HIV Controllers HLA-DRB1*13 and HLA-DQB1*06. Have Strong, Polyfunctional Mucosal CD4+ T-cell Responses

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1 JVI Accepts, published online ahead of print on 1 August 0 J. Virol. doi:./jvi.000- Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 HIV Controllers HLA-DRB1* and HLA-DQB1*0 Have Strong, Polyfunctional Mucosal CD+ T-cell Responses April L. Ferre 1, Peter W. Hunt, Delandy H. McConnell 1, Megan M. Morris 1, Juan C. Garcia, Richard B. Pollard, Hal F. Yee, Jr, Jeffrey N. Martin, Steven G. Deeks, and Barbara L. Shacklett 1,* 1 Department of Medical Microbiology and Immunology; Division of Gastroenterology; Division of Infectious Diseases, School of Medicine, University of California, Davis, CA Positive Health Program, Department of Medicine; Division of Gastroenterology; Department of Epidemiology and Biostatistics San Francisco General Hospital, University of California, San Francisco, CA Running Title: Mucosal CD Responses in HIV Controllers *Corresponding Author: Barbara L. Shacklett, PhD, Associate Professor Dept. of Medical Microbiology and Immunology, and Div. of Infectious Diseases, Dept. of Internal Medicine School of Medicine, University of California, Davis, CA 1 Tel: (0) - Fax: (0) - blshacklett@ucdavis.edu Word Count: Abstract, words Word Count: Text,,0 Downloaded from on September 1, 01 by guest 1

2 ABSTRACT A small percentage of HIV-infected individuals, termed elite controllers, are able to spontaneously control HIV replication in blood. As the gastrointestinal mucosa is an important site of HIV transmission and replication, as well as CD+ T-cell depletion, it is important to understand the nature of the immune responses occurring in this compartment. Although the role of the HIV-specific CD+ T-cell responses in mucosal tissues has been described, few studies have investigated the role of mucosal HIV-specific CD+ T-cells. In this study, we assessed HIV-specific CD+ T-cell responses in the rectal mucosa of controllers (VL<,000 copies/ml), 1 non-controllers (VL<,000 copies/ml), and individuals on HAART (VL<0 copies/ml). Controllers had higher magnitude Gag-specific mucosal CD+ T-cell responses than individuals on HAART (p<0.0), as measured by their ability to produce IFN, IL-, TNF, and MIP-1. The frequency of polyfunctional mucosal CD+ T-cells was also higher in controllers compared to non-controllers or individuals on HAART (p<0.0). Controllers with the strongest HIV-specific CD+ T-cell responses possessed class II HLA alleles, HLA-DR* and/or HLA-DQ*0, previously associated with a non-progression phenotype. Strikingly, individuals with both HLA-DR* and HLA-DQ*0 had highly polyfunctional mucosal CD+ T-cells compared to individuals with HLA-DQ*0 alone or other class II alleles. The frequency of polyfunctional CD+ T-cells in rectal mucosa positively correlated with the magnitude of the mucosal CD+ T-cell response (Spearman r=0., p=0.00), suggesting that increased CD+ T-cell help may be important in maintaining strong CD+ T-cell responses in the gut of HIV controllers. Downloaded from on September 1, 01 by guest

3 INTRODUCTION The study of individuals who are able to achieve durable control over HIV replication in the absence of antiretroviral therapy has become increasingly important in light of recent vaccine trials which have failed to induce protective immunity. Understanding the correlates of protection in these HIV controllers would aid in both the rational design of potential vaccine candidates and in the testing of their efficacy. Cell mediated immune responses, in particular HIV-specific CD+ T-cell responses, have been shown to be critical in decreasing the initial viremia after acute infection and determining the chronic infection set point (,, ). These responses have proven to be too little and too late to prevent the establishment of chronic infection; however, studies of HIV-specific CD+ T-cell responses in controllers suggest that strong, polyfunctional responses may be important in long-term virologic control (1,,, 1, 1, ). In addition, the association of controller status with class I HLA alleles, predominantly HLA-B and HLA-B, has been well documented in multiple cohorts (,,, ). The maintenance of robust HIV-specific CD+ T-cell responses may also favor longterm control of HIV replication in untreated persons. One of the primary roles of CD+ T-cells is to provide help to CD+ T-cells. Many studies have shown that proper functioning of CD+ T-cells is necessary for high quality antigen specific CD+ T-cell responses (,,,, ). HIV-specific CD+ T-cells in the peripheral blood of long-term non-progressors have been shown to be polyfunctional, producing both IFN and IL-, whereas those from progressors tend to be monofunctional, secreting only IFN (, 1, 1,, ). Additionally, the ability of CD+ T-cells to proliferate appears to be preserved in controllers (, ). The maturation status of CD+ T-cells that function in the context of HIV infection may also be important, as individuals Downloaded from on September 1, 01 by guest

