease via slit lamps. Clinical assessments were made immediately before inoculation and on days 1, 3, 5, 7, 10, 14, and 21 thereafter. The examination was performed by investigators blinded to the treatment regimen of the mice and scored according to a standard 0–4 scale: 0, no disease; 1, 25%; 2, 50%; 3, 75%; and 4, 100% disease, as previously described (43, 44). Although some mice remained ASYMP (exhibited no symptoms or disease), others developed symptoms and were considered SYMP. At a given and same time point postinfection (PI), mice were segregated into ASYMP mice that are infected but never developed any corneal herpetic disease (score of 0) and SYMP mice that are infected and developed corneal herpetic disease with corneal neovascularization and opacity development of a score of 1–4. To quantify replication and clearance of HSV-1 from the eyes, mice were swabbed daily with moist, type 1 calcium alginate swabs. Swabs were placed in 1.0 ml titration media (Media 199, 2% penicillin/streptomycin, 2% newborn calf serum) and frozen at −80°C until titrated on rabbit skin cell monolayers, as described previously (43, 44).

Statistical analyses

Data for each assay were compared by ANOVA and Student t test using GraphPad Prism version 5.03. Differences between the groups were identified by ANOVA and multiple comparison procedures, as we previously described (45). Data are expressed as the mean ± SD. Results were considered statistically significant at p < 0.05.

Results

HSV-seropositive ASYMP individuals have frequent HSV-1 VP11/12220–228 epitope-specific CD73+CD8+ T cells compared with SYMP individuals

The characteristics of the SYMP and ASYMP study population used in this present study, with respect to gender, age, HLA-A*02:01 frequency distribution, HSV-1/HSV-2 seropositivity, and status of ocular herpetic disease, are presented in Table I and detailed in the Materials and Methods section. Because HSV-1 is the main cause of ocular herpes, only individuals who are HSV-1 seropositive and HSV-2 seronegative were enrolled in the current study. HSV-1–seropositive individuals were divided into two groups: 1) 10 HLA-A*02:01–positive, HSV-1–infected ASYMP individuals who have never had any clinically detectable herpes disease, and 2) 10 HLA-A*02:01–positive HSV-1–infected SYMP individuals with a history of numerous episodes of well-documented recurrent clinical herpes diseases, such as herpetic lid lesions, herpetic conjunctivitis, dendritic or geographic keratitis, stromal keratitis, and iritis consistent with recurrent HSK, with one or more episodes per year for the past 5 y. One patient had over two severe recurrent episodes during the last 10 y that necessitated multiple corneal transplantations. Only SYMP patients who were not on Acyclovir or other antiviral or anti-inflammatory drug treatments at the time of blood sample collections were enrolled.

View this table:
Table I. Cohorts of HLA-A*02:01–positive, HSV-seropositive SYMP and ASYMP individuals enrolled in the study

We first compared the frequency of HSV-specific CD73+CD8+ T cells, using HLA-A*02:01–specific immunodominant VP220–228 epitope tetramer/anti-CD73/CD8 mAbs, in the peripheral blood of nine HLA-A*02:01–positive, HSV-1–seropositive ASYMP and nine HLA-A*02:01–positive, HSV-1–seropositive SYMP individuals and 10 seronegative healthy donors (controls). The low frequencies of PBMC-derived HSV-specific CD8+ T cells complicate a direct ex vivo detection with tetramers using a typical number of PBMC (∼106 cells), and a prior expansion of CD8+ T cells by HSV-1 or peptide stimulation in an in vitro culture will hamper a reliable determination of the frequency, phenotype, and function of epitope-specific CD8+ T cells. We therefore opted to measure the frequencies of VP220–228 epitope-specific CD8+ T cells ex vivo using a large number of PBMCs (∼10 × 106) per tetramer/CD8 mAbs panel.

The representative dot plots shown in Fig. 1A indicate that although similar frequencies of VP220–228 epitope tetramer(+) CD8+ T cells were detected in ASYMP versus SYMP individuals, higher frequencies of CD73+CD8+ T cells specific to the HSV-1 immunodominant VP220–228 epitope were detected in one ASYMP individual (55.8%, left two panels) compared with one SYMP individual (33.2%, right two panels). Fig. 1B shows median frequencies of CD73+CD8+ T cells detected in nine SYMP and nine ASYMP individuals. The highest and most significant frequencies of VP220–228-specific CD73+CD8+ T cells were consistently detected in ASYMP individuals (49.5% ± 10.5%). The frequencies of VP220–228-specific CD73+CD8+ T cells were significantly lower in SYMP individuals compared with ASYMP individuals (49.5% ± 5.5% versus 30.5% ± 10.5%, respectively; p = 0.01). Despite repeated attempts, with and without in vitro expansions, VP220–228-specific CD73+CD8+ T cells were consistently undetectable in seronegative healthy donors (data not shown).

FIGURE 1.
FIGURE 1.

Frequent HSV-1 VP11/12220–228 epitope-specific CD73+CD8+ memory T cells detected in ASYMP individuals compared with SYMP individuals. The frequency of CD73+CD8+ T cells specific to the VP220–228 peptide/tetramer complex was analyzed in HLA-A*02:01–positive HSV-1–seropositive ASYMP and SYMP individuals. (A) Representative FACS data of the frequencies of CD73+CD8+ T cells, specific to VP11/12220–228 epitope, detected in PBMCs from one HLA-A*02:01–positive HSV-1–seropositive ASYMP individual (left two panels) and one HLA-A*02:01–positive HSV-1–seropositive SYMP individual (right two panels). (B) Average frequencies of PBMC-derived CD8+ T cells, specific to VP11/12220–228 epitope, detected from nine HLA-A*02:01–positive HSV-1–seropositive ASYMP individuals compared with nine HLA-A*02:01–positive HSV-seropositive SYMP individuals. The CD73 molecule expression level on CD8+ T cells specific to VP220–228 epitope was analyzed in HLA-A*02:01–positive HSV-1–seropositive ASYMP and SYMP individuals. (C) Representative FACS data of the CD73 expression level on CD8+ T cells specific to VP220–228 epitope detected from one HLA-A*02:01–positive HSV-1–seropositive ASYMP individual (left panels) and one HLA-A*02:01–positive HSV-1–seropositive SYMP individual (right panels). (D) Average CD73 expression level on CD8+ T cells, specific to VP11/12220–228 epitope, detected from eight HLA-A*02:01–positive HSV-1–seropositive ASYMP individuals compared with six HLA-A*02:01–positive HSV-seropositive SYMP individuals. The frequency of A2AR+CD8+ T cells specific to VP220–228 epitopes complex was analyzed in ASYMP and SYMP individuals. (E) Representative FACS data of the frequencies of A2AR+CD8+ T cells detected from one ASYMP (left panel) and one SYMP individual (right panel). (F) Average frequencies of A2AR+CD8+ T cells from nine ASYMP compared with nine SYMP individuals. The results are representative of two independent experiments in each individual. The indicated p values, calculated using unpaired t test, show statistical significance between SYMP and ASYMP individuals.

