Rhesus Cytomegalovirus-Specific CD8+ Cytotoxic T Lymphocytes Do Not Become Functionally Exhausted in Chronic SIVmac239 Infection

Abstract

CD8⁺ cytotoxic T lymphocytes (CTLs) exert potent antiviral activity after HIV/SIV infection. However, efforts to harness the antiviral efficacy of CTLs for HIV/SIV prophylaxis and therapy have been severely hindered by two major problems: viral escape and exhaustion. By contrast, CTLs directed against human cytomegalovirus (HCMV), a ubiquitous chronic herpesvirus, seldom select for escape mutations and remain functional and refractory to exhaustion during chronic HCMV and HIV infection. Recently, attempts have been made to retarget HCMV-specific CTLs for cancer immunotherapy. We speculate that such a strategy may also be beneficial in the context of HIV/SIV infection, facilitating CTL-mediated control of HIV/SIV replication. As a preliminary assessment of the validity of this approach, we investigated the phenotypes and functionality of rhesus CMV (RhCMV)-specific CTLs in SIVmac239-infected Indian rhesus macaques (RMs), a crucial HIV animal model system. We recently identified two immunodominant, Mamu-A^∗02-restricted CTL epitopes derived from RhCMV proteins and sought to evaluate the phenotypic and functional characteristics of these CTL populations in chronic SIVmac239 infection. We analyzed and directly compared RhCMV- and SIVmac239-specific CTLs during SIVmac239 infection in a cohort of Mamu-A^∗01⁺ and Mamu-A^∗02⁺ RMs. CTL populations specific for at least one of the RhCMV-derived CTL epitopes were detected in ten of eleven Mamu-A^∗02⁺ animals tested, and both populations were detected in five of these animals. Neither RhCMV-specific CTL population exhibited significant changes in frequency, memory phenotype, granzyme B expression, exhaustion marker (PD-1 and CTLA-4) expression, or polyfunctionality between pre- and chronic SIVmac239 infection timepoints. In chronic SIVmac239 infection, RhCMV-specific CTLs exhibited higher levels of granzyme B expression and polyfunctionality, and lower levels of exhaustion marker expression, than SIVmac239-specific CTLs. Additionally, compared to SIVmac239-specific CTLs, greater proportions of RhCMV-specific CTLs were of the terminally differentiated effector memory phenotype (CD28^- CCR7^-) during chronic SIVmac239 infection. These results suggest that, in contrast to SIVmac239-specific CTLs, RhCMV-specific CTLs maintain their phenotypes and cytolytic effector functions during chronic SIVmac239 infection, and that retargeting RhCMV-specific CTLs might be a promising SIV immunotherapeutic strategy.

Keywords: cytomegalovirus; cytotoxic T lymphocytes; human immunodeficiency virus; rhesus macaques; simian immunodeficiency virus.

FIGURE 1

SIVmac239 viral loads for rhesus macaques in this study. (A) Mamu-A*01⁺ RMs. (B) Mamu-A*02⁺ RMs. Viral loads for rh2306 are shown in both graphs, since this animal was Mamu-A*01⁺ Mamu-A*02⁺.

FIGURE 2

Frequencies of RhCMV-specific CD8⁺ T cells before and during SIVmac239 infection. PBMCs from SIVmac239-infected Mamu-A*02⁺ animals were stained with (A–C) the indicated pMHCI tetramers or (D–F) stimulated with the corresponding minimal optimal peptides in a CD107a degranulation assay with ICS. Graphs depict the frequencies of antigen-specific CD8⁺ T cells among all CD8⁺ T cells (defined as live CD3⁺ CD8⁺ CD14^– CD20^– CD159a^– lymphocytes). Flow cytometry plots depict all CD8⁺ T cells for the animals with the highest frequencies of RhCMV-specific CD8⁺ T cells in chronic SIVmac239 infection by tetramer staining (B,C) or CD107a degranulation assay with ICS (E,F). None of the differences in tetramer or CD107a/ICS frequencies between pre- and chronic infection timepoints were statistically significant (all p > 0.05 by Welch’s t-test).

