A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4⁺ T-Cells to Recognition by Cytotoxic T-Lymphocytes

Resting CD4⁺ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8⁺ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8⁺ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8⁺ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8⁺ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8⁺ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam₃CSK₄. In contrast, we did not observe CD8⁺ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist ‘ALT-803’, an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8⁺ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8⁺ T-cells in HIV eradication strategies.United States. National Institutes of Health (AI111860

A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes.

Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist \u27ALT-803\u27, an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies

Recognition of latently-infected primary CD4⁺ T-cells by virus-specific CD8⁺ T-cell clones following exposure to candidate LRAs.

<p>HIV-, CMV- and HERV-K-specific CD8⁺ T-cell clones were derived from the HIV-infected participant OM9. Latently-infected and productively-infected target cells were prepared and characterized as in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.g001" target="_blank">Fig 1A</a></b>. The designated CD8⁺ T-cell clones were co-cultured with the indicated target CD4⁺ T-cells (autologous) immediately after the depletion of activated cells. Cells were stained and fixed after 16 hour co-cultures. <b>A.</b> Shown are flow cytometry data gated on CD3⁺CD8⁺ lymphocytes and depicting CD107a (degranulation)–y-axis, by IFN-γ –x-axis. Latently-infected cells did not induce CD107a exposure. <b>B.</b> Summary flow cytometry data of the same experiment depicted in <b>A</b>. P-values were calculated by ANOVA with Holm-Sidak’s multiple comparison test (comparing each condition with latent-mock). All conditions/replicates tested are shown. Latent-mock and latent-HIV conditions of MHC-I mismatch were omitted from the HIV-Nef-spec CD8⁺ T-cells due to insufficient cell numbers, as were replicates of MHC-I mismatch conditions for the HERV-K-Env-specific CD8⁺ T-cell clone. CD107a exposure by an HIV-specific CD8⁺ T-cell clone was only induced by productive HIV infection. <b>C</b>. In a separate experiment, latently-infected target cells were prepared in the same manner as <b>A,</b> rested for 72 hours, and then combined with an autologous HIV-Gag-specific CD8⁺ T-cell clone (upper panels) or an autologous CMV-pp65-specific CD8⁺ T-cell clone (lower panels) for a 24 hour co-culture period. Candidate latency-reversing drugs were added as indicated above the corresponding panels, and left in for the duration of co-cultures. Shown are flow cytometry data gated on CD3⁺CD8⁺ lymphocytes and depicting CD137 (4-1BB)–y-axis by CD8 x-axis. CD137 expression by an HIV-specific CD8⁺ T-cell clone was induced following treatment with IL-15, IL-2, and prostratin, but not SAHA.</p

ALT-803 induces HIV transcription from both a primary cell model of latency and from <i>ex vivo</i> patient samples.

<p>Primary CD4⁺ T-cells from healthy donor PBMC were activated, infected with a luciferase reporter HIV virus, and allowed to return to a resting state. This is similar to a post-activation latency model that has been previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.ref017" target="_blank">17</a>] with additional minor modifications (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#sec008" target="_blank">Methods</a>). After 1 week, infected cells were treated with the agents at concentrations indicated for 2 days. <b>A.</b> Shown are luminescence values from luciferase, indicating dose-dependent HIV reactivation by each of the compounds tested. <b>B.</b> Compound associated cytotoxicity was determined in latently-infected cells in parallel with the virus activation assay using Cell Titer Glo (CTG) Dose dependent cytotoxicity was observed with SAHA and romidepsin but not with IL-15 or ALT-803. <b>C and D.</b> PBMC were isolated from HIV-infected subjects who had been suppressed by ART for at least 2 years. Cultures were maintained in the presence of ARVs. Either 1 nM of ALT-803 or 5 ng/ml PMA + 500 ng/ml ionomycin were added for 7 days. HIV activation was measured by quantitating viral RNA in cell-free supernatant using COBAS qPCR. <b>C.</b> The geometric mean of viral copies/ml for each donor are indicated for control (DMSO) and ALT-803 treated wells, following 7 days of treatment. <b>D</b>. The fold HIV activations of all donors are plotted as the ratio of the viral copies/ml for ALT-803 treated or PMA/ionomycin treated to control wells. These results indicate that ALT-803 reactivates virus from the natural patient reservoir, though to a lesser degree than PMA/ionomycin. <b>E-I.</b> Cryopreserved CD4⁺ T-cells from ARV-treated HIV-infected subjects were thawed, CFSE-labeled, and treated with either 1 nM ALT-803 or 200 ng/ml of PHA for 7 days in the presence of ARVs. <b>E</b>. Activation of CD4⁺ T-cells within PBMCs was measured at day 7 by flow cytometry, gating on CD3⁺CD4⁺ T-cells and assessing CD69 expression. Each line represents a different subject. P values were calculated by RM one-way ANOVA with Holm-Sidak’s multiple comparison test. <b>F</b>. Proliferation of CD4+ T-cells within PBCs was measured at day 7 by flow, gating on CD3⁺CD4⁺ T-cells and assessing proliferation as %CFSE^dim cells. P values were calculated by RM one-way ANOVA with Holm-Sidak’s multiple comparison test. G-I. Cell associated DNA was isolated from purified CD4⁺ T-cells following 6 days of stimulation. Absolute copies of HIV-Gag and RPP30 were determined by droplet digital PCR and used to calculate copies of HIV per 10⁶ CD4⁺ T-cells. Shown are results from three different subjects. Quantifications were determined in triplicate (<b>G</b>) or quadruplicate (<b>H & I</b>). Means and SEM are shown and the P value was calculated by one-way ANOVA with Holm-Sidak’s multiple comparison test.</p