We next determined whether there were any differences in the level of expression of CD73 on VP220–228-specific CD73+CD8+ T cells from the peripheral blood of eight HLA-A*02:01–positive HSV-1–seropositive ASYMP and six HLA-A*02:01–positive, HSV-1–seropositive SYMP individuals. The representative histograms shown in Fig. 1C indicated higher levels of the CD73 molecules detected on CD8+ T cells, specific to the HSV-1 immunodominant VP220–228 epitope, from one ASYMP individual (mean fluorescence intensity [MFI] of 3614, left panel) compared with one SYMP individual (MFI of 1371, right panel). Fig. 1D shows the median expression of CD73 on CD8+ T cells (expressed as MFI) detected in eight ASYMP and six SYMP individuals. The highest and most significant levels of CD73 on VP220–228-specific CD73+CD8+ T cells were consistently detected in ASYMP individuals (MFI of 3550 ± 355). The level of CD73 on VP220–228-specific CD73+CD8+ T cells was significantly lower in SYMP individuals compared with ASYMP individuals (MFI of 1250 ± 165 versus MFI of 3550 ± 355, respectively; p = 0.03).

The frequency of A2A adenosine receptors (A2AR)+CD8+ T cells specific to VP220–228 epitopes complex was analyzed among ASYMP and SYMP individuals. Fig. 1E shows representative FACS data of the high frequency of A2AR+CD8+ T cells detected in one ASYMP (27.1% ± 3.5%, left panel) as compared with one SYMP individual (12.7% ± 1.5%, right panel). Fig. 1F shows median frequencies of A2AR+CD8+ T cells detected in eight ASYMP and six SYMP individuals. The highest and most significant frequencies of VP220–228-specific A2AR+CD8+ T cells were consistently detected in ASYMP individuals (25.5% ± 5.5% versus 11.5% ± 1.5%, respectively; p < 0.05).

Altogether, these results 1) indicate that although HSV-seropositive SYMP and ASYMP individuals have similar frequencies of HSV-1 VP11/12220–228 epitope-specific CD8+ T cells, ASYMP individuals develop frequent HSV-specific CD73+CD8+ T cells compared with SYMP individuals, 2) suggest that herpetic disease is not a consequence of a clonal deletion of specific repertoires of CD8+ T cells in SYMP individuals, and 3) suggest a positive role for the CD73 costimulatory molecule in the protection against ocular herpes.

ASYMP individuals have frequent HSV-1 VP11/12220–228 epitope-specific effector memory CD73+CD8+ TEM and CD73+CD8+ TEMRA cells

Next, we studied the expression levels of CD73 on the memory CD8+ T cell subpopulations at various stages of differentiation: naive T cells (CD45RAhighCCR7highCD8+ TNAIVE cells), central memory T cells (CD45RAlowCCR7highCD8+ TCM cells), and effector memory T cells (CD45RAhighCCR7lowCD8+ TEMRA cells and CD45RAlowCCR7lowCD8+ TEM cells). In the peripheral blood of 10 HLA-A*02:01–positive, HSV-1–seropositive ASYMP and 10 HLA-A*02:01–positive, HSV-1–seropositive SYMP individuals, we compared the CD73 expression in CD8+ T cells specific to VP11/12220–228 epitopes and divided them into TNAIVE, TCM, TEM, and TEMRA phenotypes (Fig. 2A–H). Similar percentages of CD73+CD45RAhighCCR7highCD8+ TNAIVE cells were detected in ASYMP and SYMP individuals (Fig. 2A, 2B). There was an increase in the CD73+CD45RAlowCCR7highCD8+ TCM cells detected in ASYMP and SYMP individuals (Fig. 2C, 2D). Significantly higher percentages of CD73+CD45RAhighCCR7lowCD8+ TEMRA cells (Fig. 2E, 2F) and CD73+CD45RAlowCCR7lowCD8+ TEM cells (Fig. 2G, 2H) were detected in ASYMP individuals as compared with SYMP individuals (p = 0.001).

FIGURE 2.
FIGURE 2.

Frequent HSV-1 VP11/12220–228 epitope-specific CD8+ T cells with effector memory phenotype (CD73+CD8+ TEMRA and CD73+CD8+ TEM cells) detected in ASYMP individuals compared with SYMP individuals. The CD8+ T cells specific to VP11/12220–228 peptide/tetramer representing shown in Fig. 1 were analyzed in terms of TNAIVE, TCM, TEMRA, and TEM phenotypes. Representative FACS data of the frequencies of (A) CD45RAhighCCR7highCD8+ TNAIVE cells, (C) CD45RAlowCCR7highCD8+ TCM cells, (E) CD45RAhighCCR7lowCD8+ TEMRA cells, and (G) and CD45RAlowCCR7lowCD8+ TEM cells detected in one ASYMP individual and one SYMP individual. Representative FACS data of the frequencies of (B) CD45RAhighCCR7highCD8+ TNAIVE cells, (D) CD45RAlowCCR7highCD8+ TCM cells, (F) CD45RAhighCCR7lowCD8+ TEMRA cells, and (H) CD45RAlowCCR7lowCD8+ TEM cells detected from 10 ASYMP and 10 SYMP individuals. The results are representative of two independent experiments in each individual. The indicated p values, calculated using unpaired t test, show statistical significance between SYMP and ASYMP individuals.

Altogether, the phenotypic properties of HSV-1 VP11/12220–228 epitope-specific memory CD8+ T cells revealed a clear dichotomy in memory CD8+ T cell subpopulations in SYMP versus ASYMP individuals. ASYMP individuals appeared to develop frequent HSV-specific effector memory CD73+CD8+ TEMRA and CD73+CD8+ TEM cells, as compared with SYMP individuals. By maintaining high frequencies of the “experienced” HSV-specific CD73+CD8+ TEMRA cells and CD73+CD8+ TEM cells, the ASYMP individuals should be better protected against infection and/or disease. The higher expression of CD73 seen on CD8+ T cells in ASYMP individuals may favor effector to memory CD8+ T cell transition (effector>>>memory) and the formation of more HSV-specific CD73+CD8+ TEMRA cells and CD73+CD8+ TEM cells (46). Thus, these results suggest that during a second pathogen encounter (e.g., following HSV-1 reactivation from latency), ASYMP individuals, but not SYMP individuals, would mount much faster and stronger protective antiviral CD73+CD8+ TEMRA and CD73+CD8+ TEM cell responses, allowing for better clearance of infection and disease.