FIGURE 3

Phenotypic and functional characteristics of RhCMV-specific CD8⁺ T cells before and during SIVmac239 infection. Unstimulated PBMCs were stained with pMHCI tetramer and fluorophore-conjugated mAbs to determine the frequencies of (A) T_EM2 (CD28^– CCR7^–) and (B) granzyme B⁺ cells among tetramer⁺ CD8⁺ T cells (defined as live CD3⁺ CD8⁺ tetramer⁺ CD14^– CD20^– CD159a^– lymphocytes). (C) CTL polyfunctionality was evaluated by CD107a/ICS assay, in which PBMCs were stimulated with the corresponding minimal optimal peptides. A responding CD8⁺ T cell (a live CD3⁺ CD8⁺ CD14^– CD20^– CD159a^– lymphocyte) was defined as a cell staining positive for CD107a or IFN-γ or TNF-α. Polyfunctionality was defined as the proportion of responding CD8⁺ T cells staining for all three markers simultaneously (CD107a⁺ IFN-γ⁺ TNF-α⁺). None of the differences between pre- and chronic-phase CD8⁺ T cells in panels (A–C) were statistically significant (all p > 0.05 by Welch’s t-test).

FIGURE 4

Exhaustion marker expression by RhCMV-specific CD8⁺ T cells before and during SIVmac239 infection. (A) Frequencies of PD-1⁺ CTLs and (B) PD-1 median fluorescence intensities (MFIs) were determined by pMHCI tetramer staining of unstimulated PBMCs. PD-1 expression was analyzed in tetramer⁺ CD8⁺ T cells (defined as live CD3⁺ CD8⁺ tetramer⁺ CD14^– CD20^– CD159a^– lymphocytes). (C) Frequencies of CTLA-4⁺ CTLs and (D) CTLA-4 MFIs were evaluated by CD107a/ICS assay, in which PBMCs were stimulated with the corresponding minimal optimal peptides. CTLA-4 expression was analyzed in responding CD8⁺ T cells, which were defined as live CD3⁺ CD8⁺ CD14^– CD20^– CD159a^– lymphocytes staining positive for CD107a or IFN-γ or TNF-α. None of the differences between pre- and chronic-phase CD8⁺ T cells in panels (A–D) were statistically significant (all p > 0.05 by Welch’s t-test).

FIGURE 5

Frequencies of T_EM2 lymphocytes among RhCMV- and SIVmac239-specific CD8⁺ T cells during chronic SIVmac239 infection. Graphs depict the frequencies of T_EM2 (CD28^– CCR7^–) cells among tetramer⁺ CD8⁺ T cells (defined as live CD3⁺ CD8⁺ tetramer⁺ CD14^– CD20^– CD159a^– lymphocytes). Statistical analysis was conducted using Welch’s t-test. Comparison of T_EM2 frequencies between Tat SL8- and Gag CM9-specific CD8⁺ T cells yielded a p-value of 0.0407 (Welch’s t-test; not shown in figure). All other comparisons of RhCMV- and/or SIVmac239-specific CD8⁺ T cells without p-values listed in figure yielded non-significant p-values (p > 0.05).

FIGURE 6

Granzyme B expression in RhCMV- and SIVmac239-specific CD8⁺ T cells during chronic SIVmac239 infection. Graph depicts the frequencies of granzyme B⁺ cells among tetramer⁺ CD8⁺ T cells (defined as live CD3⁺ CD8⁺ tetramer⁺ CD14^– CD20^– CD159a^– lymphocytes). Statistical analysis was conducted using Welch’s t-test. The following comparisons of granzyme B frequencies in SIVmac239-specific CD8⁺ T cell populations also yielded statistically significant p-values: Tat SL8- and Gag CM9-specific CD8⁺ T cells (p = 0.0103); Tat SL8- and Nef YY9-specific CD8⁺ T cells (p = 0.0150). All other comparisons of RhCMV- and/or SIVmac239-specific CD8⁺ T cells without p-values listed in figure yielded non-significant p-values (p > 0.05).