4 who are able to preserve central memory T-cells and sustain an activated effector memory CD+ T-cell population are better able to suppress viral replication (). Much less in known about the association of class II HLA alleles and controller status; however, a few studies have cited a relationship between HLA-DRB1* and/or HLA-DQB1*0 and HIV control (, 1,, ). Previously, we have shown that CD+ T-cells from the rectal mucosa of controllers with protective class I alleles (HLA-B, B, B, B, and B1) are highly polyfunctional compared to CD+ T-cells from either controllers or non-controllers lacking these alleles (1). Additionally, we found that mucosal CD+ T-cell responses from individuals who had protective class I alleles in combination with the class II alleles, HLA-DRB1* and/or HLA-DQB1*0, were of greater magnitude than mucosal CD+ T-cell responses from those who had protective class I alleles alone (1). Therefore, we wanted to specifically examine HIV-specific mucosal CD+ T-cell responses among controllers with and without these potentially protective class II HLA alleles. Our hypothesis was that controllers, particularly those who possessed HLA-DRB1* and/or HLA-DQB1*0, would have more robust and polyfunctional HIV-specific CD+ T-cell responses than non-controllers or subjects on HAART, and that these responses would correlate with strong CD+ T-cell responses in the same individuals. We found that indeed, controllers generally had stronger HIV-specific CD+ T-cell responses than other groups in rectal mucosa, and among controllers, those with the haplotype HLA-DRB1*/HLA-DQB1*0 had particularly high percentages of polyfunctional HIV-specific CD+ T-cells in rectal mucosa. Furthermore, the proportion of polyfunctional mucosal CD+ T-cells directly correlated with the total magnitude of the mucosal CD+ T-cell response. These data collectively suggest that the Downloaded from on September 1, 01 by guest

5 preservation or expansion of both HIV-specific CD+ and CD+ T-cells may be an important mechanism whereby certain controllers maintain durable control of HIV replication MATERIALS AND METHODS Subjects and Tissue Collection. Subjects were recruited through ongoing studies of chronic HIV infection at San Francisco General Hospital and the Center for AIDS Research, Education, and Services (CARES) Clinic in Sacramento, California, and have been previously described (1). Subjects were classified in one of five categories based on viral load (VL) measurements and treatment status: elite controllers (EC; VL<0 copies/ml, off therapy, n=1), viremic controllers (VC; VL<,000 copies/ml, off therapy, n=), non-controllers (NC; VL>,000 copies, off therapy, n=1), highly active antiretroviral therapy (HAART) suppressed (VL<0 copies, on HAART, n=) and HIV negative controls (n=), as described by Deeks and Walker (). Written informed consent for phlebotomy and flexible sigmoidoscopy was obtained from all subjects in accordance with the Declaration of Helsinki, and with study protocols approved by the Institutional Review Board, University of California Davis, and the Committee on Human Subjects Research, University of California San Francisco. Approximately 0mL of blood were obtained by sterile venipuncture and collected into tubes containing ethylenediaminetetraacedic acid. Rectal biopsy tissue (0- pieces) was obtained by flexible sigmoidoscopy at cm from the anal verge using a sigmoidoscope equipped with a biopsy channel and single use biopsy forceps. This procedure has been well documented to cause only minimal discomfort and provide enough cells to perform cellular immunology assays (mean x cells) (, 1,, 1). Tissue was placed in RPMI 10 Downloaded from on September 1, 01 by guest

6 supplemented with 1% fetal calf serum, penicillin (0U/mL), streptomycin (0 g/ml) and L- glutamine (nm), herein referred to as R1. Specimens were immediately transported to the University of California, Davis for same day processing Peripheral Blood and Rectal Biopsy Tissue Processing. Peripheral blood was layered onto a Ficoll-Hypaque (Pfizer, New York, NY) density gradient to isolate mononuclear cells (PBMC). Rectal biopsy tissue was processed according to a previously published protocol designed to maximize viable lymphocyte yield (, 1,, 1). Briefly, biopsy tissue was subjected to three rounds of collagenase type II digestion (0.mg/mL; Sigma-Aldrich, St. Louis, MO), followed by mechanical disruption using an 1-gauge blunt end needle, and passage through a 0 m cell strainer. Cells were then washed in R1 and then layered on a %/% Percoll gradient (Sigma-Aldrich). Mononuclear cells were harvested from both the media-% Percoll interface and the %/% Percoll interface to maximize yield. PBMC and rectal mononuclear cells (RMC) were rested overnight at o C and % CO, and piperacillin-tazobactam (Zosyn, 0.mg/mL; Wyeth-Ayerst, Princeton, NJ) was added to RMC cultures to prevent bacterial growth. HLA Class II Typing. Genomic DNA was isolated from -x PBMC using the QIAamp DNA blood mini kit (Qiagen, Valencia, CA) and quantified by spectrophotometer. Low resolution HLA-DR and HLA-DQ typing was performed using polymerase chain reaction (PCR) and the DR/DQ T Downloaded from on September 1, 01 by guest locus SSP (sequence specific primer) Unitray kit (Invitrogen, Carlsbad, CA) PCR products

7 were resolved by electrophoresis on a % agarose gel, photographed, and analyzed using UniMatch Plus software (Invitrogen) Antibodies and Peptide Pools. Fluorochrome-labeled monoclonal antibodies to CD (clone UCHT-1), CDa (clone HA), interferon- (IFN- ; clone B), macrophage inflammatory protein-1 (MIP-1 ; clone D1-1), tumor necrosis factor- (TNF- ; clone MAb), interleukin- (IL-; clone.1), and unlabeled antibodies to CD (clone L) and CDd (clone L) were purchased from BD Biosciences (San Jose, CA). Fluorochrome-labeled antibodies to CD (clone SFCITD) were purchased from Beckman Coulter (Fullerton, CA) and CD (clone SK1) from Invitrogen. All antibodies were titrated to determine optimum concentrations for assay conditions (data not shown). HIV Gag (p, HXB sequence) peptide pools containing 1-mer peptides overlapping by amino acids were purchased from BD Biosciences. Intracellular Cytokine Staining and Flow Cytometry. Intracellular cytokine staining was performed as previously described on newly isolated PBMC or RMC without prior expansion (1). Briefly, 1-x PBMC or RMC were stimulated for hours with HIV Gag p pool (. g/ml) in the presence of anti-cd (. g/ml) and anti-cdd ( g/ml) costimulatory antibodies, anti-cda, Golgi Stop TM (1 M; BD Biosciences), and brefeldin A ( g/ml; Sigma-Aldrich). Dimethyl Sulfoxide (DMSO) and Staphylococcus enterotoxin B ( g/ml; SEB) served as negative and positive controls respectively. Cells were then stained with antibodies to surface antigens CD and CD and with -amino-actinomycin D (-AAD; BD Biosciences) to label dead cells. Thereafter, all buffers Downloaded from on September 1, 01 by guest