HSV-specific CD73(+)CD8+ T cells from ASYMP individuals are multifunctional compared with HSV-specific CD73(−)CD8+ T cells

We next compared the effector functions of HSV-1 VP11/12220–228 epitope-specific CD73(+)CD8+ T cells versus CD73(−)CD8+ T cells from ASYMP and SYMP individuals. VP11/12220–228 epitope-primed CD8+ T cells from ASYMP individuals were divided into CD73+CD8+ T cells and CD73−CD8+ T cells (Fig. 3A) and their functions analyzed.

FIGURE 3.
FIGURE 3.

Frequent HSV-1 VP11/12220–228 epitope-specific CD73+CD8+ polyfunctional T cells detected in SYMP individuals. (A) VP11/12220–228 epitope-primed CD8+ T cells from ASYMP individuals were divided into CD73+CD8+ T cells and CD73−CD8+ T cells, and their functions were compared. (B) FACS was used to determine the expression level of CD107a/b on tetramer-gated CD8+ T cells specific to VP11/12220–228 epitope, as described in the Materials and Methods section. VP11/12220–228 epitope-specific CD73+CD8+ T cells express high levels of CD107a/b cytotoxic degranulation compared with CD73−CD8+ T cells. The numbers on the top of each histogram represent the MFI depicting the expression level of CD107a/b molecules. Numbers in bold represent MFI on CD73+CD8+ T cells from ASYMP individuals. Representative FACS data (C) and average percentage (D) of VP11/12220–228 epitope-specific CD107a/b+CD73+CD8+ T cells versus CD107a/b+CD73−CD8+ T cells. (E) Level of CD107a/b molecules expression on VP11/12220–228 epitope-specific CD8+ T cells following costimulation with 1) mAbs anti-CD3 alone, 2) mAbs anti-CD3 plus mAbs anti-CD28, and 3) anti-CD3 plus mAbs anti-CD73. Representative FACS data (F) and average percentage (G) of VP11/12220–228 epitope-specific IFN-γ+CD73+CD8+ T cells versus IFN-γ+CD73−CD8+ T cells are shown. (H) IFN-γ expression level on VP11/12220–228 epitope-specific CD8+ T cells following costimulation on with 1) mAbs anti-CD3 alone, 2) mAbs anti-CD3 plus mAbs anti-CD28, and 3) anti-CD3 plus mAbs anti-CD73. Representative FACS data (I) and average percentages (J) of VP11/12220–228 epitope-specific TNF-α+CD73+CD8+ T cells versus TNF-α+CD73−CD8+ T cells are shown. (K) TNF-α expression levels on VP11/12220–228 epitope-specific CD8+ T cells following costimulation on with 1) mAbs anti-CD3 alone, 2) mAbs anti-CD3 plus mAbs anti-CD28, and 3) anti-CD3 plus mAbs anti-CD73. The average frequencies of CD8+ T cells from 10 HLA-A*02:01–positive ASYMP and 10 SYMP individuals in response to stimulation with the VP11/12220–228 peptide are shown. The results are representative of two independent experiments in each individual. The indicated p values, calculated using one-way ANOVA test, show statistical significance between SYMP and ASYMP individuals.

We first compared the expression levels of the CD107a/b cytotoxic degranulation molecules on gated VP11/12220–228 epitope-specific CD73+CD8+ T cells and CD73−CD8+ T cells (Fig. 3B). We found high levels of CD107 expressed on VP11/12220–228 epitope-specific CD73+CD8+ T cells from ASYMP individuals as compared with VP11/12220–228 epitope-specific CD73−CD8+ T cells, suggesting a positive correlation of strong HSV-specific CD73+CD8+ T cell cytotoxic responses with protection from ASYMP ocular herpetic disease. A significant increase in the expression level of CD107, as determined by MFI (Fig. 3B, p ≤ 0.05), and higher percentages of VP11/12220–228 epitope-specific CD107highCD73+CD8+ T cells were consistently detected in ASYMP individuals as compared with SYMP individuals (Fig. 3C, 3D, p = 0.01). Cross-linking of the CD73 and CD3 molecules did not lead to increased levels of CD107 molecules on CD8+ T cells from ASYMP individuals (Fig. 3E), suggesting that the expression level of CD107 had already reached its maximum.

We next determined the ability of HSV-specific CD73+CD8+ T cells versus CD73−CD8+ T cells to produce IFN-γ. Freshly isolated CD8+ T cells from ASYMP individuals were stimulated in vitro for 6 h with the immunodominant VP11/12220–228 epitope peptide, as described in the Materials and Methods section. The percentages and numbers of IFN-γ+CD8+ T cells were compared among gated VP11/12220–228 epitope-specific CD73+CD8+ T cells and CD73−CD8+ T cells by intracellular FACS staining. Significantly higher percentages of VP11/12220–228 epitope-specific CD73+CD8+ T cells producing IFN-γ were detected in ASYMP individuals, as compared with lower percentages of VP11/12220–228 epitope-specific IFN-γ+CD73−CD8+ T cells (Fig. 3F, 3G, p = 0.03). Cross-linking of the CD73 and CD3 molecules significantly increased the level of IFN-γ by CD8+ T cells produced from ASYMP individuals following VP11/12220–228 peptide stimulation (Fig. 3H).

To confirm the polyfunctional nature of CD73+CD8+ T cells, we further studied the production of TNF-α on VP11/12220–228 epitope-specific CD8+ T cells. Freshly isolated CD8+ T cells from ASYMP individuals were stimulated in vitro for 6 h with the immunodominant VP11/12220–228 epitope peptide, as described in the Materials and Methods section. Percentages and numbers of TNF-α+CD8+ T cells were compared among gated VP11/12220–228 epitope-specific CD73+CD8+ T cells and CD73−CD8+ T cells by intracellular FACS staining. Significantly higher percentages of VP11/12220–228 epitope-specific CD73+CD8+ T cells producing TNF-α were detected in ASYMP individuals, as compared with lower percentages of VP11/12220–228 epitope-specific TNF-α+CD73−CD8+ T cells (Fig. 3I, 3J, p = 0.01). Following VP11/12220–228 peptide stimulation and cross-linking of CD73 and CD3 molecules, there was significant increase in the levels of both IFN-γ and TNF-α produced by CD8+ T cells from ASYMP individuals (Fig. 3H, 3K).

In ASYMP individuals, we detected a positive correlation between the frequency of VP11/12220–228 epitope-specific CD73+CD8+ T cells and the expression of CD107a/b (p = 0.3, Fig. 4A) and the production of IFN-γ (p = 0.01, Fig. 4B). However, no correlation was found between the frequency of VP11/12220–228 epitope-specific CD73+CD8+ T cells and the production of TNF-α (Fig. 4C) (p > 0.5). Moreover, no correlation was found between the frequency of VP11/12220–228 epitope-specific CD73−CD8+ T cells and any of T cell functions studies (Fig. 4D–F) (p > 0.5).

FIGURE 4.
FIGURE 4.