Effects of latency-reversing agents on CD8⁺ T-cell elimination of HIV-infected target cells.

<p><b>A—C.</b> CD4⁺ T-cells were enriched from HIV-infected subjects, stimulated with anti-CD3/anti-CD28 for 48 hours, and infected with HIV-LAI. Three different CD8⁺ T-cell clones (HIV-Gag-KK9-specific, HIV-Pol-TY9-specific, and CMV-pp65-specific) were washed and then cultured in the presence of: i) RPMI-10 ii) RPMI-10 + 50 U/ml IL-2 iii) RPMI-10 + 1.4 nM ALT-803 iv) RPMI-10 + 50 U/ml IL-2 + 1.4 nM ALT-803 v) RPMI-10 + 50 U/ml IL-2 + 6.6 μM Pam₃CSK₄ vi) RPMI-10 + 50 U/ml IL-2 + 25 nM romidepsin vii) RPMI-10 + 50 U/ml IL-2 + 390 nM prostratin. Cells were cultured 60 hours. For the romidepsin treatment, cells were washed after 3 hours and medium was replaced with RPMI-10 + 50U/ml IL-2 for the remaining 57 hours. For the prostratin treatment cells were washed after 40 hours and given a 20 hour ‘rest’ in RPMI-10 + 50U/ml IL-2. For all other conditions LRAs were left in throughout the 60 hours and then washed repeatedly. CD8⁺ T-cell clones were then co-cultured with autologous HIV-infected target cells at the indicated clone:target ratios for 16 hours. Levels of infection were assessed by flow cytometry by gating on CD4⁺CD8^- viable lymphocytes and then measuring %HIV-Gag⁺CD4dim (%Infected, y-axis). All conditions were tested in triplicate. Shown are means ± SEM. P values were calculated by 2-way ANOVA with Tukey’s multiple comparison test, comparing each condition to every other condition. For both clones, P < 0.0001 for all comparisons except for the following non-significant cases: i) no treatment vs IL-2 + ALT-803 ii) no treatment vs IL-2 + prostratin iii) IL-2 vs ALT-803 iv) IL-2 vs IL-2 + ALT-803 v) ALT-803 vs IL-2 + ALT-803 vi) IL-2 + romidpesin vs IL-2 + prostratin. The results showed that IL-2, ALT-803, and Pam₃CSK₄ enhance killing of infected cells by CD8⁺ T-cell clones, whereas prostratin impaired killing. Non-specific killing was not observed with the CMV-pp65 <b>D</b>. CD4⁺ T-cells from an ARV-treated HIV-infected subject were stimulated with anti-CD3/anti-CD28 for 48 hours, and infected with HIV-LAI. Autologous bulk CD8⁺ T-cells were enriched by negative selection and cultured with the indicated LRAs for 16 hours at the following concentrations: ALT-803–1.4 nM, IL-2–1.3 nM, IL-7–1 nM, panobinostat– 20 nM, Pam₃CSK₄−3.3 μM. For ALT-803, IL-2, IL-7, and Pam₃CSK₄ LRAs were left in throughout the co-culture period. For panobinostat, cells were treated with LRAs for 3 hours and then drugs were washed out for remaining co-culture period. In either case, bulk CD8⁺ T-cells were washed 3 additional times prior to co-culture with autologous infected CD4⁺ T-cells for 6 hours. Cells were then stained with viability dye and antibodies to CD4, CD3, CD8, and HIV-Gag (intracellularly) and analyzed by flow cytometry. Shown are mean ± SEM of % infected cells remaining (normalized to no CD8⁺ T-cell control). Statistical significance was calculated by ANOVA with Dunnett’s multiple comparisons test. These data shown that ALT-803 enhanced the abilities of <i>ex vivo</i> CD8+ T-cells to kill HIV-infected cells.</p

Induction of HIV by LRAs in a primary CD4+ T-cell direct infection latency model.