8 and reagents contained actinomycin D to saturate sites with the potential to bind -AAD. Cells were fixed in % paraformaldehyde and then permeabilized using FACS Perm (BD Biosciences). Intracellular cellular staining for CD, IFN, IL-, MIP-1, and TNF followed permeabilization. Finally, the cells were placed in 1% paraformaldehyde for at least 1 hour, but no longer than hours, before being read on an LSRII flow cytometer equipped with a 0,, and nm laser (BD Biosciences). Polyclonal CD+ T-cell Expansion of Peripheral Blood and Rectal Mononuclear Cells. Polyclonal expansion was performed in order to obtain sufficient CD+ T-cells for epitope mapping using the ELISpot method (). This approach has been shown to expand T- cells non-specifically without significantly altering T-cell receptor clonotypes, patterns of epitope recognition, or the ability of T-cells to respond to peptide stimulation by secreting IFN (,, ). It should be noted that in the present study, polyclonal expansion was utilized only to generate sufficient cells to map the peptide specificity of responses that had previously been identified by intracellular cytokine staining of fresh PBMC and RMC. One million PBMC or RMC were placed in ml R1 in a well plate with 1 g/ml CD- bispecific antibody (generously provided by Dr. Johnson Wong, Harvard University), which preferentially expands CD+ T-cells while promoting apoptosis in CD+ T-cells (). PBMC and RMC cultures were supplemented with ril- (0U/mL; R&D Systems, Minneapolis, MN). When initial attempts to culture RMC (but not PBMC) gave a low success rate, we supplemented cultures with 1 ng/ml human recombinant IL- (R&D Systems, Minneapolis, MN), based on a previously published protocol. This cytokine promotes survival of memory T- cells, in part by inactivating pro-apoptotic pathways (). Amphotericin B (1. g/ml; MP Downloaded from on September 1, 01 by guest

9 Biomedicals, Solon, OH), and piperacillin-tazobactam (Zosyn, 0. g/ml; Wyeth-Ayerst, Princeton, NJ) were added to prevent fungal and bacterial growth, respectively. After the first week, IL- (and IL- for RMC cultures) was added twice weekly, as well as additional amphotericin B and Zosyn to compensate for any added R1 volume during the expansion. Two to three days following the initial CD- antibody stimulation, x irradiated heterologous PBMC (000 RAD) from a seronegative donor were added to cultures. Cultures were expanded and maintained for to weeks. After weeks of culture, cells were restimulated with 0.1 g/ml F anti-cd antibody (also provided by Dr. Johnson Wong, Harvard University). CD+ T-cell Response Mapping By to weeks, polyclonally expanded cultures were composed of primarily CD+ T- cells; however, approximately half of the cultures included 0-0% CD+ T-cells. Therefore, CD+ T-cells were depleted from cultures using magnetic bead separation prior to epitope mapping. Anti-CD microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used to label CD+ T-cells. They were then removed from the CD+ T-cells using the MACS system (Miltenyi Biotec) with an LD column. CD+ T-cell response mapping was performed using an IFN ELISpot assay as previously described for CD+ T-cell response mapping (0). Briefly, pools of HIV HXB consensus clade B Gag, Env, and Nef peptides (1-mers overlapping by ; NIH AIDS Research & Reference Reagent Program, Rockville MD) were created so that each peptide was contained in exactly two pools. This allowed the creation of a peptide matrix for screening HIV peptides. Peptides common to pools positive for a CD+ T-cell response (>0 spot forming cells/million, Downloaded from on September 1, 01 by guest

10 SFC/million, after the subtraction of background) were then tested individually using a standard IFN ELISpot assay Data Analysis. Flow cytometry data were analyzed with FlowJo software (TreeStar, Ashland, OR). Boolean gate analysis allowed the separation of CD+ T-cell responses into 1 individual combinations of the four functional parameters (IFN, IL-, MIP-1, and TNF Data generated from Boolean gate analysis was then visualized in SPICE software (v...; Mario Roederer, Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD). Standard bivariate plots were also constructed to confirm the placement of individual Boolean gates. IFN ELISpots were read on an AID plate reader (Autoimmun Diagnostika GMBH, Stra erg, Germany) and pool or peptide-specific CD+ T-cell responses were quantified as SFC/million after the subtraction of background. Responses >0 SFC/million were considered as positive. Statistical Analysis. CD+ T-cell flow cytometry data were analyzed using a previously described formula which considers whether or not antigen-specific responses differ significantly from background (, 1). This step was important for evaluating mucosal T-cell responses as they are highly responsive to costimulation (data not shown) and can produce low levels of cytokines and chemokines in the absence of antigen-specific stimulation. The formula assumes a Poisson distribution and takes into account the actual number of collected events rather than percentages Downloaded from on September 1, 01 by guest