HSV-1 VP11/12220–228 epitope-specific CD73+CD8+ T cells are more functional. Correlation of VP11/12220–228 epitope-specific CD73+CD8+ T cell percentage in PBMC with (A) expression of CD107a/b cytotoxic degranulation molecules and production of (B) IFN-γ and (C) TNF-α. Correlation of VP11/12220–228 epitope-specific CD73−CD8+ T cells percentage in PBMC with (D) expression of CD107a/b cytotoxic degranulation molecules and production of (E) IFN-γ and (F) TNF-α. The nominal p values show statistical significance between the percentage of VP11/12220–228 epitope-specific CD8+ T cells and production of IFN-γ function.

Altogether, these results suggest that in contrast to HSV-specific effector CD73−CD8+ T cells, the HSV-specific effector CD73+CD8+ T cells from ASYMP individuals are multifunctional. Overall, ASYMP individuals had higher proportions of multifunctional CD73+CD8+ T cells with expression of three functions (IFN-γ, TNF-α, and/or CD107a/b).

HSV-specific CD73(−)CD8+ T cells are exhausted compared with HSV-specific CD73(+)CD8+ T cells

VP11/12220–228 epitope-specific CD8+ T cells from ASYMP and SYMP individuals were divided into CD73+CD8+ T cells and CD73−CD8+ T cells, and FACS was used to determine the expression level of PD-1, a marker of T cell exhaustion, on tetramer-gated CD8+ T cells specific to VP11/12220–228 epitope in CD73+CD8+ and CD73−CD8+ T cell populations (Fig. 5A, 5B). The VP11/12220–228 epitope-specific CD73−CD8+ T cells expressed high level of PD-1 marker of exhaustion compared with CD73+CD8+ T cells (Fig. 5A, 5B). The numbers on the top of each histogram represent the MFI depicting the expression level of PD-1 molecule on CD73+CD8+ T and CD73−CD8+ T cells (Fig. 5A). The percentages of PD-1highCD73+CD8+ T cells and PD-1highCD73−CD8+ T cells were also compared between the two T cell subpopulations (Fig. 5C). Significantly higher frequency of VP11/12220–228 epitope-specific PD-1highCD73−CD8+ T cells was detected in SYMP individuals, as compared with lower frequency in ASYMP individuals (Fig. 5C). In both SYMP and ASYMP individuals, an inverse correlation was detected between the frequency of VP11/12220–228 epitope-specific CD73+CD8+ T cells and the level expression of PD-1 (Fig. 5D). Moreover, we compared the frequency of PD-1highTIM-3highCD73+CD8+ T cells and PD-1highTIM-3highCD73−CD8+ T cells side-by-side in SYMP and ASYMP individuals (Supplemental Fig. 1). Significantly higher percentages of VP11/12220–228 epitope-specific PD-1highTIM3highCD73−CD8+ T cells were detected in SYMP individuals compared with ASYMP individuals (Supplemental Fig. 1C–F).

FIGURE 5.
FIGURE 5.

HSV-1 VP11/12220–228 epitope-specific CD73−CD8+ T cells are dysfunctional (exhausted). VP11/12220–228 epitope-specific CD8+ T cells from ASYMP and SYMP individuals were divided into CD73+CD8+ T cells and CD73−CD8+ T cells, and their function was compared. VP11/12220–228 epitope-specific CD73−CD8+ T cells express high level of PD-1 marker of exhaustion compared with CD73+CD8+ T cells. FACS was used to determine the expression level of PD-1 on tetramer-gated CD8+ T cells specific to VP11/12220–228 epitope, as described in the Materials and Methods section. The numbers on the top of each histogram represent MFI depicting the expression level of PD-1 molecule. (A) Representative FACS data, (B) average MFI, (C) and average percentage of VP11/12220–228 epitope-specific PD-1highCD73+CD8+ T cells versus PD-1highCD73−CD8+ T cells are shown. (D) An inverse correlation of VP11/12220–228 epitope-specific CD73+CD8+ T cell percentage in PBMC with PD-1 expression in SYMP and ASYMP individuals. The results are representative of two independent experiments in each individual. The indicated p values, calculated using unpaired t test, show statistical significance between SYMP and ASYMP individuals.

These results confirm that 1) more HSV-specific effector CD73−CD8+ T cells from SYMP individuals are dysfunctional (exhausted); in contrast, HSV-specific effector CD73+CD8+ T cells from ASYMP individuals are mostly multifunctional, 2) the increase in the frequency of HSV-specific CD73−CD8+ T cells from SYMP individuals expressing PD-1 and TIM-3 markers of T cell exhaustion is consistent with continuous antigenic stimulation (47), and 3) a higher expression of PD-1 on HSV-specific CD8+ T cells from SYMP individuals also suggests more exposure to Ag compared with ASYMP individuals, leading to partial dysfunction.

Because of the obvious ethical and practical considerations of obtaining tissue-resident CD8+ T cells (i.e., from the cornea or from TG, the site of acute and latent infections, respectively), our investigations in humans were limited to human PBMC-derived CD8+ T cells. To gain more insight into the nature of protective versus nonprotective cornea and TG-resident HSV-specific CD8+ T cell subpopulations in SYMP and ASYMP herpes, the remainder of the study used a mouse model of ocular herpes.

Frequent functional HSV-1 gB498–505 epitope-specific CD73+CD8+ T cells are present in the cornea of ASYMP mice compared with the cornea of SYMP mice

We first compared, in a kinetic study, the frequency of HSV-specific CD8+ T cells expressing CD73 molecule from the cornea and TG of HSV-1–infected B6 mice. Representative FACS data of the percentages of CD73+CD8+ T cells (Fig. 6A) and average percentages of CD73+CD8+ T cells detected on 0, 8, 14, 23, and 41 d PI (Fig. 6B) showed a peak frequency for gB498–505 epitope-specific CD73+CD8+ T cells, which was reached in the cornea of HSV-1–infected mice around day 23 PI. However, this peak was reached in infected TG as early as day 14 PI. The numbers outlined in the top left corner of each dot plot in Fig. 6A indicate the highest percentages of gB498–505 epitope-specific CD73+CD8+ T cells were at 44% in cornea on day 23 PI and 62% in TG on day 14 PI. Kinetics of the average percentages of CD73+CD8+ T cells showed a decline of gB498–505 epitope-specific CD73+CD8+ T cells in the cornea starting around day 23 PI and around day 14 PI in the TG (Fig. 6B).

FIGURE 6.
FIGURE 6.

