The Role of Cytokines which Signal through the Common γ Chain Cytokine Receptor in the Reversal of HIV Specific CD4+ and CD8+ T Cell Anergy

Background HIV specific T cells are putatively anergic in vivo. IL-2, a member of a class of cytokines that binds to receptors containing the common gamma chain (γc) has been shown to reverse anergy. We examined the role of γc cytokines in reversing HIV specific T cell anergy. Methods PBMC from untreated HIV-infected individuals were briefly exposed to a panel of γc cytokines, and frequencies of gag specific T cells were enumerated by intracellular IFN-γ flow cytometry. Results Of the γc cytokines, brief exposure to IL-2, IL-15, or combined IL-15/IL-7 significantly enhanced (range 2–7 fold) the CD4+ and CD8+ T cell IFN-γ responses to HIV gag, with IL-15 giving the greatest enhancement. The effects of cytokines were not due to enhanced proliferation of pre-existing antigen specific cells, but were due to a combination of enhanced cytokine production from antigen specific T cells plus activation of non-epitope specific T cells. Conclusions These observations support the notion that a significant number of HIV specific T cells are circulating in an anergic state. IL-2, IL-7 and particularly IL-15 as an immune modulator to reverse HIV-1 specific T cell anergy should be investigated, with the caveat that non-specific activation of T cells may also be induced.


INTRODUCTION
Although a potent CD4 + and CD8 + T cell immune response has been shown to control most virus infections, Human Immunodeficiency Virus (HIV) replication persists in the majority of infected individuals despite the presence of a detectable T cell immune response [1][2][3][4][5]. Analysis of T cell immune responses in HIVinfected individuals have revealed a number of apparent functional defects whose severity correlate closely with the degree of viral replication in the plasma. When compared to T cells of other viral specificities, HIV-specific T cells have been shown to have defects in proliferative capacity, cytokine production and effector function [6][7][8][9][10][11][12][13][14]. More specifically, using MHC-class I peptide-tetramer technology, HIV and SIV specific CD8 + T cells have been shown to be impaired in their ability to produce interferon-gamma (IFN-c) in which less than 25% of tetramerstaining cells are able to produce cytokine in response to their cognate antigen [10,11,15,16]. Similar defects have also been proposed in HIV-specific CD4 + T cells, in which, viral replication is associated with a defect in IL-2 production [7,9,17]. Since a direct comparison of HIV-specific CD4 + T cells by tetramer analysis and cytokine production is yet to be reported due to the difficulties in producing stable peptide-MHC class II tetramers, it is unknown what proportion of HIV-specific CD4 + T cells are defective in IL-2 or IFN-c production in acute and chronic infection [18]. A number of mechanisms for this anergic or 'stunned' state of antigen specific T cells in HIV infection have been proposed, which include excessive activation from high antigen loads [7,19], direct effects of gp120 [20], or altered peptide ligands encoded due to viral mutants [21].
The common-gamma (cc) chain cytokines have been shown to be important growth factors for T cells [22] and of which IL-2, has been shown to reverse anergy in vitro [23,24]. The common cytokine receptor gamma chain is essential for the function of at least six cytokines including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. IL-2 is the most well characterized of these, and has been used in clinical trials to enhance CD4 counts in HIV infected individuals [25][26][27][28], however, its direct effect on HIV-specific T cells has not been well characterized. Although IL-2 is a T-cell growth factor, it also enhances apoptosis of mature T cells and induces tolerance by expanding CD25 + regulatory CD4 + T cells [29,30]. IL-7 is important in T cell homeostasis by enhancing the survival of central memory T cells. IL-15 has been demonstrated to ensure the survival and proliferation of memory CD4 + and CD8 + T cells and NK cells. IL-4 primarily mediates the development of humoral immunity by promoting TH2 CD4 + T cells and B cell proliferation [22]. Thus, although members of the common cc cytokine family show considerable overlap, many differences in their functional characteristics are also found. In addition, it is unknown whether IL-7, IL-4 or IL-15 are capable of reversing anergy via signaling through the cc chain.
In the current study, we asked whether brief treatment of ex vivo HIV-specific T cells with various members of the cc cytokine family could reverse their anergic state. We sought to determine the relative effectiveness of these cytokines in reversing anergy as well as possible mechanisms as to how these cytokines work on antigen specific T cells.

