NK and CD4⁺ T cell co-operative immune responses correlate with control of disease in a macaque SIV infection model

Abstract

Control of infectious disease may be accomplished by successful vaccination, or by complex immunologic and genetic factors favoring antigen-specific multicellular immune responses. Using a rhesus macaque model, we evaluated antigen-specific T cell-dependent NK cell immune responses in SIV-infected macaques, designated controlling or non-controlling based on long-term chronic viremia levels, to determine if NK cell effector functions contribute to control of SIV infection. We observed that Gag stimulation of macaque PBMCs induced subset-specific NK cell responses in SIV-controlling, but not non-controlling animals, and that circulatory NK cell responses were dependent on antigen-specific IL-2 production by CD4⁺ central memory T cells. NK cell activation was blocked by anti-IL-2 neutralizing antibody and by CD4⁺ T cell depletion which abrogated the Gag-specific responses. Among tissue-resident cells, splenic and circulatory NK cells displayed similar activation profiles, whereas liver and mucosal NK cells displayed a decreased activation profile, similar in SIV controlling and non-controlling macaques. Lack of T cell-dependent NK cell function was rescued in SIV non-controlling macaques through drug-mediated control of viremia. Our results indicate that control of disease progression in SIV controlling macaques is associated with co-operation between antigen-specific CD4⁺ T cells and NK cell effector function, highlight the importance of such cell-to-cell co-operativity in adaptive immunity and suggest this interaction should be further investigated in HIV vaccine development and other prophylactic vaccine approaches.

INTRODUCTION

Natural killer (NK) cells are key components of the immune system. Due to their rapid response potential and broad biodistribution, they impact innate and adaptive anti-viral immune responses (1). They are specialized in detection and elimination of pathogen-infected and neoplastic cells, and modulate immune responses through production of inflammatory and regulatory cytokines and chemokines (2, 3). The cytotoxic activity of NK cells is exerted by both antibody-dependent and –independent mechanisms, illustrating the ability of NK cells to bridge innate and adaptive immunity (4). Further evidence of this bridging comes from reports of an antigen-specific IL-2-dependent co-operation between human CD4 T cells and NK cells (5, 6). Following vaccination against either P. falciparum or rabies virus, antigen-specific IL-2 production by memory CD4⁺ T cells is correlated with, and necessary for, NK cell activation (7-9). In some individuals the NK cell response to P. falciparum can represent up to 70% of IFN-γ-producing lymphocytes in such antigen-specific recall assays (8). Thus, mechanisms which lead to efficient T cell-mediated NK cell effector function are of interest for both prophylactic and therapeutic vaccine development (7).

Recent evidence suggests that innate immunity may play a crucial role in the control of HIV infection at all stages of disease (10). NK cell functions such as production of IFN-γ, β-chemokines, and direct killing of HIV-infected cells have all been hypothesized as potential correlates of protection in HIV-1 highly exposed seronegative subjects (11). The possibility that co-operation with the adaptive immune system may impact NK cells, providing a de facto potential for T cell-dependent effector responses, has important implications for HIV/SIV vaccine development, and would provide yet another mechanism available for prophylactic and/or therapeutic protection. Here we studied rhesus macaques, the model of choice for evaluating SIV vaccines (12), to determine if T cell-dependent NK cell immune responses contribute to control of SIV infection. We asked if memory CD4 T cells co-operate with NK cells, and whether such an interaction affects SIV replication in controlling versus non-controlling SIV-infected macaques. We found that subpopulation-specific circulatory and tissue NK cell responses were observed only in SIV-controlling animals. These responses were directly correlated with and dependent on antigen-specific IL-2 production by SIV-specific memory CD4⁺ T cells and inversely correlated with viral load. Our results suggest that NK and CD4⁺ T cell responses co-operate in the control of SIV replication and disease progression, providing another potential correlate of protective immunity.

MATERIALS AND METHODS

Animals and cell collection

Assays used freshly isolated (n=25) and frozen (n=20) peripheral blood mononuclear cells (PBMCs) from naïve or SIV _mac251-infected rhesus macaques (Macaca mulatta). Macaques were catalogued as SIV controllers if they exhibited a chronic viral load of ≤10⁴ copies/mL plasma for at least 16 weeks prior to sample collection. SIV non-controlling animals had a chronic viral load of ≥10⁵ copies/mL plasma. Frozen cells were obtained from previously identified SIV controlling and non-controlling macaques (13, 14). Animals were housed at Bioqual, Inc. (Gaithersburg, MD), or at Advanced BioScience Laboratories, Inc. (ABL; Kensington, MD), and maintained according to institutional Animal Care and Use Committee guidelines. Blood samples were collected by venipuncture of anesthetized animals into EDTA-treated collection tubes. PBMCs were obtained by centrifugation on Ficoll-Paque PLUS gradients (GE Healthcare, Piscataway, NJ). Cells were washed and resuspended in R-10 medium (RPMI 1640 containing 10% FBS, 2 mM L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate and antibiotics). Frozen cells were thawed and rested for 8 h in R-10 medium before stimulation. For evaluation of tissue-specific T cell-dependant NK cell responses, 11 macaques (3 naïve, 4 SIV controlling and 4 SIV non-controlling) were sacrificed 64 to 76 weeks after initial SIV infection for collection of peripheral blood, spleen, liver, jejunum and colon biopsies. Spleen and liver biopsies were minced, passed through a 40 μm cell strainer, and lysed to remove contaminating red blood cells. Jejunum and colon biopsies were teased apart with 23G needles, and then digested for two 30 min intervals on an orbital shaker in R-10 medium containing 1 mg/mL of collagenase II (Sigma, St Louis, MO). Liver, jejunum and colon mononuclear cells were further enriched by centrifugation on Percoll gradients.