Frequent functional gB498-505 epitope-specific CD73+CD8+ T cells detected in the cornea and TG of HSV-1–infected ASYMP B6 mice. (A and B) Kinetics of CD73 molecule expression in cornea- and TG-derived CD8+ T cells. Flow cytometry of the frequency of gB498–505 epitope-specific CD73+CD8+ T cells in the cornea and TG of B6 mice (n = 10) following ocular infection with HSV-1 (McKrae, 2 × 105 PFU per eye). (C) Representative FACS data (left panel) and average frequency (right panel) of the gB498–505 epitope-specific CD73+CD8+ T cells in the cornea and TG of ASYMP (n = 5) versus SYMP (n = 5) mice. (D) Representative FACS data (left panel) and average frequency (right panel) of the gB498–505 epitope-specific IFN-γ+CD8+ T cells (E) and CD107+CD8+ T cells in the cornea and TG of ASYMP (n = 5) versus SYMP (n = 5) mice. Data are representative of two independent experiments. The indicated p values, calculated using unpaired t test, show statistical significance between SYMP and ASYMP individuals.

We next compared the frequency of the HSV-specific CD73+CD8+ T cells in the cornea and TG of HSV-1–infected SYMP versus ASYMP B6 mice (Fig. 6C). The low frequencies of HSV-specific CD8+ T cells in the TG and cornea of ocularly infected SYMP and ASYMP B6 mice complicate a direct ex vivo detection of gB498–505 epitope-specific CD73+CD8+ T cells from a typical number of ∼106 cells using gB498–505/tetramers. Moreover, a prior expansion of CD8+ T cells by HSV-1 or gB498–505 peptide stimulation in an in vitro culture would hamper determination of a reliable frequency of gB498–505 epitope-specific CD8+ T cells. We therefore opted to measure the frequencies of gB498–505 epitope-specific CD8+ T cells ex vivo by pooling 10 corneas and 10 TG from SYMP or from ASYMP mice and then using a large number of cells (∼10 × 106) per tetramer/CD8 mAbs panel.

The representative dot plots shown in Fig. 6C indicate that a significantly higher frequency of CD73+CD8+ T cells specific to the HSV-1 gB498–505 epitope was present in the cornea of ASYMP mice (61.9%, top left panel) as compared with SYMP mice (43.7%, p = 0.04, top right panel). However, similar frequencies of CD73+CD8+ T cells were detected in TG of SYMP and ASYMP mice (49.2 and 56.4%, p > 0.05, respectively, two bottom panels). Despite repeated attempts, with and without in vitro expansions, gB498–505-specific CD73+CD8+ T cells were consistently undetectable in mock-infected B6 mice (data not shown).

We then compared the effector functions of the HSV-1 gB498–505 epitope epitope-specific CD73(+)CD8+ T cells from cornea and TG ASYMP versus SYMP mice (Fig. 6D). We found a significantly higher frequency of gB498–505 epitope-specific IFN-γ+CD73+CD8+ T cells in the corneas of ASYMP mice as compared with corneas of SYMP mice (10.9 and 5.6%, respectively, p = 0.03, top panels). However, similar frequencies of IFN-γ+CD73+CD8+ T cells were detected in the TG of SYMP and ASYMP mice (12.4 and 9.8%, respectively, p > 0.05, bottom panels). Moreover, significantly higher percentages of gB498–505 epitope-specific CD107+CD73+CD8+ T cells were detected in both the corneas and TGs of ASYMP mice, as compared with lower percentages of gB498–505 epitope-specific CD107+CD73+CD8+ T cells in the corneas and TGs of SYMP mice (Fig. 6E, p = 0.03 and p = 0.04, respectively).

Altogether, these results indicate that similar to ASYMP humans, 1) HSV-1–infected ASYMP mice developed frequent HSV-specific CD73+CD8+ T cells compared with SYMP mice, 2) the HSV-specific effector CD73+CD8+ T cells from ASYMP mice produce IFN-γ and have cytotoxic activity, and 3) the phenotypic and functional studies in both humans and mice suggest a positive role for TEM expressing the CD73 costimulatory molecule in protection against ocular herpes infection and disease.

CD73−/− deficient mice develop significantly less memory CD8+ cells in the cornea and TG and more recurrent herpetic infection and disease

Next, we determined whether the lack of CD73 would directly affect HSV-specific CD8+ T cells mobilization and function in the cornea and TG and whether this would affect recurrent herpetic infection and disease. CD73−/− deficient and WT mice were infected ocularly with 2 × 105 PFU of HSV-1 (strain McKrae) without corneal scarification, as described in the Materials and Methods section. On day 35 (i.e., during latent phase), the eyes of 15 infected animals that survived acute infection were exposed to UV-B irradiation to induce reactivation of HSV-1 from latently infected TG. UV-B irradiation led to virus shedding in tears and recurrent corneal herpetic disease, as we recently described (43, 44). The timeline of HSV-1 infection, UV-B irradiation, and subsequent immunological and virological assays is illustrated in Fig. 7A.

FIGURE 7.
FIGURE 7.

CD73−/− deficient mice developed more corneal infection and severe herpetic disease associated with less functional HSV-specific CD8+ T cells in cornea and TG compared with WT B6 mice. (A) Schematic representation of the timeline of HSV-1 infection and UV-B–induced recurrent disease in WT B6 mice and CD73−/− deficient mice. A group of CD73−/− deficient mice and WT mice (6–8 wk old) were ocularly infected on day 0 with 2 × 105 PFU of HSV-1 (strain McKrae) following scarification. After establishment of latency (35 d PI), reactivation of latent virus was induced following irradiation with UV-B. Tears were collected daily for 6 d after UV-B, and recurrent corneal disease was observed daily for eye disease for 25 d after UV-B exposure. (B) Presence of infectious virus in the tears of WT and CD73−/− mice after UV-B treatment. Viral titer estimation detected in tear samples 6 d after UV-B irradiation expressed as mean of virus load. The data are expressed as mean of virus load (PFU per milliliter). (C) Recurrent corneal herpetic disease detected for up to 25 d following UV-B irradiation and scored on a scale of 0 to 4. (D) Representative slit lamp images of WT and CD73−/− mice corneas. Mice were euthanized on day 60 (25 d post–UV-B exposure), and single-cell suspensions from cornea and TG was obtained after collagenase treatment and stained for markers of CD8+ T cells, IFN-γ, and CD107 and analyzed by immunostaining and FACS. Sections of the cornea (E) and TG (I) from WT and CD73−/− mice were costained using DAPI (blue; DNA stain) and mAb specific to CD103 (red) and CD8 Infiltration (green). Original magnification ×40. Single-cell suspension from the cornea was obtained after collagenase treatment at 37°C for an hour and stained for markers of CD8+ TRM cells, IFN-γ, and CD107a/b and analyzed by FACS. Representative FACS plot of the frequency of HSV-specific CD103+CD8+ T cells (F and J), IFN-γ+CD8+ T cells (G and K), and CD107a/b+CD8+ T cells (H and L) detected in cornea and TG of WT and CD73−/− deficient mice. The results are representative of two independent experiments. The indicated p values, calculated using an unpaired t test, show statistical significance between WT and CD73−/− deficient mice.