11 of responding cells, as lymphocyte yield from rectal mucosa tends to be low. When a determination was made that antigen-specific values were significantly different from background, net responses were then calculated by subtracting unstimulated control values from antigen-specific values. Responses that were not determined to be significantly different from background were dropped (, 1). For response data, comparisons between two groups were made using a two-tailed Mann- Whitney test in GraphPad Prism software (GraphPad Software, San Diego, CA). Comparisons between polyfunctionality pie charts utilized a permutation test based on statistics, and comparisons between two individual functional categories employed a Wilcoxon rank test (SPICE Software). A two-tailed Fisher s exact test was used for comparing the distribution of HLA-DRB1* and HLA-DQB1*0 among subject groups. Correlations between two variables were done using the Spearman correlation, and linear regression was used to graph a best-fit line to the data (GraphPad Prism Software). RESULTS Baseline Characteristics. The individuals in this study have been described in an earlier report (1). Briefly, HIV positive individuals were assigned to one of four groups as described in Materials and Methods: elite controllers, viremic controllers, non-controllers, and HAART-suppressed patients. Eight HIV-negative subjects were included as controls. The percentage of CD+ T-cells as a proportion of CD+ T-cells in peripheral blood and rectal mucosa was previously determined by flow cytometry (1). Elite and viremic controllers had significantly higher percentages of CD+ T-cells in rectal mucosa compared to non- Downloaded from on September 1, 01 by guest

12 controllers (medians.% and 0.1% vs. 1.1% respectively, p<0.0); rectal CD percentages in controllers were comparable to those in subjects on HAART (median.%). All HIV positive individuals had lower percentages of CD+ T-cells than HIV negative controls (median %; p<0.01) (1). High magnitude HIV-specific CD+ T-cell responses in rectal mucosa. The ability of CD+ T-cells to respond to HIV Gag stimulation by producing IFN, IL-, MIP-1, and/or TNF was measured by flow cytometry using fresh PBMC and RMC without prior expansion (see reference (1) for gating strategy). CD+ T-cell responses were generally much higher, in terms of total percentage of responding cells, in rectal mucosa than peripheral blood. This likely reflects the predominance of antigen-experienced memory cells in rectal mucosa, and was previously observed for HIV-specific CD+ T-cells (1). The median total HIV-specific mucosal CD+ T-cell response (cells positive for one or more of the above functions, all responses combined) was significantly higher in elite controllers compared to individuals on HAART (Figure 1A;.% vs. 0.% respectively; p=0.0). While some elite controllers had unusually strong Gag-specific mucosal CD+ T-cell responses (as high as %), responses within the group were highly heterogeneous, and therefore the median total CD+ T- cell response was not significantly different between elite controllers and non-controllers (.% vs..% respectively, p=0.1). While predominantly tissue-specific differences (i.e. between peripheral blood and rectal mucosa) were found in the overall magnitude of HIV-specific CD+ T-cell responses, differences were found in CD+ T-cell responses between subject groups when assessed for individual functions. Elite controllers and viremic controllers had higher frequencies of mucosal Downloaded from on September 1, 01 by guest

13 CD+ T-cells cells producing IFN Figure 1B) and MIP-1 (Figure 1C) than either noncontrollers (NC) or HAART-suppressed individuals. In general, <0.% of peripheral blood CD+ T-cells from controllers, non-controllers, or HAART-suppressed subjects responded in any way to Gag stimulation (Figure 1D). There was no consistent difference in the ability of mucosal CD+ T-cells to produce IL- or TNF across subject groups; however, controllers generally had higher levels of IL- producing cells in peripheral blood (Figure 1E), as previously described by our group and others (, 1, 1, ), as well as higher levels of TNF (Figure 1F). HIV controllers have complex Gag-specific mucosal CD+ T-cell responses. Boolean gate analysis was used to assess the complexity of the HIV-specific CD+ T-cell response by looking at all possible combinations of four functional parameters: IFN-, IL-, TNF and MIP-1. Cells that simultaneously expressed three or four cytokines in response to HIV peptide stimulation were considered polyfunctional. As there were no significant differences in polyfunctionality between elite and viremic controllers (data not shown), the two groups were combined into a single controller group in order to simplify the analysis. Controllers showed a trend towards an overall more polyfunctional mucosal CD+ T-cell response compared to non-controllers, as represented by the pie charts in Figure A (p=0.01). Broken down into the 1 separate functional categories, controllers had higher frequencies of four-functional mucosal CD+ T-cells than non-controllers and more three-functional CD+ T- cells (primarily IFN- +MIP-1 +TNF + and lacking IL-) than non-controllers or individuals on HAART (Figure A; p<0.0). Interestingly, controllers showed high levels of MIP-1 single Downloaded from on September 1, 01 by guest

14 positive cells in rectal mucosa, while non-controllers had high levels of TNF single positive cells compared to other subject groups (p<0.0). The response complexity in peripheral blood of HIV controllers appeared to be more polyfunctional than in non-controllers or HAART-suppressed subjects, but these differences did not reach statistical significance (Figure B). Similar to responses seen in rectal mucosa, controllers had higher percentages of Gag-specific CD+ T-cells cells capable of three or four functions than non-controllers (p<0.0); however, the dominant three-function category (in terms of median response) in blood was IFN- +IL-+TNF +, rather than IFN- +MIP-1 +TNF + as seen in rectal mucosa Figure B). Individuals with HLA-DRB1* and HLA-DQB1*0 have stronger, more polyfunctional mucosal CD+ T cell responses. It was interesting to note that some elite controllers had unusually high magnitude CD+ T-cell responses in rectal mucosa (Figure 1A). We therefore wished to determine if there was an association between these high responses and specific class II HLA alleles. Since a previous study had found that the haplotype HLA-DRB1*/HLA-DQB1*0 was enriched in a cohort of long-term non-progressors (LTNP) and that p lymphoproliferative responses in these individuals were more robust than those without the haplotype (), we initially focused on the role of this specific haplotype. In our cohort, HLA-DRB1* was only found in controllers (n=); likewise, 1 out of 1 individuals with HLA-DQB1*0 were controllers (Figure ; Table S1). Six controllers (1%) had the combined haplotype of HLA-DRB1*/HLA-DQB1*0, controllers (%) had HLA- DQB1*0 alone, one controller (%) had the HLA-DRB1* allele alone, and controllers Downloaded from on September 1, 01 by guest 1