FIGURE 7

Frequencies of polyfunctional lymphocytes (CD107a⁺ IFN-γ⁺ TNF-α⁺) among RhCMV- and SIVmac239-specific CD8⁺ T cells during chronic SIVmac239 infection. Graph depicts the frequencies of polyfunctional (CD107a⁺ IFN-γ⁺ TNF-α⁺) CD8⁺ T cells responding to minimal optimal peptide in a CD107a degranulation assay with ICS. A responding CD8⁺ T cell was defined as a live CD3⁺ CD8⁺ CD14^– CD20^– CD159a^– lymphocyte staining positive for CD107a or IFN-γ or TNF-α. Statistical significance was evaluated using Welch’s t-test. None of the comparisons between SIVmac239-specific CD8⁺ T cell populations yielded statistically significant p-values (not shown on graph, all p > 0.05).

FIGURE 8

PD-1 expression by RhCMV- and SIVmac239-specific CD8⁺ T cells during chronic SIVmac239 infection. Graph depicts (A) frequencies of PD-1⁺ cells among tetramer⁺ CD8⁺ T cells and (B) PD-1 median fluorescence intensities (MFIs) for tetramer⁺ CD8⁺ T cells (defined as live CD3⁺ CD8⁺ tetramer⁺ CD14^– CD20^– CD159a^– lymphocytes). Statistical analysis was conducted using Welch’s t-test. The following comparisons of PD-1⁺ frequencies between SIVmac239-specific CD8⁺ T cell populations yielded statistically significant p-values: Tat SL8- and Gag CM9-specific CD8⁺ T cells (p = 0.0066); Tat SL8- and Nef YY9-specific CD8⁺ T cells (p = 0.0160). The following comparisons of PD-1 MFIs for SIVmac239-specific CD8⁺ T cells yielded statistically significant p-values: Tat SL8- and Gag CM9-specific CD8⁺ T cells (p = 0.0048); Gag CM9- and Nef YY9-specific CD8⁺ T cells (p = 0.0134); Gag CM9- and Gag GY9-specific CD8⁺ T cells (p = 0.0159). All other comparisons of RhCMV- and/or SIVmac239-specific CD8⁺ T cells without p-values listed in figure yielded non-significant p-values (p > 0.05).

FIGURE 9

CTLA-4 expression by RhCMV- and SIVmac239-specific CD8⁺ T cells during chronic SIVmac239 infection. Graph depicts (A) frequencies of CTLA-4⁺ lymphocytes among CD8⁺ T cells responding to minimal optimal peptide and (B) CTLA-4 median fluorescence intensities (MFIs) for responding CD8⁺ T cells in a CD107a/ICS assay. A responding CD8⁺ T cell was defined as a live CD3⁺ CD8⁺ CD14^– CD20^– CD159a^– lymphocyte staining positive for CD107a or IFN-γ or TNF-α. Statistical significance was evaluated using Welch’s t-test. None of the comparisons of CTLA-4 frequencies between SIVmac239-specific CD8⁺ T cell populations yielded statistically significant p-values (all p > 0.05). The following comparisons of CTLA-4 MFIs for SIVmac239-specific CD8⁺ T cells yielded statistically significant p-values: Tat SL8- and Gag CM9-specific CD8⁺ T cells (p = 0.0172); Gag CM9- and Nef YY9-specific CD8⁺ T cells (p = 0.0089); Gag CM9- and Gag GY9-specific CD8⁺ T cells (p = 0.0217). All other comparisons of RhCMV- and/or SIVmac239-specific CD8⁺ T cells without p-values listed in figure yielded non-significant p-values (p > 0.05).