<p>CD4⁺ T-cells were isolated from PBMC by negative selection, cultured with CCL19 and then magnetofected with HIV, or mock infected (no virus). Two days later, cells were depleted of activated cells. We defined activated cells as those expressing at least one of CD69, HLA-DR, or CD25 based on a study that defined CD4+ T-cells with the triple-negative phenotype as quiescent[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.ref029" target="_blank">29</a>]. In parallel, CD4⁺ T-cells were activated using anti-CD3/anti-CD28 antibodies and infected with HIV (productively infected). <b>A</b>. Depletion of activated cells by anti-PE microbeads. Shown are flow cytometry data gated on CD3⁺CD4⁺ lymphocytes (95% pure) and depicting CD25/CD69/HLA-DR staining (all pooled on PE channel)–y-axis, by HIV-Gag–x-axis. The left and middle panels represent pre- and post-depletion of activated cells, respectively to generate latently-infected cells. The right panel shows productively-infected target cells. <b>B</b>. Latently infected or mock-infected resting CD4⁺ T-cells were prepared as in <b>A</b> and then either stimulated with 1.5 nM IL-15, with 2.6 μM prostratin, or left unstimulated for 36 hours. Shown are flow cytometry data, gated on lymphocytes (SSC/FSC) and depicting CD4 –y-axis, by HIV-Gag–x-axis. <b>C</b>. In a separate experiment analogous to <b>B</b> latently-infected or mock-infected cells were treated with the indicated concentrations of IL-15 for 16 hours. The percentage of HIV-Gag⁺ cells (gated as in <b>B</b>) is plotted against the concentration of IL-15 (red circles = infected, green squares = uninfected). <b>D.</b> Latently-infected cells were stimulated with the indicated LRAs for 36 hours and HIV p24 in supernatants was quantified by ELISA. Shown are background-subtracted mean ± SEM values (duplicates). P-values were calculated by ANOVA with Holm-Sidak’s multiple comparison test (comparing each condition with no treatment [No Tx]) * p < 0.05, ** p < 0.01.</p

A subset of latency-reversing agents prime latently-infected CD4⁺ T-cells for CD8⁺ T-cell recognition in a continuous co-culture assay.

<p><b>A.</b> An HIV-Gag-SLYNTVATL (SL9) specific CD8⁺ T-cell clone was isolated from subject OM9. To confirm specificity, this clone was co-cultured with an autologous B lymphoblastoid cell line (BLCL) that had been pulsed with 1 μg/ml of SL9 peptide or with an unpulsed control. Shown are flow cytometry data gated on CD8⁺ lymphocytes, depicting CD107a staining (degranulation)–y-axis by IFN-γ –x-axis. These data indicate that the CD8⁺ T-cell clone to be used in subsequent panels was highly specific. <b>B</b>. CD4⁺ T-cells latently infected with HIV-JR-CSF, or mock infected, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.g001" target="_blank">Fig 1</a>, using leukapheresis material autologous to the CD8⁺ T-cell clone in <b>A.</b> Total and integrated HIV DNA were quantified by qPCR. These cells were treated with the indicated drugs at the following concentrations: IL-2 1.3 nM, IL-7 1 nM, IL-15 1.4 nM or SAHA 500 nM for 72 hours in the presence of nevirapine. These concentrations of IL-2, IL-7, and IL-15 equate to 20 ng/ml, and were selected based on concentrations used in previous studies of latency reversal [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.ref032" target="_blank">32</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.ref033" target="_blank">33</a>]. Target cells were then co-cultured with the HIV-Gag-specific CD8⁺ T-cell clone from <b>A</b> for an additional 72 hours. Shown are IFN-γ levels quantified in supernatants (mean ± SEM). These results indicate that IL-2 and IL-15 primed latently-infected cells for recognition by CD8⁺ T-cells. <b>C.</b> An experiment was setup in an identical manner to <b>B</b>, co-culturing with an HIV-Gag-SL9-specific CD8⁺ T-cell clone in the presence of the indicated single of combinations of drugs at the following concentrations: SAHA 500 nM; IL-15SA 1.4 nM. Shown are mean ± IFN-γ quantifications (ELISA).</p

A subset of latency-reversing agents primes latently-infected CD4⁺ T-cells for CD8⁺ T-cell recognition in a pulse-wash assay.