15 (%) lacked any of these alleles. Non-controllers in our study did not possess HLA-DRB1* and only one had HLA-DQB1*0. Controllers more frequently possessed HLA-DRB1* and/or HLA-DQB1*0 than non-controllers (Figure ; Fisher s exact test, p=0.00). Mucosal CD+ T-cells in controllers with one or both of these class II alleles were found to have high magnitude responses compared to either controllers or non-controllers lacking those alleles (Figure ;.% vs..% (p=0.0) and.1% (p=0.0) respectively). Controllers with both alleles also had highly polyfunctional mucosal CD+ T-cell responses (Figure ). Mucosal CD+ T-cells from controllers with the HLA-DRB1*/HLA- DQB1*0 haplotype had significantly higher frequencies of -function cells (IFN +MIP- 1 +TNF +) than controllers with only the HLA-DQB1*0 allele or other class II HLA alleles (Figure ; p<0.0). There was also a trend towards higher percentages of -function (IFN +IL- +MIP-1 +TNF +), -function (IL-+MIP-1 +TNF +), and dual function (IL-+TNF +), cells in the rectal mucosa of the controllers who had HLA-DRB1*/HLA-DQB1*0 as compared to controllers without the haplotype. Controllers that had only HLA-DQB1*0 exhibited less complex responses, similar to subjects that had neither HLA-DRB1* nor HLA- DQB1*0 (Figure ). While there was only one controller who possessed HLA-DRB1* in the absence of HLA-DQB1*0, this individual had the most polyfunctional mucosal CD+ T-cell response with 1% of the total HIV Gag-specific CD+ T-cell response consisting of or factors (data not shown). Downloaded from on September 1, 01 by guest 1

16 CD+ T-cell response mapping from four controllers with HLA-DRB1* and/or HLA- DQB1*0. CD+ T-cells from individuals with high magnitude total CD+ T-cell responses in either blood or mucosa and possessing HLA-DRB1* and/or HLA-DQB1*0 (Figure ) were polyclonally expanded and their responses to HIV Gag, Env, and Nef peptides mapped using IFN ELISpot. Four controllers had measurable responses to HIV Gag and Nef peptides (Table 1). No CD+ T-cell responses to HIV Env peptides were detected in either blood or rectal mucosa. All four subjects responded to Gag p aa -0 (DYVDRFYKTLRAEQA), an immunodominant peptide also described by Malhotra et al (). Three of four subjects also had robust responses to a Gag peptide that encompasses the protease cleavage site between p nucleocapsid and the p1 linker region (aa -, RQANFLGKIQPSHKGRPGN). There were fewer CD+ T-cell responses to HIV Nef in these individuals, and these tended to be of lower magnitude compared to the majority of HIV Gag responses (Table 1). While these subjects possess the HLA-DRB1* and/or HLA0DQB1*0 alleles, the actual MHC restrictions of these responses are currently unknown and will be addressed in future studies. It should be noted that some low-frequency T-cell populations may fail to expand in response to polyclonal stimulation, leading to underestimation of antigen-specific response frequency and/or breadth (,, ). In addition, the use of clade B consensus peptides in this study means that responses to autologous viral sequences were not measured. Nevertheless, our previous studies as well as those of other groups suggest that this approach can reliably detect and map most immunodominant and many subdominant T-cell responses (, 1,, ). Downloaded from on September 1, 01 by guest 1

17 Polyfunctional CD+ T-cell responses correlate with the magnitude of the total CD+ T-cell response in rectal mucosa. There was a positive correlation between the magnitude of the total CD+ T-cell response and the total CD+ T-cell response in rectal mucosa (Figure A; Spearman s r=0.1, p<0.0001). There was also a significant positive correlation between polyfunctional CD+ T- cell responses ( or functions) and total CD+ T-cell responses (Figure B, Spearman s r=0., p=0.00), and there was a strong trend towards a positive correlation between polyfunctional CD+ T-cell vs. polyfunctional CD+ T-cell responses (, or functions) (Figure C, Spearman s r=0.0, p=0.0). One outlier, a controller, had no polyfunctional CD+ T-cell response, yet had the highest polyfunctional CD+ T-cell response (Figure C). So while in general, stronger and more polyfunctional CD+ T-cell responses correlated with stronger, more polyfunctional CD+ T-cell responses, this was not true for every individual. DISCUSSION While many studies have described the complex functional responses of HIV-specific CD+ T-cells, fewer reports have focused on HIV-specific CD+ T-cell responses. Of those studies that have focused on HIV-specific CD+ T-cells, to our knowledge, all have investigated peripheral blood rather than mucosal CD+ T-cell responses. Here we present HIV-specific CD+ T-cell functional responses in both peripheral blood and rectal mucosa of controllers, noncontrollers, and individuals on HAART. Our findings show that HIV controllers have highly polyfunctional HIV-specific mucosal CD+ T-cell responses compared to non-controllers. When considered individually, levels of Gag-specific IFN and MIP-1 production in the mucosa were higher in controllers than non-controllers (Figure 1B and C). Responses in blood Downloaded from on September 1, 01 by guest 1