Antiretroviral Treatment

To study the impact of viral load on T cell-dependant NK cell activation, 7 SIV non-controlling macaques were treated for a period of 8 weeks with a triple cocktail of antiretroviral therapy (ART) containing: Didanosine (Videx, Bristol-Myers Squibb Co, Princeton, NJ), Stavudine (Zerit, Bristol-Myers Squibb Co.) and 9-[2-(R)-[[bis[[(isopropoxycarbonyl)-oxy]methoxy]phosphinoyl]methoxy]propyl]adenine fumarate (PMPA, Gilead Sciences Inc., Foster City, CA). Videx was given intravenously as a single daily dose of 10 mg/kg, Zerit was administered twice a day orally at a 1.2 mg/kg dose, and PMPA was given as a single daily subcutaneous dose of 20 mg/kg. To prevent damage to the pancreas, the Videx dose was decreased to 5 mg/kg after 3 weeks of treatment.

Flow cytometry

Anti-human fluorochrome-conjugated monoclonal antibodies known to cross-react with rhesus macaque antigens were used, including: FITC anti-CD69 (FN50), V450 anti-IFN-γ (B27), PE-Cy5 anti-CD95 (DX2), PE-Cy7 anti-CD56 (B159), Alexa Fluor 700 anti-CD3 (SP34-2), APC-Cy7 anti-IL-2 (MQ1-17H12), and APC-Cy7 anti-CD16 (3G8), all from BD Biosciences (San Jose, CA); Alexa Fluor 647 anti-CD107a (eBioH4A3), PE-Cy7 anti-CD28 (CD28.2), and eFluor 650NC anti-CD8α (RPA-T8), all from eBioscience (San Diego, CA); QDot605 anti-CD4 (custom conjugation of clone L200 from BD Biosciences), and QDot605 anti-CD8α (3B5) from Invitrogen (Carlsbad, CA); PE anti-NKG2A (Z199) from Beckman Coulter (Fullerton, CA); APC anti-α4β7 (rhesus recombinant), and QDot655 anti-CD4 (T4/19Thy5D7) from the NIH NHP Reagent Resource; Pacific Blue anti-CCR7 (TG8/CCR7), and PerCP/Cy5.5 anti-TNF-α (Mab11) from BioLegend (San Diego, CA). The yellow and aqua LIVE/DEAD viability dyes (Invitrogen) were used to exclude dead cells. After SIV peptide stimulation, PBMCs and tissue mononuclear cells were stained for specific surface molecules, fixed and permeabilized with a Cytofix/Cytoperm Kit (BD Biosciences), and then stained for specific intracellular molecules. At least 500,000 singlet events (PBMCs) or 30,000 CD3⁺ singlet events (tissue mononuclear cells) were acquired on a LSR II (BD Biosciences) and analyzed using FlowJo Software (TreeStar Inc). For all samples, gating was established using a combination of isotype and fluorescence-minus-one controls.