<p>Latently-infected resting CD4⁺ T-cell targets were prepared as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.g001" target="_blank">Fig 1</a>, using cells from three different HIV-infected subjects: OM9, OM292, OM265. Cells were infected with HIV-LAI, HIV-LAI-SL9-escape (three mutations in epitope), or mock infected. Specificities of the following CD8⁺ T-cell clones were confirmed by degranulation assays (CD107a): HIV-Gag-RV9 (OM292), HIV-Env-VL9 (OM292), HIV-Gag-SL9 (OM265), HIV-Pol-TY9 (OM265), CMV pool (OM265), and HIV-Gag-SL9 (OM9). Target cells were plated in ARVs and treated with the following concentrations of putative LRA for 56 hours: no treatment, 0.5% DMSO, 0.5 mM HMBA in 0.5% DMSO, 6.7 μM Pam₃CSK₄, 500 nM SAHA in 0.5% DMSO, 25 nM romidepsin, 25 nM panobinostat, 1.3 nM IL-2, 1 nM IL-7, 1.4 nM IL-15, 1.4 nM <b>ALT-803</b>, 39 nM prostratin. These concentrations of IL-2, IL-7, and IL-15 equate to 20 ng/ml, and were selected based on concentrations used in previous studies of latency reversal [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.ref032" target="_blank">32</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.ref033" target="_blank">33</a>]. The concentration of ALT-803 was set equimolar to IL-15. Concentrations of the HDAC inhibitors were selected based on previous reports indicating latency reversal without substantial toxicity to resting CD4⁺ T-cells [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.ref017" target="_blank">17</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.ref031" target="_blank">31</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.ref037" target="_blank">37</a>]. The concentration of prostratin was selected to be on the low end of its active range against HIV latency to challenge the sensitivity of the assay. Target cells were then washed 3x to remove LRAs, and CD8⁺ T-cell clones were added. Following a 16 hour CD8⁺ T-cell + target cell co-culture, supernatants were harvested for IFN-γ ELISA. <b>A.</b> Shown are mean ± SEM IFN-γ ELISA data for an HIV-Gag-specific CD8⁺ T-cell clone (left) and a CMV-pp65-specific CD8⁺ T-cell clones (right). In each panel, the drugs to the left of the dotted vertical line were dissolved in 0.5% DMSO and thus are comparable to the DMSO control while those to the right of the dotted vertical line are in PBS (Il-2, IL-7, IL-15, ALT-803, Pam₃CSK₄) or < 0.01% DMSO (romidepsin, panobinostat, prostratin) and thus are comparable to the no treatment (No Tx) control. P values refer to differences in the wild-type infected condition and were calculated by ANOVA with Holm-Sidak’s multiple comparison test. The results indicate that a subset of LRAs prime latently-infected cells for recognition by this representative CD8⁺ T-cell clone. <b>B.</b> Shown are summary ELISA data with each line representing a single CD8⁺ T-cell clone tested against autologous target cells with the indicated LRAs or controls. P values were calculated by paired t-tests. The results indicate that a subset of LRAs consistently prime latently-infected cells for recognition by a panel of CD8⁺ T-cell clones.</p

HIV-specific CD8⁺ T-cells sense ALT-803 mediated viral reactivation from the natural HIV reservoir.

<p>PBMC from 5 HLA-A02⁺ and 5 HLA-A02^- ARV-treated HIV-infected subjects were depleted of CD8⁺ T-cells, and of cells expressing CD69, CD25, or HLA-DR and then stimulated with 1.4 nM ALT-803 or maintained as untreated controls for 72 hours in the presence of ARVs. These target cells were then washed and co-cultured with an HIV-Gag-SL9-specific CD8⁺ T-cell clone (HLA-A02 restricted) for 18 hours with the addition of either an anti-MHC-I blocking antibody or an isotype control. Shown are ELISA data measuring concentrations of IFN-γ in supernatants at the end of this co-culture period. Each condition was tested in duplicate and mean values were plotted. P values were calculated by one-tailed Wilcoxon matched pairs tests. The results indicate that ALT-803 primes latently-infected cells from the natural reservoir for recognition by an HIV-specific CD8⁺ T-cell clone.</p