Study participants
17 treatment naive HIV infected individuals were recruited for this study (see Table 1). Eight individuals were recently infected by HIV within 6 months of study (diagnosed by recent seroreactivity). Nine individuals with chronic HIV infection with disease progression were studied. This was defined as documented HIV infection .1 year, with evident CD4 + T cell decline of .50 CD4 cells/ mm 3 and a viral load .10,000 copies/ml. All investigational protocols were approved by the University of Toronto and St. Michael's hospital Institutional Review Boards.

Source of antigens
Forty-nine overlapping 20mer peptides spanning the gag region of HIV-IIIB (amino acids 1-500), overlapping by 10 amino acids, were obtained from the NIH AIDS Research Reagent program (catalog # 3992), dissolved in DMSO and used as a pool with each peptide at a final concentration of 1.0 mg/ml. DMSO in appropriate dilutions was used as a negative control.

Flow cytometry and intracellular cytokine determination
The procedure for intracellular staining of cytokines in PBMCs was performed using the Cytofix/cytoperm Plus kit according to the manufacturer's instructions (Cat# 554715, BD Biosciences, San Diego, CA). Cells were washed, fixed and permeabilized in FACS permeabilization buffer and were stained by a panel of conjugated antibodies (fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP) and allophycocyanin (APC) including antibodies to human CD4, CD8, CD69 (Pharmingen), and IFN-c and respective isotype controls. Cells were then washed and resuspended in 1% paraformaldehyde/PBS and then analyzed the following day on a FACSCalibur (BD Biosciences, San Diego, CA). All antibodies were obtained from BD Biosciences. In selected experiments, PBMC from HLA-A *0201 individuals who had CD8 + T cell responses to the SLYNTVATL epitope of HIV gag were also stained with the MHC-I tetramer to this peptide (iTag, Beckman-Coulter, Fullerton, CA). In the latter experiments cells were washed three times with PBS to ensure removal of peptide prior to tetramer staining. Data were acquired by Cell Quest software (BD Biosciences, San Diego, CA) and analyzed using FloJo (Treestar Inc., San Carlos, CA). From 100,000 to 200,000 events in the lymphocyte gate were acquired/sample.

Proliferation assays
To more specifically determine the effect of cytokines on the proliferative activity of antigen specific cells in our assays, stable incorporation of the intracellular fluorecent dye 5-(and-6)carboxyfluorescein diacetate succinimidyl ester (CFSE) was employed. PBMCs were suspended in PBS containing 5% FBS and were stained at room temperature for 5 min with 5 mM CFSE. Staining was terminated by adding PBS containing 5% fetal bovine serum (FBS) and subsequent washing with PBS. Cells were then treated similarly as above for antigen specific assays. Proliferation was assessed by measuring dilution of CFSE staining on antigen specific cells that were co-stained with antibodies to CD4, CD8 and IFN-c.

Statistical analysis
Data were compared using the Wilcoxon signed rank test for paired samples (two-tailed).