18 generally did not reveal the same magnitude or complexity seen in the gut mucosa. The one consistent exception to these trends was the finding that in peripheral blood, CD+ T-cells from controllers generally produced more IL- and TNF than CD+ T-cells from non-controllers (Figure 1E & F), as previously reported by Emu et al. (1) and Harari et al. (1, ). Additionally, controllers had stronger and more complex HIV-specific CD+ T-cell responses than HAART-suppressed individuals with a similarly low antigen burden, suggesting that the augmented CD+ T-cell responses in HIV controllers are not simply a consequence of low viral load. While the total HIV-specific CD+ T-cell response magnitude was similar for many controllers and non-controllers, we noticed that some individuals who maintained viral control had unusually strong mucosal HIV Gag-specific responses, as high as % of the rectal CD+ T- cell population. We hypothesized that individuals with these high responses might be enriched for certain class II HLA alleles. Indeed, controllers with HLA-DRB1* and/or HLA- DQB1*0, class II HLA alleles previously associated with HIV control and long-term nonprogression (, 1,, ) had the strongest mucosal CD+ T-cell responses in our cohort. Moreover, subjects with the combined haplotype had exceptionally polyfunctional Gag-specific CD+ T-cell responses in rectal mucosa. When CD+ T-cell responses were analyzed with respect to paired CD+ T-cell responses, there was a direct correlation between polyfunctional CD+ T-cell responses and both the magnitude and quality of CD+ T-cell responses in rectal mucosa. These findings were consistent with prior studies highlighting the importance of CD+ T-cell help in maintaining functional CD+ T-cell populations during chronic viral infection (,,,, ). It has been shown that individuals with strong Gag-specific CD+ T-cell responses in conjunction with Downloaded from on September 1, 01 by guest 1

19 strong CD+ lymphoproliferative responses are better able to control HIV-1 viremia (). Nevertheless, some controllers in our cohort appeared to lack polyfunctional CD+ T-cell responses, yet had strong, polyfunctional CD+ T-cell responses. This suggests that high quality, robust CD+ T-cell help contributes to, but may not be not essential for, robust and polyfunctional CD+ T-cell responses. It is likely that non-t-cell based mechanisms of control may be a factor in those controllers who lack strong, polyfunctional CD+ and CD+ T-cell responses, as suggested by Deeks and Walker (). In addition, we cannot rule out the possibility that low-level ongoing viral replication in the gut mucosa of controllers could be driving the expansion of both HIV-specific CD+ and CD+ T-cells. Therefore, CD+ and CD+ T-cell responses could be related without being causally associated. Although a role for MHC class I alleles in HIV control and long-term non-progression has been unequivocally established, there is as yet no consensus on the role of specific MHC class II alleles in long-term HIV control in humans. Several previous reports have suggested an association of HLA-DRB1* and/or HLA-DQB1*0 with slow disease progression (, 1,, ). In a study by Malhotra et al, both long-term non-progressors and individuals receiving antiretroviral therapy who had the HLA-DRB1*/DQB1*0 haplotype revealed stronger Gagspecific proliferative responses and IFN secretion than individuals lacking this haplotype (). Additionally, SIV controllers that maintain stable viral loads <1,000 copies/ml are more likely than non-controllers to possess the MHC class II alleles Mamu-DRB1*0 and Mamu- DRB1*00 (1). It should be noted that in our study all of the subjects with HLA DRB1*/HLA- DQB1*0 also had class I alleles associated with control (five with HLA-B, and one with HLA-B1) (1). Accordingly, this study was not powered to distinguish the effects of class I Downloaded from on September 1, 01 by guest 1

20 versus class II alleles. However, in the Malhotra study none of the HLA-DRB1* subjects had protective class I alleles (). Large-scale genetics studies will be required to address the relative contributions of class I and class II alleles to HIV control (). It is also possible that HLA-DRB1*/HLA-DQB1*0 are in linkage disequilibrium with another factor that strongly contributes to immune control, thereby indirectly leading to relative preservation of mucosal CD+ T-cells and maintenance of strong HIV-specific CD+ T-cell responses. This hypothesis may be supported by the observation that HLA-DRB1*01 and/or 0 alleles have also been associated with protection against persistent HBV infection, cervical carcinoma related to HPV infection, and Plasmodium falciparum malaria (,, ). An intriguing finding of the Malhotra study () was that HLA-DRB1*-restricted CD+ T-cell clones recognized and bound with high affinity the Gag epitopes EIYKRWIILG (EG: p aa 0-) and DYVDRFYKTLRAEQA (DA1: p aa -0), that immediately precede and partially overlap immunodominant CD+ T-cell epitopes restricted by HLA-B (KK:KRWIILGLNK, p aa -) and HLA-B (QW: QASQEVKNW, p, aa 0-1) (1, 0). Gag p is highly conserved among HIV-1 subtypes, and analogous regions are found in the Gag proteins of SIV and FIV (,, 1, ). Mutations in these regions often come at a fitness cost to the virus (0, ), and generally involve compensatory mutations upstream of an epitope (,, ). Another recent report showed that the CD+ T- cells of controllers that responded to the Gag peptide FRDYVDRFYKTLRAEQASQE (p, aa -), which overlaps with DA1, had both high functional avidity and diverse TCR repertoires (). Highly avid HIV-specific CD+ T-cell responses directed at conserved regions within Gag may persist longer to help maintain a healthy CD+ T-cell population, thereby aiding in the maintenance of viral suppression. Downloaded from on September 1, 01 by guest 0