NK and T cell Activation Assays

T cell recall responses were assayed by stimulating 2 × 10⁶ PBMCs with 1 μg/mL SIV_mac239 Gag or Env peptide pools (complete sets of 15-mer peptides, overlapping by 11 and spanning the entire protein; NIH AIDS Research & Reference Reagent Program) for 6 h. Stimulation was performed in the presence of 10 μg/mL Brefeldin A, 2 μg/mL anti-CD49d and 0.375 μg/mL PE-Cy7 anti-CD28 (all from BD Biosciences). To determine T cell-dependant NK cell responses, 2 × 10⁶ PBMCs or tissue mononuclear cells were stimulated with 1 μg/mL SIV₂₃₉ Gag or Env peptide pools for 24 h. Stimulation was performed in the presence of 2 μg/mL unconjugated anti-CD49d and anti-CD28 (BD Biosciences). BD GolgiPlug, BD GolgiStop and Alexa Fluor 647 anti-CD107a were added for the last 5 h of culture at the manufacturer's recommended concentrations. Subsequently, cells were washed and stained with a panel of monoclonal antibodies allowing detection of multiple parameters of T and NK cell activation. CD3⁺ T cells were divided into CD4⁺ and CD8⁺ populations and for each, cells were further subdivided into CD28⁺CD95⁺ central (CM) and CD28⁻CD95⁺ effector memory (EM) cells. Percent cytokine secreting cells among each memory subset was then determined. NK cells (CD3^-CD8⁺NKG2A⁺) were further subdivided by their CD16 and CD56 expression patterns (Supp. Fig. 1A). Next, the up-regulation of IFN-γ, TNF-α, CD107a and CD69 in each subpopulation was calculated (Supp. Fig. 1B). For both T and NK cell activation assays, non-stimulated and SEB-treated (5μg/mL; Sigma) tubes were used as negative and positive controls, respectively. In some assays, 10 μg/mL of an anti-IL-2 monoclonal antibody (BioLegend) was added to neutralize IL-2. Non-human primate CD3 and CD4 MicroBead Kits (Miltenyi Biotec, Auburn, CA) were used to deplete these cells from PBMCs. Recombinant macaque IL-15 (150 ng/ml) and IL-2-Fc (a fusion of macaque IL-2 and IgG₂ Fc, 400 ng/ml), both obtained from the NIH/NCRR funded Resource for Nonhuman Primate Immune Reagents (Emory University, Atlanta, GA) were used for activation of enriched NK cells (PBMCs depleted of CD3⁺ T cells). In order to increase the number of animals assayed, 20 viably frozen PBMC samples (3 naïve, 9 SIV controllers and 8 non-controllers) were evaluated for T and NK cell activation. Experimental results using fresh or frozen PBMCs yielded comparable data and were therefore combined for analysis (data not show).

Statistical Analysis

Results are shown as means ± standard errors of the mean. Data were analyzed using Prism (v5.03, GraphPad Software). A p value of ≤0.05 was considered statistically significant for each test.

RESULTS

Gag-specific IFN-γ production by lymphocytes of SIV controlling macaques

SIV_mac251-infected macaques were categorized as controlling (Cont) or non-controlling (Non-Cont) based on their chronic viral load levels (Fig. 1A). No difference was observed in the percentage of NK cells (CD3^-CD8⁺NKG2A⁺) in either group of SIV-infected macaques compared to naïve animals (Fig. 1B). As expected, CD4⁺ T cells were significantly decreased in both groups of infected macaques, more so in non-controllers, while CD8⁺ T cells were proportionally increased (as percent of CD3⁺ T cells) in both SIV-infected groups. Stimulation of PBMCs of SIV-infected animals with Gag or Env peptide pools for 24 h significantly up-regulated Gag-specific IFN-γ producing cells only in SIV controlling macaques (Fig. 2A and C), although all groups responded equally to SEB. IFN-γ-producing cells included NK, CD4⁺ and CD8⁺ T cells (Fig. 2B and D). Among SIV controllers, the proportion of IFN-γ producing cells was significantly higher in NK and CD8⁺ T cells only in response to Gag stimulation (Fig. 2D). Both the proportion (Fig. 2D) and the amount of IFN-γ produced per cell (MFI, data not shown) were significantly higher in NK cells after Gag stimulation of PBMCs. Similarly, NK and CD8⁺ T cells from SIV controllers significantly increased the expression of CD107a (Fig. 2E) and TNF-α (p<0.05, data not shown) in response to Gag stimulation. To rule out lack of response in NK cells of non-controllers due to an intrinsic SIV-associated cell dysfunction, PBMCs of SIV controlling and non-controlling macaques were depleted of CD3⁺ T cells to generate enriched NK cells. On average, CD3⁺ T cell depletion resulted in a 2.5-fold enrichment of NK cells (from 4.5 ± 3.3% to 11.2 ± 6.6 % of live lymphocytes). Neither the enriched NK cells of SIV controlling or non-controlling macaques responded to direct Gag peptide stimulation, indicating a lack of direct recall response capability, and implicating T cells for SIV peptide recognition (Fig. 2F). Nevertheless, NK cells of both groups were equally capable of producing IFN-γ and up-regulating CD69 and CD107a expression in response to IL-2 plus IL-15 stimulation.

Fig. 1. Viral loads and NK, CD4⁺ and CD8⁺ T cell frequencies in blood.

(A) Geometric mean viral loads of macaques (13 naïve, 14 SIV controllers and 18 SIV non-controllers). Viral loads in naïve animals are plotted as 50 copies/mL, the lower detection limit of the assay. ***, p<0.001 indicates statistically significant differences between the compared groups by one-way ANOVA (Kruskal-Wallis test). (B) Percentages of NK (CD3^-CD8⁺NKG2A⁺), CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells in fresh samples (13 naïve, 5 SIV controllers and 10 SIV non-controllers) determined by flow cytometry. *, p<0.05, and *, p<0.05 indicate statistically significant differences between the compared groups by one-way ANOVA. Cont, SIV controllers; Noncont, SIV non-controllers.