Effect of brief exposure to cc cytokines on HIV specific T cell responses
Since gag is the most conserved protein of HIV and elicits the most frequent T cell responses in HIV infected individuals, we elected to study the effects of cc cytokines on the T cell response to HIV gag in a cohort of untreated HIV infected individuals with recent seroconversion or chronic progressive disease (see Table 1). Individuals with recent seroconversion were studied because of the high levels of immune activation and viremia associated with this stage of infection [31][32][33][34]. We asked whether short term overnight treatment of HIV-gag specific T cells could reverse any defect in IFN-c production that may be occurring in vivo. Freshly obtained ex vivo PBMC were treated overnight in plain medium or with the following cytokines or combination, thereof: IL-2, IL-15, IL-4 or IL-7+IL-15. The latter combination of IL-7+IL-15 was previously shown to have a synergistic effect on inducing the proliferation of memory T cells [22]. The following day, PBMC were washed extensively to remove residual cytokines, exposed to HIV gag or control antigen, and then assessed for their ability to produce IFN-c as measured by intracellular flow cytometry. An example of a representative experiment from Participant #11, a chronic progressor, is shown in Figure 1a and b depicting both control and antigen stimulated conditions in CD4 + (Fig. 1a) and CD8 + T (Fig. 1b) cells. The frequency of HIV gag specific T cells was calculated by measuring IFN-c/CD69 expressing cells observed in HIV gag containing conditions and subtracting from DMSO (control antigen) containing conditions, similar to as previously described [35]. In this individual, when PBMC were cultured in plain medium the measured frequency of gag specific IFN-c producing CD4 + T was .082%. However, if the PBMCs were briefly exposed to IL-2, the measured frequency increased to 0.326%, if they were exposed to IL-15, the frequency was 0.45%, to both IL-15/IL7 it was 0.66%, whereas, if cells were briefly exposed to IL-4, we only detected a frequency of 0.047%. Similarly, for CD8 + T cells, we detected a 0.02% frequency of gag specific IFN-c producing cells if PBMC were briefly cultured in plain medium, whereas the frequencies increased to 0.46%, 0.6%, 0.6% or 0.06% if they were briefly cultured in the presence of IL-2, IL-15, IL-15/IL-7, or IL-4, respectively. Data from participant #5, a recent seroconverter is also depicted in Figure 2. When combining data from all 17 individuals (  Effect of brief exposure of cc cytokines on proliferation of HIV specific cells We saw a dramatic enhancement (range 2-7 fold) in the frequency of IFN-c producing T cell responses after exposure to IL-2 or IL-15. One possibility is that exposure to these cytokines allowed proliferation of antigen specific cells overnight in vitro prior to their detection by intracellular cytokine staining. In order to address this, in selected participants, PBMC were stained with CFSE prior to performing experiments in order to assess proliferation of antigen specific cells via the effect of proliferation on diluting the CFSE stain. A representative experiment comparing medium and IL-15 conditions in Participant #7 is shown in Figure 3. We find that IFN-c producing T cells do not dilute CFSE in the presence of IL-15 indicating that the enhanced frequency of IFN-c producing cells is not due to proliferation overnight.

Antigen specificity of enhanced responses due to cc cytokines
An alternative possibility of the enhanced responses observed with IL-2 and IL-15 pretreatment may be related to activation of cells not specific for the antigen. In order to address this, we examined the effect of overnight IL-2 and IL-15 treatment on CD8 + T cells specific for the HLA-A2*0201 restricted epitope of HIV gag, SLYNTAVTL, using tetramer co-staining experiments. PBMC from three HLA-A2*0201 positive individuals with chronic progressive HIV infection, who previously showed detectable IFN-c responses to the this epitope, were treated overnight with medium, IL-2, or IL-15, and then were stimulated for 6 hours with the SLYNTVATL peptide, and assessed for intracellular IFN-c production by flow cytometry. A representative experiment is depicted in Figure 4a, with summary data from all three individuals shown in Figures 4b-d. As demonstrated with the CFSE experiments, brief cytokine treatment did not enhance the proliferation of antigen specific T cells since the numbers of tetramer staining cells were similar with all three conditions (Figure 4a and d). SLYNTAVTL specific CD8 + T cells from these three individuals showed severe anergy, as ,10% of tetramer staining cells could produce IFN-c upon stimulation. Brief treatment with IL-2 or IL-15 was able to partially reverse this anergy, with IL-15 being more effective than IL-2 at increasing the proportion of tetramer staining cells to produce IFN-c (Figure 4a and b). The detection of increased IFN-c expressing cells after SLYNTVATL stimulation was however, also associated with an increase frequency of tetramer negative but IFN-c positive cells, which was most significantly seen in the IL-15 containing condition, indicating that a portion of the IL-15 effect is not SLYNTVATL specific (Figure 4a and c). Since a portion of the enhancing effect of IL-2 and in particular, IL-15 was due to cells that were not antigen specific, we looked at the expression of the activation marker, CD69, in cells that were pretreated with cc cytokines in the absence of HIV antigen. As can be observed in Figure 1a