21 In the current study, we found that controllers with HLA-DRB1* and/or HLA- DQB1*0 had strong responses to the DA1 peptide; however, out of of these individuals had HLA-DQB1*0 alone, suggesting that these responses may not be restricted solely by HLA- DRB1*. This would not be surprising, since class II MHC alleles are known to be highly promiscuous, and in particular the Gag peptide spanning residues 1-0 was previously noted to bind of of the most common HLA-DR alleles worldwide (0,, ). Determining the class II MHC restriction of the DA1 peptide in these individuals is the focus of ongoing studies. We also observed Nef-specific responses in these four controllers, consistent with previous reports showing the immunodominance of HIV Gag and Nef among HIV-specific CD+ T-cell responses (0). Our data, taken together with previous findings () support the hypothesis that CD+ T- cell responses act in concert with CD+ T-cell responses as immune correlates of HIV control. High magnitude, polyfunctional HIV-specific CD+ T-cells that reside in the rectal mucosa appear to be a strong and consistent predictor of long-term control of HIV replication in the absence of therapy. These populations are consistently more abundant in controllers as compared to individuals on long-term antiretroviral therapy. Since the level of viremia is comparable in these two groups (), it is unlikely that the observed increase in HIV-specific CD+ T-cell responses is simply a consequence of virus control. Moreover, the finding that among HIV controllers there is an association between the presence of certain class II HLA alleles and strong CD+ T-cell responses argues for an active role of these cells in contributing to virus control. Collectively, these data suggest that vaccine and therapeutic strategies aimed at expanding these mucosal T-cell responses may prove beneficial. Downloaded from on September 1, 01 by guest 1

22 ACKNOWLEDGMENTS The authors thank Rebecca Hoh (University of California, San Francisco) and Melissa Schreiber (University of California, Davis) for assistance with patient recruitment and enrollment. This study was supported by the National Institutes of Health (grant AI000, BLS; grants AI0, AI1 and AI01, SGD); grant AI0, PWH; grant AI0, the University of California, San Francisco Center for AIDS Research), the California HIV/AIDS Research Program (grant CH0-D-0; RBP), the American Foundation for AIDS Research (-0-RGRL; SGD), and the University of California, San Francisco Clinical and Translational Science Institute (UL1 RR01). This investigation was conducted in a facility constructed with support from the Research Facilities Improvement Program (grant C0 RR- 0-01) from the National Center for Research Resources, National Institutes of Health. The LSR-II violet laser was upgraded with funding from the James B. Pendleton Charitable Trust. Downloaded from on September 1, 01 by guest

23 REFERENCES 1. Almeida, J. R., D. A. Price, L. Papagno, Z. A. Arkoub, D. Sauce, E. Bornstein, T. E. Asher, A. Samri, A. Schnuriger, I. Theodorou, D. Costagliola, C. Rouzioux, H. Agut, A. G. Marcelin, D. Douek, B. Autran, and V. Appay. 00. Superior control of HIV-1 replication by CD+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J Exp Med 0:-.. Altfeld, M., E. T. Kalife, Y. Qi, H. Streeck, M. Lichterfeld, M. N. Johnston, N. Burgett, M. E. Swartz, A. Yang, G. Alter, X. G. Yu, A. Meier, J. K. Rockstroh, T. M. Allen, H. Jessen, E. S. Rosenberg, M. Carrington, and B. D. Walker. 00. HLA Alleles Associated with Delayed Progression to AIDS Contribute Strongly to the Initial CD(+) T Cell Response against HIV-1. PLoS Med :e0.. Altfeld, M., and E. S. Rosenberg The role of CD(+) T helper cells in the cytotoxic T lymphocyte response to HIV-1. Curr Opin Immunol :-0.. Altfeld, M., J. van Lunzen, N. Frahm, X. G. Yu, C. Schneider, R. L. Eldridge, M. E. Feeney, D. Meyer-Olson, H. J. Stellbrink, and B. D. Walker. 00. Expansion of preexisting, lymph node-localized CD+ T cells during supervised treatment interruptions in chronic HIV-1 infection. J Clin Invest :-.. Apple, R. J., H. A. Erlich, W. Klitz, M. M. Manos, T. M. Becker, and C. M. Wheeler. 1. HLA DR-DQ associations with cervical carcinoma show papillomavirustype specificity. Nat Genet :1-.. Betts, M. R., M. C. Nason, S. M. West, S. C. De Rosa, S. A. Migueles, J. Abraham, M. M. Lederman, J. M. Benito, P. A. Goepfert, M. Connors, M. Roederer, and R. A. Downloaded from on September 1, 01 by guest

24 Koup. 00. HIV nonprogressors preferentially maintain highly functional HIV-specific CD+ T cells. Blood :1-.. Boaz, M. J., A. Waters, S. Murad, P. J. Easterbrook, and A. Vyakarnam. 00. Presence of HIV-1 Gag-specific IFN-gamma+IL-+ and CD+IL-+ CD T cell responses is associated with nonprogression in HIV-1 infection. J Immunol 1:-.. Brockman, M. A., A. Schneidewind, M. Lahaie, A. Schmidt, T. Miura, I. Desouza, F. Ryvkin, C. A. Derdeyn, S. Allen, E. Hunter, J. Mulenga, P. A. Goepfert, B. D. Walker, and T. M. Allen. 00. Escape and compensation from early HLA-B- mediated cytotoxic T-lymphocyte pressure on human immunodeficiency virus type 1 Gag alter capsid interactions with cyclophilin A. J Virol 1:0-1.. Carrington, M., and S. J. O'Brien. 00. The influence of HLA genotype on AIDS. Annu Rev Med :-1.. Chen, Y., R. Winchester, B. Korber, J. Gagliano, Y. Bryson, C. Hutto, N. Martin, G. McSherry, A. Petru, D. Wara, and A. Ammann. 1. Influence of HLA alleles on the rate of progression of vertically transmitted HIV infection in children: association of several HLA-DR alleles with long-term survivorship and the potential association of HLA-A*01 with rapid progression to AIDS. Long-Term Survivor Study. Hum Immunol :1-.. Critchfield, J. W., D. H. Young, T. L. Hayes, J. V. Braun, J. C. Garcia, R. B. Pollard, and B. L. Shacklett. 00. Magnitude and complexity of rectal mucosa HIV-1- specific CD+ T-cell responses during chronic infection reflect clinical status. PLoS One :e. Downloaded from on September 1, 01 by guest