Virus replication in the cornea (the site of HSV acute replication) and corneal herpetic disease were compared in CD73−/− deficient mice and WT B6 mice (Fig. 7B–D). The frequency of HSV-specific CD73+CD8+ T cells and the function of CD8+ T cells were compared in the cornea (Fig. 7E–H) and TG (Fig. 7I–L) of CD73−/− deficient mice and WT mice.

Viral titers measured in corneal swabs taken on day 8 after UV-B exposure demonstrated significantly less control of virus replication (p < 0.05, Fig. 7B) leading to significantly more recurrent ocular herpetic disease (p = 0.04, Fig. 7C, 7D) in CD73−/− deficient mice compared with WT mice. CD73−/− mice developed severe cornea lesions (average score of 3 on a scale of 0 to 4) compared with WT mice (average score of 1.5). This was associated with a significant decrease in CD103+CD8+ T cells infiltrating the cornea (p < 0.05, Fig. 7E, 7F) and TG (p < 0.05, Fig. 7I, 7J) of CD73−/− mice compared with WT mice. However, similar frequencies of HSV-1 gB498–505-specific IFN-γ+CD8+ T cells were detected in the cornea (Fig. 7G) and TG (Fig. 7K) of CD73−/− and WT mice. Moreover, we detected significant decreases in HSV-1 gB498–505-specific CD107+ CD8+ T cytotoxic T cells infiltrating the cornea (Fig. 7H) and TG (Fig. 7L) of CD73−/− mice as compared with WT mice.

These results indicated that the lack of CD73 led to a decreased function of HSV-specific CD8+ T cells in the cornea and TG. In addition, this was associated with severe recurrent herpes infection and disease in both humans and mice, confirming a positive role for TEM expressing the CD73 costimulatory molecule in the protection against ocular herpes infection and disease. The association of HSV-specific CD73+CD8+ T cell response with ASYMP herpes makes this memory subpopulation desired in T cell–based immunotherapies against ocular herpes, as it might be involved, with a yet-to-be determined mechanism, in the protection against recurrent herpes infection and disease.

Discussion

Characterizing the phenotype and function of HSV-specific effector and memory CD8+ T cells associated with ASYMP herpes infection is critical for the design of T cell–based herpes immunotherapies. In this study, we report that HSV-specific effector memory CD73+CD45RAhighCCR7lowCD8+ TEMRA and CD73+CD45RAlowCCR7lowCD8+ TEM cells are present in significantly higher proportions in HSV-1–infected ASYMP individuals. In contrast, SYMP individuals had significantly lower frequencies of HSV-specific CD73−CD8+ TCM cells. Similar to ASYMP individuals, ASYMP B6 mice developed polyfunctional HSV-specific CD73+CD8+ T cells in the cornea and TG following ocular infection with HSV-1, suggesting a protective role of CD73 pathway in herpes infection and disease. The functional results of CD73+CD8+ T cells from ASYMP individuals suggest that compared with SYMP individuals, ASYMP individuals are better prepared to mount protective cytotoxic and IFN-γ–producing cytotoxic CD8+ T cell responses, which are the two effector arms of immunity that protect against herpes. The role of CD73 in herpes protection was further clarified following ocular infection of WT and CD73−/− deficient mice with HSV-1. In contrast to WT mice, HSV-1–infected CD73−/− mice were highly susceptible to immune-mediated pathology. The CD8+ T cell phenotypic and functional studies, in both humans and mice, suggest a positive role for memory CD8+ T cells expressing the CD73 costimulatory molecule in protection against ocular herpes infection and disease. The association of HSV-specific CD73−CD8+ T cell response with SYMP herpes makes memory CD73−CD8+ T cell subpopulation undesired in immunotherapeutic strategies against herpes, as CD73−CD8+ T cells might be involved, with a yet-to-be determined mechanism, in the pathogenesis of herpetic disease. Our findings should hopefully guide a successful design of effective T cell–based vaccines and immunotherapeutic strategies.

Memory CD8+ T cells play a major role in host defense by enabling the immune system to respond more rapidly and vigorously to previously encountered infectious pathogens by rapid recognition and lysis of virus-infected cells. The importance of both TCM and TEM subsets for the control of infectious diseases and the effectiveness of vaccines has been shown in several human and murine studies (48–51). Our understanding of the population size, the phenotype, and the function of protective versus nonprotective HSV-specific CD8+ T cell subpopulations mainly comes from studies of B6 mouse model of herpes infection (18, 29, 36, 52–54). To gain insight into the nature of human protective versus nonprotective HSV-specific CD8+ T cell subpopulations, we used several human-focused immunological assays to compare the protective versus nonprotective HSV-1 VP11/12220–228 epitope-specific CD8+ T cells in the peripheral blood of ASYMP and SYMP individuals at both effector and memory levels (1). Our findings from this study indicate that ASYMP and SYMP individuals have similar frequencies of HSV-1 VP11/12220–228 epitope-specific CD8+ T cells, thereby suggesting that herpetic disease is not a consequence of a clonal deletion of specific repertoires of CD8+ T cells in SYMP individuals. This result is in agreement with a recent study which exemplifies that once the clonal repertoire of HSV-specific memory CD8+ T cells is established, it is kept constant for several years (55). Nevertheless, there are phenotypic and functional differences in T cells between ASYMP and SYMP individuals. Within the overall memory CD8+ T cell population, two distinct major subpopulations have been described and can be recognized by the differential expression of chemokine receptor CCR7 and l-selectin (CD62L). TCM cells express CD62L and CCR7 and secrete IL-2 but not IFN-γ or IL-4. TEM cells do not express CD62L or CCR7; rather, the cells produce effector cytokines like IFN-γ and IL-4, which was consistent with our findings. A lack of CCR7 expression, which is required for T cell egress and recirculation, impairs the capacity of TEM cells for homing to lymphoid tissues (56). We previously reported on phenotypic and functional differences of HSV-specific effector and memory CD8+ T cells from ocular herpes ASYMP and SYMP individuals. ASYMP individuals had a significantly higher proportion of differentiated polyfunctional HSV–specific CD8+ TEM cells. Conversely, SYMP patients had significantly higher frequencies of less-differentiated monofunctional HSV-specific CD8+ TCM cells (39, 41, 57). The present study extends those findings by demonstrating that the naturally protected ASYMP individuals have significantly higher proportions of multifunctional HSV-specific TEM (CD73+CD45RAhighCCR7lowCD8+ TEMRA and CD73+CD45RAlowCCR7lowCD8+ TEM cells) compared with monofunctional HSV-specific TCM (CD73+CD45RAlowCCR7highCD8+ TCM cells) in SYMP individuals. In like manner, TEM and TEMRA appeared predominantly in most ASYMP individuals and were most effector-like, with a high expression of CD107a/b and high production of IFN-γ. With less effector-like conditions, TEM and TEMRA in SYMP individuals, comparatively, possessed a low expression of CD107a/b and less production of IFN-γ. We are aware that the information gained from peripheral blood T cells may not be completely reflective of tissue-resident T cells. However, owing to the obvious ethical and practical considerations of obtaining tissue-resident CD8+ T cells from the cornea or from the TG, the sites of acute and latent infections, our investigations in humans were limited to peripheral blood–derived CD8+ T cells. Nevertheless, the results obtained from the B6 mouse model showed there was a significant increase in the frequency of CD73+CD8+ T cells in the corneas (but not TG) of ASYMP mice compared with the corneas of SYMP mice. The reason why we found high frequency of CD73+CD8+ T cells in peripheral blood of ASYMP individuals (which is away from the sites of latency) but not in TG of ASYMP mice remains to be determined. Despite this, our results indicate that maintaining high frequencies of the experienced HSV-specific CD73+CD45RAhighCCR7lowCD8+ TEMRA and CD73+CD45RAlowCCR7lowCD8+ TEM cells can better arm ASYMP individuals to prevent and/or efficiently clear new infections and reactivations. At a second pathogen encounter (e.g., following HSV-1 reactivation from latency), ASYMP individuals, but not SYMP individuals, can mount markedly stronger and faster protective HSV-specific CD73+CD45RAhighCCR7lowCD8+ TEMRA and CD73+CD45RAlowCCR7lowCD8+ TEM cell responses, allowing for better clearance of herpes infection and disease.