Fig. 2. Up-regulation of IFN-γ production by NK and T cells of SIV controller macaques in response to Gag.

Freshly isolated PBMCs were purified and stimulated with Gag and Env peptide pools for 24 h prior to flow cytometry analysis. (A) Representative dot blots depicting IFN-γ production by lymphocytes of a SIV controller macaque in response to different stimuli. (B) Representative dot blots of IFN-γ production in NK, CD4⁺ and CD8⁺ T cells in response to Gag stimulation. (C) Percentage of peptide-specific IFN-γ ⁺ cells among total lymphocytes of SIV controlling and non-controlling macaques. Percentage of IFN-γ⁺ (D) and CD107a⁺ (E) cells among NK, CD4⁺ and CD8⁺ T cells in SIV controlling macaques following peptide stimulation. Data pooled from 11 macaques (3 naïve, 4 SIV controllers and 4 SIV non-controllers).***, p<0.001, and **, p<0.01 indicate statistically significant differences when compared to the non-stimulated (NS) group by two-way ANOVA. (F) Enriched NK cells (CD3-depleted PBMCs) from 3 SIV controlling and 3 non-controlling macaques were stimulated with Gag or IL-2 plus IL-15. Percentage of responding NK cells for each stimuli and parameter measured are shown.

Subset-specific NK cell activation in SIV controlling macaques

Three subpopulations of macaque circulatory NK cells exist based on CD16 and CD56 expression patterns (Supp. Fig. 1A). CD16⁺CD56^-/dim cells (CD16⁺ NK Cells) represent ~85% of circulatory NK cells, have low cytokine-producing capacity and are considered mostly cytotoxic effectors. CD16^-CD56⁺ cells (CD56⁺ NK cells), ~5% of circulatory NK cells, are considered mostly cytokine-producing cells. Double negative (DN, CD16^-CD56^-) NK cells (~10% of circulatory NK cells) do not have a human counterpart and appear to have both cytolytic and cytokine-producing potential (15). In accord with previous results from our lab and others, CD16⁺ NK cells were significantly reduced in SIV non-controlling macaques, which in turn led to a significant increase in the proportion of DN NK cells (Supp. Fig. 2A) (16-18). Upon stimulation of PBMC with Gag or Env peptides, IFN-γ producing NK cells were significantly increased only in SIV controlling animals in comparison to unstimulated PBMC (Fig. 3A). Although all subsets were capable of IFN-γ production, the highest proportion was observed in CD56⁺ NK cells in response to Gag (Fig. 3A, center column). TNF-α-producing NK cells were up-regulated at comparable levels both in CD16⁺ and CD56⁺ NK cells of SIV controlling macaques in response to Gag stimulation, and in SIV non-controlling macaques in response to Env stimulation (Fig. 3B, left and center columns). Up-regulation of CD107a, a surrogate marker for NK cell cytotoxicity, was only observed in DN NK cells of SIV controlling macaques in response to Gag (Fig. 3C, right column). Finally, increased proportions of the early cell activation marker CD69 were observed in all NK cell subsets of SIV controlling macaques, and only in response to Gag (Fig. 3D).

Fig. 3. Effector function in different NK cell subsets is increased in SIV controllers in response to Gag stimulation.

Fresh and frozen PBMCs were stimulated for 24 h in the presence of SIV Gag or Env peptides. Up-regulation of IFN-γ (A), TNF-α (B), CD107a (C), and CD69 (D) was measured in CD16⁺ (left column), CD56⁺ (center column), and CD16^-CD56^- (DN, right column) NK cell subpopulations. Data pooled from 36 macaques (8 naïve, 13 SIV controllers and 15 SIV non-controllers). *, p<0.05, **, p<0.01, and ***, p<0.001 indicate statistically significant differences between compared groups by two-way ANOVA. NS, non-stimulated.

NK cell activation correlates with IL-2 production by CD4⁺ Central Memory T cells

As enriched macaque NK cells didn't respond to Gag peptide stimulation in the absence of T cells (Fig. 2F), we evaluated the T cell contribution to the NK cell responses. Antigen-specific IL-2 producing T cells were evaluated in a 6 h stimulation assay, as T cells elicit a faster response to peptide stimulation than NK cells. A significant up-regulation of IL-2 producing cells among total CD4⁺ T cells of SIV controlling macaques was observed in response to Gag but not Env stimulation (Supp. Fig. 3A). CD8⁺ T cells did not produce any IL-2 in response to either Gag or Env stimulation (Supp. Fig. 3B). Gag-specific IL-2-producing CD4⁺ T cells were confined to the central memory (CM, CD28⁺CD95⁺) compartment (Fig. 4A). No significant IL-2 production was observed in effector memory (EM, CD28^-CD95⁺) CD4⁺ T cells (Supp. Fig. 3C). A positive correlation was seen between both IFN-γ (Fig. 4B, p=0.0004) and TNF-α (Fig. 4C, p=0.0257) producing CD56⁺ NK cells with IL-2 producing CM CD4⁺ T cells, supporting the role of IL-2 in activating NK cells during the T cell recall responses. IL-2 production by CM CD4⁺ T cells also positively correlated with IFN-γ production by CD16⁺ (p=0.0278) and DN (p=0.0373) NK cells (data not shown).