DISCUSSION
In the current study, we show that brief treatment of ex vivo PBMCs obtained from untreated HIV infected individuals, with the cc cytokines IL-2, IL-15 or IL-15 plus IL-7 can to varying extents, enhance the ability of HIV-specific CD4 + and CD8 + T cells to produce IFN-c. Our findings support those of others in which the addition of IL-15 could enhance IFN-c ELISpot responses to CMV, or PPD in healthy donors [36], to HIV in treated HIV-infected children [37], and to SIV in SIV infected macaques [38]. These observations support the notion that a significant number of HIV-1 specific CD4 + and CD8 + T cells are circulating in an anergic or unresponsive state. This was confirmed by finding that cc cytokines increase the proportion of HIV specific CD8 + T cells directed against the SLYNTVATL epitope (p17) to produce IFN-c, and that this effect occurred independantly of any effect on proliferation of these cells. IL-15 or the combination of IL-15 plus IL-7 was the most potent at reversing anergy, whereas, IL-4 had no effect. Although IL-15 could enhance the ability of p17-specific CD8 + T cells to produce IFN-c, the majority of tetramer positive cells still could not secrete IFN-c in response to peptide stimulation, indicating the intense anergic state of these cells in the individuals studied. It is possible that more prolonged culture of cells (e.g., 1-2 weeks) in the presence of cytokines may have more extensively reversed this defect. We used a short (12 hour) exposure in our study in order to avoid any effect of these cytokines on proliferation of antigen specific cells in vitro, and because we were interested in determining whether ex vivo antigen specific cells were indeed anergic. The lack of significant effects with IL-4 suggests that signaling through the common cc alone is not sufficient to reverse anergy but that signaling through additional cytokine specific chains like IL-2Rb and IL-15b may also be necessary. All of the cc cytokines, activate the jak1 and jak3 kinases upon binding to their specific and the common gamma chains, respectively. However, downstream to jak activation, IL-2, IL-15 and IL-7 signaling then specifically phosphorylate stat 3 and stat 5, whereas, IL-4 signaling phosphorylates stat 6. Thus, it appears likely that stat 3 and stat 5 activation are responsible in part for the effects observed. The mechanisms as to how IL-2 and IL-15 enhance the effect of signaling through the TCR are beginning to emerge. Recently Liu et al. [39] showed by genetic profiling microarray analysis, that 73% of genes upregulated by IL-15 on memory T cells overlap with genes upregulated by TCR signals on the same cells. These findings suggest that signals induced through the TCR could be amplified via IL-15 as similar genes are being upregulated, with the resulting effect being decreasing the threshold for T cell activation through the TCR after exposure to antigen. This is consistent with what we observed in our cohort, as, IL-2, IL-15 and IL-15/7 enhanced the baseline activation state of T cells by enhancing CD69 expression, a marker of activation through the TCR. Thus, the beneficial effect of IL-2 or IL-15 appeared to occur at the expense of enhanced background activation of T cells. In our tetramer analysis, the effects of enhanced activation was associated with enhanced IFN-c production from cells which did not appear to be specific to the peptide stimulus. It is unclear whether this represents activation of cells with TCRs that crossreact to the peptide, and thus are activated because the threshold of activation was lowered, or reflect completely non-specific activation.
The cc cytokines, including IL-2, IL-15 and IL-7 are currently receiving consideration for treatment in HIV infection as systemic therapies. Our data suggests that all should potentially be useful in enhancing the functional capacity of HIV specific T cells in vivo, with IL-15 or combined IL-15 plus IL-7 showing the most potent effects. Clinical use of these cytokines however may also be associated with an enhanced activation state of T cells, particularly with IL-15, which may have both positive effects or negative consequences. A positive effect would be the recruitment of potentially cross reactive T cells that could deal with virus variants that escape from the immunodominant response. The negative effect would be that enhanced immune activation would facilitate further HIV viral replication. Figure 4. Effect of cc cytokines on p17 specific CD8 + T cells. PBMC from three HLA A*0201 individuals with chronic progression were cultured overnight in medium, IL-2 or IL-15 as described in Figure 1. Cells were then washed and exposed to the A*0201 restricted p17 peptide of HIV and then stained for intracellular IFN-c, CD8, and with a p17 specific tetramer. Dots plots for one individual are shown in (a) after gaiting on CD8. Large numbers (%) in upper right quadrants indicate the% of tetramer staining cells that co-stain for IFN-c. Small numbers in all quadrants represent % of total CD8 + T cells. Summary data from all three individuals are shown depicting in (b) the % of tetramer staining cells which also produce IFN-c (upper R/upper R+lower R quadrant) , in (c) the % of CD8 + T cells expressing IFN-c but do not stain for tetramer (upper L quadrant/ total CD8s), and in (d) the number of tetramer staining CD8 + T cells in all conditions (upper and lower R quadrant/ total CD8s). Bar graphs depict means and bars represent standard errors on the mean. doi:10.1371/journal.pone.0000300.g004