Naive T cells are commonly characterized by the surface expression of CD62L, CCR7, CD45RA, and the absence of memory CD45RO isoform (reviewed in Ref. 58). Although naive T cells are regularly regarded as a quiescent cell population, there is increasing evidence that naive T cells are actually heterogeneous in phenotype, function, dynamics and differentiation status, resulting in a whole spectrum of naive cells with different properties (58). For instance, some nonnaive T cells express surface markers similar to naive T cells (stem cell memory T cells) (59). TMNP, memory T cells, carry a naive phenotype (60), whereas some Ag-specific naive T cells may have lost their naive phenotype (reviewed in Ref. 61). In this study, we found Ag-specific tetramer-binding T cells expressing a high level of CD45RA and CCR7 falling into the phenotype of naive cells (Fig. 2). Nonetheless, these naive T cells could be heterogeneous and therefore must be further characterized in ASYMP and ASYMP individuals to better appreciate potential differences in the phenotype, including expression of CD73, function, dynamics, and differentiation status.

Furthermore, previous studies in mice have shown that following HSV-1 infection, CD8+ T cells persist in the cornea and TG for prolonged periods of time following clearance of corneal herpes lesions (13, 62–68). The phenotype and function of these persistent CD8+ T cells have not been fully elucidated. In this report, we investigated the association between HSV-1 disease severity and the quantity, quality, and diversity of HSV-specific memory CD8+ T cell subpopulations from blood (in human) and cornea (in mice). We found that 1) circulating and tissue-resident HSV-1 VP11/12220–228 epitope-specific memory CD8+ T cells differ among individuals with mild to nonexistent symptoms (ASYMP) versus individuals with severe symptoms (SYMP) of ocular herpetic diseases, and 2) specific phenotype and function of HSV-1 VP11/12220–228 epitope-specific, tissue-resident memory CD8+ T cells are associated with ocular herpes disease in mice. Whether the phenotype and function of CD8+ T cells found in B6 mice can be extrapolated to human corneal tissue-resident memory, as reported in other herpes systems (69, 70), is yet to be determined. Investigating the phenotype and function of CD8+ T cells in human cornea and TG samples from ASYMP and SYMP cohorts is warranted to determine the quantity, quality, and diversity of cornea- and TG-resident HSV-specific memory CD8+ T cell subpopulations, as well as their association with corneal disease severity. This information would help guide our ongoing efforts in developing a T cell based immunotherapeutic strategy to cure ocular herpes.

HSV-specific memory CD8+ T cells elicited during a primary infection persist for years after clearance of the virus and mediate recall responses that halt future attempts of virus reactivation from latency. We previously reported that circulating CD8+ T cells from HSV-1–seropositive ASYMP individuals had a greater frequency of polyfunctional HSV-specific CD8+ TEM cells compared with more CD8+ TCM cells from SYMP patients (57). The present study extends those findings by reporting that polyfunctional HSV-specific CD8+ TEM cells that are predominant in ASYMP individuals expressed higher levels of CD73 costimulatory molecules. The HSV-1 VP11/12220–228 epitope-specific CD73+CD8+ T cells, but not the HSV-1 VP11/12220–228 epitope-specific CD73−CD8+ T cells, in ASYMP individuals displayed high levels of CD107 and produced elevated IFN-γ, implying a protective role for the CD73+CD8+ T cells in ocular herpes. The present results confirm our previous reports that up to 95.5% of ASYMP individuals had CD8+ T cells that were able to produce three to five functions concurrently compared with only 32% of SYMP individuals (1, 57). Additionally, it suggests that polyfunctional CD73+CD8+ T cells, but not the CD73−CD8+ T cells, from ASYMP individuals may provide a better and faster immune surveillance to protect against recurrent herpetic disease. Based on these findings, we propose a mechanism built on a novel “SYMP/ASYMP concept,” in which the immunopathology associated with corneal lesions are the result from the balance between immunopathological CD73−CD8+ T cell responses specific to SYMP HSV-1 epitopes and immunoprotective CD73+CD8+ T cell responses specific to ASYMP HSV-1 epitopes. These mechanisms might involve CD73+CD8+ TEM and CD73+CD8+ TEMRA cell–mediated protection in ASYMP individuals. Accordingly, the polyfunctionality of HSV-specific ASYMP CD73+CD8+ TEM and CD73+CD8+ TEMRA cells is likely to be one factor, among others, that accounts for the T cell control of herpes infection and disease. Monofunctional HSV-specific SYMP CD73−CD8+ T cells, in contrast, may be involved in the immunopathological recurrent corneal disease. However, this does not mitigate or exclude the possibility of other hosts or viral factors either acting alone or in concert with nonprotective CD73−CD8+ T cell responses in determining the course of herpetic disease. Regardless of the mechanism, if ASYMP individuals develop more protective CD73+CD8+ TEMRA and CD73+CD8+ TEM cells whereas SYMP patients do not, it would be logical to exclude epitopes that do not induce protective CD73+CD8+ TEMRA and CD73+CD8+ TEM cells from future herpes vaccines.

We recently demonstrated the requirement of CD4+ T cell help for efficient priming and maintenance of HSV-specific CD8+ T cells (18, 45). Others have shown that CD4+ T cells are required to pave the way for efficient migration of memory CD8+ T cells into restricted tissues, such as the cornea and TG (71–74). We do not exclude that CD73+CD4+ T cells act directly as effector cells or as helper cells in the mobilization of effector and memory CD73+CD8+ TEM cells into HSV infected tissues of ASYMP individuals. Future studies will determine 1) whether HSV-specific effector CD73+CD4+ T cells are frequent in ASYMP compared with SYMP individuals, and 2) whether helper CD73+CD4+ T cells would contribute in the mobilization of HSV-specific effector and memory CD8+ TEM cells into HSV-1–infected cornea and TG, and the results will be the subject of future reports.