Fig. 4. Gag-specific IL-2 production by central memory T cells is essential for NK cell effector responses.

Fresh and frozen PBMCs were stimulated for 6 h in the presence of SIV Gag or Env peptides or SEB. (A) Production of IL-2 by CD28⁺CD95⁺ central memory (CM) CD4⁺ T cells. (B-C) Correlation between IL-2 production by CM CD4⁺ T cells (at 6 h) and CD56⁺ NK cell IFN-γ (B) and TNF-α (C) responses at 24 h (all Gag-specific by subtraction of non-stimulated (NS) controls). (D-E) Reduction of T cell-dependent NK cell responses in CD16⁺ (D) and CD56⁺ (E) NK cells by the addition of an IL-2 neutralizing antibody. (F-G) Suppression of IFN-γ (F) and CD107a (G) up-regulation in total NK cells by depletion of CD3⁺ or CD4⁺ T cells. Data pooled from 36 macaques (8 naïve, 13 SIV controllers and 15 SIV non-controllers) for A, B and C, from 12 SIV controllers for D and E, and from 3 SIV controllers for F and G. Spearman's correlation analysis was used to determine statistical significance in B and C. *,p<0.05, and ***, p<0.001 indicate statistically significant differences between the compared groups by two-way ANOVA in D and E.

Addition of an IL-2 neutralizing antibody abrogated IFN-γ production by CD16⁺ NK cells (Fig. 4D). Further, the same antibody also significantly (p<0.05) reduced IFN-γ producing CD56⁺ NK cells, although the highest Gag responders were only partially diminished (Fig. 4E). Depletion of either CD3⁺ or CD4⁺ T cells from macaque PBMCs reduced Gag-specific T cell-dependent NK cell responses by over 90% as assessed by IFN-γ production (Fig. 4F). CD107a up-regulation was similarly diminished (Fig. 4G), further supporting the role played by CM CD4⁺ T cells.

Immune responses by tissue-resident NK cells

We evaluated T cell-dependent NK cell responses in spleen, liver, jejunum and colon NK cells. Initially, we examined tissue homing potential and the proportional distribution of NK and T cells in the tissues of the naïve and infected macaques. In SIV non-controllers, expression of α₄β₇, the gut-homing marker, was significantly decreased in CD16⁺ and increased in DN NK cells compared to naïve animals (p<0.05, Supp. Fig. 2B). Expression patterns of CCR7, the lymph node homing marker, were similar in naïve and SIV-infected animals. Despite the subset-specific alterations in α₄β₇ expression, NK cells were present at comparable proportions in all tissues assayed in both naïve and SIV-infected macaques (Supp. Fig. 4A). Similar to PBMCs (Fig. 1B), all tissues from SIV-infected animals showed a decreased proportion of CD4⁺ T cells, most evident in SIV non-controlling macaques (Supp. Fig. 4B-E). Jejunum and colon samples were not available from all animals, but were similar in T and NK cell composition (Supp. Fig. 4A, D-E). Therefore, these two groups were combined for analysis as mucosal biopsies (Supp. Fig. 4F).

Previous reports have shown that tissue-resident NK cells in rhesus macaques are mostly CD16^-, but a fourth subpopulation, characterized by surface expression of both CD16 and CD56, also exists (15). Given the CD16/CD56 expression differences that exist between circulatory and tissue-resident NK cells, we expressed NK immune responses as a percentage of total NK cells in lymphocytes isolated from the various tissues in order to compare up-regulation of activation markers in NK cells across different compartments. Similar to circulatory NK cells, splenic NK cells of SIV controlling macaques significantly up-regulated the expression of IFN-γ (Fig. 5A), CD107a (Fig. 5B),TNF-α (Fig. 5C) and CD69 (Fig. 5D) in response to Gag stimulation. Interestingly, in the spleen of SIV controlling macaques the proportion of IFN-γ and TNF-α producing NK cells also increased in response to Env stimulation. Furthermore, in contrast to circulatory NK cells, splenic NK cells from non-controlling macaques up-regulated the expression of CD107a and CD69 in response to Gag (Fig. 5B and D). Hepatic NK cell responses were almost undetectable for all animals assayed, with the exception of SIV non-controlling animals, which exhibited a low (albeit significant) up-regulation of IFN-γ producing cells in response to Gag (Fig. 5A, Liver). We observed an up-regulation of IFN-γ producing mucosal NK cells in response to Gag in SIV controlling macaques (Fig. 5A, Mucosal).