T cell exhaustion contributes to the failure to control persistent infections, and this likely includes latent/chronic HSV-1 and HSV-2 infections. High expression of inhibitory receptors, including PD-1, and the inability to sustain functional T cell responses contribute to T cell exhaustion. We detected a higher expression of PD-1 exhaustion receptor on HSV-specific CD73−CD8+ T cells compared with HSV-specific CD73+CD8+ T cells. This suggests that functional exhaustion of PD-1highCD73−CD8+ T cells, predominantly in SYMP patients, may contribute to SYMP herpes disease. These findings do not exclude that infection with some highly pathological clinical isolates of HSV-1 in SYMP individuals leads, by a yet-to-be-determined mechanism, to functionally compromised HSV-specific PD-1highCD73−CD8+ T cells. The latter is supported by a recent report of differences in the protective and pathological properties of HSV-2 clinical isolates from the United States and South Africa (75). The mechanism that leads to phenotypic and functional exhaustion of PD-1highCD73−CD8+ T cells in SYMP individuals remains to be determined. Although continuous production of Ags does not occur during latent HSV-1 infections, antigenic stimulation during sporadic reactivations of HSV-1 from latency may lead to T cell exhaustion. We are currently testing whether blockade of the negative T cell costimulatory pathway PD-1/PDL-1 using specific mAb therapy in combination with vaccination will restore the function of exhausted HSV-specific CD8+ T cells in latently infected mice following UV-B induced virus reactivation. This might enhance the epitope-specific TEM cell responses in otherwise SYMP mice and protect against herpetic disease.

The association of HSV-specific SYMP CD73−CD8+ T cell response with herpetic disease makes this subpopulation undesired in T cell–based immunotherapies against ocular herpes, as it may be involved with a yet-to-be determined mechanism in the pathogenesis of ocular herpes. In contrast, the HSV-specific ASYMP CD73+CD8+ T cells might be involved, with a yet-to-be determined mechanism, in protection against ocular herpes. In this context, it is worth recapping that CD73 is an ecto-5′-nucleotidase, an enzyme that is critically involved in the conversion of ATP into extracellular adenosine (76–81). It is therefore likely that increased expression of CD73 on TEM cells and the high frequency of CD73+CD8+ TEM cells detected ASYMP individuals may contribute to an increase in the amount of extracellular adenosine which in turn is associated to express A2AR, expressed on other types of immune cells, including CD8+ T cells (82). Moreover, our results show that a higher proportion of CD8+ T cells in ASYMP individuals expressed A2AR, which likely allows them to bind and clear the extracellular adenosine (Fig. 1E). Our future studies will exploit available knockout mice lacking A2ARs and determine whether the protective effect of CD73+CD8+ T cells is limited to their ability to bind adenosine and whether selective A2AR agonists/antagonists can be used to effectively improve CD73+CD8+ T cell responses against herpes infection and disease. Such studies will elucidate the cellular and molecular mechanisms by which CD73 and A2AR both appear to be expressed on ASYMP HSV-specific CD8+ TEM cells, and the results from those studies will be the subject of future reports.

In this study, we show that the HSV-1–specific CD73+CD8+ T cells play a crucial role in protection against ocular herpes infection and disease. CD73, an enzyme that converts extracellular AMP into adenosine, is expressed on various types of immune cells, including CD4+ T cells, and binds to the A2AR (76–78). CD73 also plays an important role in regulating T cells migration into infected tissues (76–78, 81). Moreover, compared with SYMP individuals, in ASYMP individuals we found significantly higher frequency of HSV-specific CD8+ T cells expressing the A2AR (i.e., A2AR+CD8+ T cells) (Fig. 1C). The A2AR receptor allows expansion of T cells lacking effector functions in extracellular adenosine-rich microenvironment (83–85). T cells are targets of adenosine-mediated immunoregulation because they predominantly express A2AR (83, 86). A2AR promote expansion of HSV-2–specific CD73+CD8+ TEM cells and/or mobilize effector and memory HSV-2–specific CD73+CD8+ T cells into the infected vaginal mucosa and dorsal ganglia (the sites of acute and latent HSV-2 infections); whether this would contribute to the natural immunity seen in the genital herpes ASYMP individuals is being currently investigated, and the results will be the subjects of future reports.

In summary, our investigation provides new insights about the phenotype and the function of protective HSV-specific CD8+ T cell subpopulations that are associated with the immunologic control of herpes infection and disease in ASYMP individuals. It suggests an import role of polyfunctional HSV-specific CD73+CD8+ TEMRA cells and CD73+CD8+ TEM cells in antiviral defense against ocular HSV-1 infection and disease. The association of HSV-specific CD73−CD8+ T cell response with SYMP herpes makes memory CD73−CD8+ T cell subpopulations undesired in immunotherapeutic strategies against herpes, as CD73−CD8+ T cells might be involved, with a yet-to-be determined mechanism, in the pathogenesis of herpetic disease. Thus, the findings demonstrate quantitative and qualitative features of effective HSV-specific CD8+ T cell responses that should be taken into consideration in designing effective T cell–based ocular herpes immunotherapies.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

This work is dedicated to the memory of late Prof. Steven L. Wechsler “Steve” (1948–2016), whose numerous pioneering works on herpes infection and immunity laid the foundation to this line of research. We thank the National Institutes of Health Tetramer Facility (Emory University, Atlanta, GA) for providing the tetramers used in this study, Diane Capobianco (RN) from University of California, Irvine, Institute for Clinical and Translational Science for helping with blood drawing from HSV-1 seropositive SYMP and ASYMP individuals, and Adam BenMohamed for critical reading and editing this manuscript.

Footnotes

  • This work was supported by Public Health Service Research R01 Grants EY026103, EY019896, and EY024618 from National Eye Institute, R21 Grant AI110902 and R41 Grant AI138764-01 from the National Institute of Allergy and Infectious Diseases (to L.B.), and in part by The Discovery Center for Eye Research and a grant from Research to Prevent Blindness.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    A2AR
    A2A adenosine receptor
    ASYMP
    asymptomatic
    gB
    glycoprotein B
    gD
    glycoprotein D
    HSK
    herpetic stromal keratitis
    HSV-1
    HSV type 1
    MFI
    mean fluorescence intensity
    PI
    postinfection
    RT
    room temperature
    SYMP
    symptomatic
    TCM
    central memory CD8+ T
    TEM
    effector memory
    TEMRA
    effector memory RA
    TG
    trigeminal ganglia
    WT
    wild type.

  • Received May 23, 2018.
  • Accepted August 6, 2018.

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