Fig. 5. Activation of tissue-resident NK cells is observed mostly in SIV controlling macaques.

Lymphocyte single-cell suspensions were purified from spleen, liver and mucosal tissues (jejunum and/or colonic biopsies) and stimulated for 24 h in the presence of SIV Gag or Env peptides. Up-regulation of IFN-γ (A), CD107a (B), TNF-α (C), and CD69 (D) in response to Gag (black bars) and Env (white bars) peptides was measured within total NK cells (CD3^-CD8⁺NKG2A⁺ live lymphocytes) for each tissue. Data pooled from 11 macaques (3 naïve, 4 SIV controlers and 4 SIV non-controllers). For each parameter, activation of NK cells was determined by subtracting non-stimulated control values. N, naïve; NC, SIV Non-controlling; C, SIV controlling. *, p<0.05 (Gag), and #, p<0.05 (Env) indicate statistically significant differences when compared to the naïve group by Mann-Whitney t-Test.

Impact of Viral Load on CD4⁺-dependent NK cell responses

During chronic SIV/HIV infection, high viral loads are often associated with phenotypic and functional abnormalities of immune cell subpopulations including T cells (19), B cells (20), and NK cells (17, 21). Here, SIV non-controlling macaques displayed impaired T cell antigen-specific recall responses and CD4⁺-dependent NK cell responses. Given that both Ag-specific T cell production of IL-2 (p=0.0019, data not shown), and T cell-dependent NK cell activation levels are inversely correlated with plasma viremia (p=0.0036, Fig. 6A), we investigated whether decreasing viral loads through antiretroviral therapy (ART) would improve the responsiveness of NK and T cells. Seven SIV non-controlling macaques were treated with ART for 8 weeks which significantly reduced plasma viremia in all macaques (Fig. 6B). Four of 7 animals displayed undetectable viral loads. However, only non-significant trends towards increased proportions of CD4⁺ T cells and decreased proportions of CD8⁺ T cells resulted (Fig. 6C). Nevertheless, ART had a positive impact on the capacity of NK cells to respond in T cell-dependent assays, as the proportion of CD69⁺ and IFN-γ⁺ Gag-specific NK cells increased significantly after 8 weeks of therapy when compared to pre-ART levels (Fig. 6D). Although T cell-dependent NK cell responses were partially rescued during ART, no significant increase in the production of IL-2 by either total or CM CD4⁺ T cells in response to Gag-stimulation was observed (Fig. 6E). Despite this, the marginal Gag-specific increases in IL-2 production observed in CM CD4⁺ T cells after 8 weeks of ART significantly correlated with the increased production of IFN-γ (p=0.0004, Fig. 6F), and expression of CD69 (p=0.0456, data not shown) by NK cells at the same time point.

Fig. 6. Therapeutic control of viremia in SIV non-controlling macaques improves T cell-dependent NK cell effector responses.

7 SIV-infected non controlling macaques were treated with ART for 8 weeks. Peripheral blood was obtained before and after ART. (A) Correlation between viral load and the percentage of CD56⁺ NK cells producing IFN-γ in response to 24 h stimulation (using same animals as in 4B-C). Effect of ART on plasma viral loads (B), and on the proportion of NK and T (CD4⁺ and CD8⁺) cells in peripheral blood (C). (D) T cell-dependent Gag-specific NK cell effector responses measured after 24 h stimulation. Responses without stimulation have been subtracted. (E) Gag-specific T cell responses measured after 6 h stimulation. Correlation between Gag-specific production of IL-2 by CM CD4⁺ T cells and production of IFN-γ (F) by NK cells post- ART. Data combined from 7 macaques. Dotted line in B represents the limit of detection (50 copies per mL) for the viral load assay. *,p<0.05, and **, p<0.01 indicate statistically significant differences between the compared groups by Mann-Whitney t-Test. Spearman's correlation analysis was used to determine statistical significance in A and F. CM, central memory (CD28⁺CD95⁺).

Collectively our data illustrate that control of SIV disease progression in macaques is associated with increased T cell-dependent NK cell effector function in blood as well as in spleen and mucosal tissues. The circulatory NK cell responses correlated directly with IL-2 production by antigen-specific CM CD4⁺ T cells. Further, although viral load negatively affects NK cell function, T cell-dependent NK cell responses in SIV non-controlling macaques can be at least partially rescued by short-term ART. Overall, our data support the notion that T cell-dependent NK cell recall responses are potential immune correlates of protection and should be monitored both in vaccine development as well as therapeutic studies.

DISCUSSION

Recent studies examining vaccine-induced immunity in humans have highlighted the often overlooked importance during antigen-specific immune responses of NK cells that mediate cytotoxic and cytokine-producing effector functions (7-9). Several NK cell functional responses have been correlated with either the prevention from acquisition or the immunological control of SIV and HIV infection (10, 11, 22-24). Despite the fact that non-human primate-based vaccination models serve as surrogates for HIV/SIV vaccine development (12), few pre-clinical studies have evaluated the role played by NK cells during vaccine-elicited immune responses. Here, we examined in detail the IL-2 dependent activation of circulatory and tissue-resident NK cells, and its association with antigen-specific CD4⁺ T cell responses. We demonstrated that T cell-dependent NK cell activation is mostly dependent on IL-2 produced by antigen-specific memory CD4⁺ T cells. To the best of our knowledge, this is the first report to characterize SIV-specific T cell-dependent NK cell responses in circulatory, as well as tissue-resident NK cells. Notably, this study identifies NK cells as key players in adaptive immune responses. Although a higher proportion of NK cells compared to CD4⁺ or CD8⁺ T cells produced IFN-γ in response to Gag stimulation of PBMCs (Fig. 2B and 2D), the greater absolute numbers of CD3⁺ lymphocytes makes the CD3⁺CD8⁺IFN-γ⁺ T cell subset most abundant. Therefore, although NK cells provide an early, rapid response, it must be noted that overall control of SIV infection in macaques has been associated with strong and antigen-specific CD4⁺ and CD8⁺ T cell responses (25, 26). CD8 cells have long been known to contribute to control of SIV viremia (27). More recently, CD4⁺ T cells have been shown to be associated with the post-peak decline of viremia in SIV-infected macaques (28). Furthermore, their direct cytolytic function has been linked to clearance of SIV-infected macrophages (29), and used as a predictor of disease outcome in HIV-infected individuals (30). In addition to cellular immune control, multiple genetic correlates of protection have also been identified. For example, certain TRIM5α polymorphisms are associated with viral susceptibility/resistance, and particular MHC class I molecules are correlated with control of HIV and SIV (31, 32). Similar associations with some MHC class II molecules in both non-human primates (33, 34) and humans (35) have been reported, although not all HLA class II associations have been reproducible (36). Thus, the NK response described here is one component of a complex immune approach which combats HIV/SIV infection and disease progression.

In our study, antigen-specific T cell responses were observed mostly in SIV controlling macaques, suggesting an increased level of protective immunity. However, the higher viral loads in the SIV non-controlling group may have had an overall negative effect on antigen-specific immune function. It is important to mention that although SIV non-controlling animals had decreased numbers and antigen-specific function of CD4⁺ T cells, their response to SEB as positive control was comparable to that of SIV controlling and naïve animals (Fig. 2C and 4A). Moreover, enriched NK cell cultures from SIV controlling and non-controlling macaques were equally responsive to IL-2 plus IL-15 stimulation (Fig. 2F), although a more physiological assay (such as killing of MHC-devoid target cells) might allow for identification of more subtle functional differences between NK cells of SIV controller and non-controller animals. Taken together, these results suggest that reduced T cell-dependent NK cell effector responses observed in SIV non-controlling macaques are not due to a generalized impaired capacity of CM CD4⁺ T cells to produce IL-2, or to a direct viral load-associated NK cell dysfunction, but instead to the decrease in number of SIV-specific memory CD4⁺ T cells, which prevents effective crosstalk with NK cells. Similarly, the lack of peptide responsiveness observed in SIV non-controlling animals strongly suggests that Ag-specific CD4⁺ T cell help is required for optimal activation of not only NK cells, but also for CD8⁺ T cell priming during antigen recall assays (37).

Previous studies have evaluated NK cells during chronic SIV infection in a variety of nonhuman primates (15, 38, 39), primarily assessing NK cell function based on responses to MHC-devoid target cells (such as K562 and 721.221) or to PMA/Ionomycin stimulation. Therefore, the possible role of antigen specific T cells and crosstalk with other cell types was overlooked. Further, detailed analyses of antigen-specific T cell-dependent functional responses within each circulatory NK cell subpopulation have not been previously described. Although we observed that T cell-dependent NK cells responses were subset-specific, the capacity or threshold of each NK cell subset to interact and be activated by CD4⁺ T cells remains to be determined. Interestingly, we observed that T cell-dependent NK cell effector functions were also antigen-specific, as shown by limited responses to Env stimulation both in circulatory, as well as tissue-resident NK cells. The importance of immune cell co-operation, specifically in the context of NK cell activation has previously been reported (6-9); however our evaluation of such responses in SIV-infected macaques displaying different courses of disease progression has extended these observations, highlighting the specificity and relevance of these responses to disease control. In this regard, preliminary results from our laboratory suggest that the killing capacity of naïve NK cells against MHC-devoid 721.221 target cells is enhanced (>15%) by IL-2 pre-treatment (Vargas-Inchaustegui et. al., unpublished data). If such non-specific enhancement in killing were also observed during SIV-specific T cell-dependent NK cell activation, it would have important implications for SIV/HIV immunopathology, prophylaxis and vaccine design. Given that our experiments were conducted with chronically infected animals, detailed analysis of T cell-dependent NK cell effector function during vaccination protocols as well as during the early acute phase of SIV infection are currently underway. Similarly, we wish to evaluate whether increased frequencies of regulatory T cells or markers of immune exhaustion (such as PD-1 expression) can be correlated to the lack of SIV-specific responsiveness observed in our SIV non-controlling macaques. These future studies will elucidate the potential role of these collaborative responses in the prevention and/or early control of SIV infection.

Here, NK cell activation was dependent on antigen-specific IL-2 production by CD4⁺ CM T cells (Fig. 4A), similar to results of Horowitz et. al. who reported that in humans vaccinated against rabies virus, NK cell effector responses were mainly dependent on IL-2 produced by memory CD4⁺ T cells, although IL-12 and IL-18 produced by myeloid accessory cells were also necessary for NK cell activation (7). In our study, IL-2 appeared to be the major activation molecule for two reasons: 1) antigen-specific responses were measured using peptide pools for short stimulation periods, making T cell receptor-positive cells the most likely candidates to respond in such short time periods, and 2) IL-2 neutralization, as well as CD4⁺ T cell depletion almost completely abrogated NK cell recall responses (Fig. 5D-G). Although our results suggest that 95% percent of effective NK cell activation is dependent on CM CD4⁺ T cell-derived IL-2, we cannot exclude a potential contribution of other NK cell activatory cytokines such as IL-12, IL-15 or IL-18. Similarly, given that depletion of CD8⁺ T cells was not technically possible (macaque NK cells express CD8α), further experiments are needed to formally conclude that CD8⁺ T cells are not necessary for the NK cell effector functions described in this study. Finally, whether specific epitopes in Gag and/or Env are associated with CD4⁺ CM T cell development and IL-2 production, thus controlling T cell-dependent NK effector responses, will require further analysis, as peptide pools representing the entire Gag and Env proteins were used in this study for stimulation.

Although we detected an improvement in NK cell function after 8 weeks of ART, we did not observe significantly increased T cell function, namely Gag-specific IL-2 production. Furthermore, only 2 out of 4 markers of NK cell activation measured, CD69⁺ and IFN-γ, were significantly increased after 8 weeks of ART, and only in the animals with the highest recovery in IL-2 production (Fig. 6F). Collectively, our results suggest that earlier treatment initiation and/or longer treatment periods may be required for full recovery of both T and NK cell function. In this regard, it has been reported that the kinetics of T and NK cell immune reconstitution during ART are very different (40), and that complete functional recovery of these cells is directly dependent on the level of viral suppression achieved (41).

Although the phenotype of rhesus macaque tissue-resident NK cells has been previously described (15), the functional characteristics of these cells during SIV infection have not been reported. Surprisingly, immune responses by tissue-resident NK cells were location- and antigen-specific. We observed similar activation profiles in splenic and circulatory NK cells, but such responses were only partially present in mucosal NK cells and non-existent in hepatic NK cells of SIV controlling macaques (Fig. 5). We hypothesize that during SIV-infection, tissue-resident NK cells may be functionally restricted due to their anatomic location, or alternatively, cells with which they react may regulate their activity. In support of this hypothesis, it has been shown that intra-hepatic levels of IL-10 maintain murine NKG2A⁺Ly49^- liver NK cells in a functionally hyporesponsive state, as evidenced by their lack of IFN-γ-production in response to IL-12/IL-18 stimulation (42). Furthermore, dampening of hepatic and mucosal NK cell activation can be controlled through the 2B4 inhibitory pathway, either by differential glycosylation of 2B4 (43), or by interaction with its ligand CD48 during NK/DC cross-talk (44). In recent years, a new subset of innate lymphocytes that express some NK cell markers and produce the cytokine IL-22 (NKp44⁺ NK cells) have been identified in different species (45, 46). Interestingly, IL-22 plays a crucial role in modulating tissue responses during inflammation (47), and IL-22-producing NKp44⁺ NK cells were found to be depleted during chronic SIV infection (46). Therefore, investigation of NKp44⁺ NK cell function using CD4-dependent antigen-specific and/or antibody-mediated functional assays could further improve our understanding of the role played by different types of tissue-resident NK cells in the control of SIV/SHIV infection.

In summary, this study has uncovered a novel effector function of NK cells during an SIV-specific adaptive immune response which is dependent on IL-2 produced by antigen-specific CD4⁺ CM T cells. The NK cell immune responses described are associated with strong and durable control of viremia. Therefore, T cell-dependent NK cell activation should be considered in vaccine strategies for HIV and other infectious diseases as a potential indicator of protective immunity.