25 Deeks, S. G., and B. D. Walker. 00. Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy. Immunity :0-1.. den Uyl, D., I. E. van der Horst-Bruinsma, and M. van Agtmael. 00. Progression of HIV to AIDS: a protective role for HLA-B? AIDS Rev :-. 1. Emu, B., E. Sinclair, D. Favre, W. J. Moretto, P. Hsue, R. Hoh, J. N. Martin, D. F. Nixon, J. M. McCune, and S. G. Deeks. 00. Phenotypic, Functional, and Kinetic Parameters Associated with Apparent T-Cell Control of Human Immunodeficiency Virus Replication in Individuals with and without Antiretroviral Treatment. J Virol : Emu, B., E. Sinclair, H. Hatano, A. Ferre, B. Shacklett, J. N. Martin, J. M. McCune, and S. G. Deeks. 00. HLA Class I-Restricted T Cell Responses May Contribute to the Control of HIV Infection, but Such Responses are Not Always Necessary for Long-term Virus Control. J Virol. 1. Ferre, A. L., P. W. Hunt, J. W. Critchfield, D. H. Young, M. M. Morris, J. C. Garcia, R. B. Pollard, H. F. Yee, Jr., J. N. Martin, S. G. Deeks, and B. L. Shacklett. 00. Mucosal immune responses to HIV-1 in elite controllers: a potential correlate of immune control. Blood :-. 1. Ferre, A. L., D. Lemongello, P. W. Hunt, M. M. Morris, J. C. Garcia, R. B. Pollard, H. F. Yee, Jr., J. N. Martin, S. G. Deeks, and B. L. Shacklett. 0. Immunodominant HIV-Specific CD+ T-cell Responses are Common to Blood and Gastrointestinal Mucosa, and Gag-specific Responses Dominate in Rectal Mucosa of HIV Controllers. J Virol. in press [Epub 0 July ]. Downloaded from on September 1, 01 by guest

26 Frahm, N., B. T. Korber, C. M. Adams, J. J. Szinger, R. Draenert, M. M. Addo, M. E. Feeney, K. Yusim, K. Sango, N. V. Brown, D. SenGupta, A. Piechocka-Trocha, T. Simonis, F. M. Marincola, A. G. Wurcel, D. R. Stone, C. J. Russell, P. Adolf, D. Cohen, T. Roach, A. StJohn, A. Khatri, K. Davis, J. Mullins, P. J. Goulder, B. D. Walker, and C. Brander. 00. Consistent cytotoxic-t-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities. J Virol : Giraldo-Vela, J. P., R. Rudersdorf, C. Chung, Y. Qi, L. T. Wallace, B. Bimber, G. J. Borchardt, D. L. Fisk, C. E. Glidden, J. T. Loffredo, S. M. Piaskowski, J. R. Furlott, J. P. Morales-Martinez, N. A. Wilson, W. M. Rehrauer, J. D. Lifson, M. Carrington, and D. I. Watkins. 00. The major histocompatibility complex class II alleles Mamu- DRB1*0 and -DRB1*00 are enriched in a cohort of simian immunodeficiency virus-infected rhesus macaque elite controllers. J Virol : Goulder, P. J., M. Bunce, P. Krausa, K. McIntyre, S. Crowley, B. Morgan, A. Edwards, P. Giangrande, R. E. Phillips, and A. J. McMichael. 1. Novel, crossrestricted, conserved, and immunodominant cytotoxic T lymphocyte epitopes in slow progressors in HIV type 1 infection. AIDS Res Hum Retroviruses : Harari, A., S. Petitpierre, F. Vallelian, and G. Pantaleo. 00. Skewed representation of functionally distinct populations of virus-specific CD T cells in HIV-1-infected subjects with progressive disease: changes after antiretroviral therapy. Blood :-.. Harari, A., F. Vallelian, P. R. Meylan, and G. Pantaleo. 00. Functional heterogeneity of memory CD T cell responses in different conditions of antigen exposure and persistence. J Immunol 1:-. Downloaded from on September 1, 01 by guest

27 Hatano, H., E. L. Delwart, P. J. Norris, T. H. Lee, J. Dunn-Williams, P. W. Hunt, R. Hoh, S. L. Stramer, J. M. Linnen, J. M. McCune, J. N. Martin, M. P. Busch, and S. G. Deeks. 00. Evidence for persistent low-level viremia in individuals who control human immunodeficiency virus in the absence of antiretroviral therapy. J Virol :-.. Hill, A. V., C. E. Allsopp, D. Kwiatkowski, N. M. Anstey, P. Twumasi, P. A. Rowe, S. Bennett, D. Brewster, A. J. McMichael, and B. M. Greenwood.. Common west African HLA antigens are associated with protection from severe malaria. Nature :-00.. Ibarrondo, F. J., P. A. Anton, M. Fuerst, H. L. Ng, J. T. Wong, J. Matud, J. Elliott, R. Shih, M. A. Hausner, C. Price, L. E. Hultin, P. M. Hultin, B. D. Jamieson, and O. O. Yang. 00. Parallel human immunodeficiency virus type 1-specific CD+ T- lymphocyte responses in blood and mucosa during chronic infection. J Virol :-.. Johnson, R. P., A. Trocha, L. Yang, G. P. Mazzara, D. L. Panicali, T. M. Buchanan, and B. D. Walker.. HIV-1 gag-specific cytotoxic T lymphocytes recognize multiple highly conserved epitopes. Fine specificity of the gag-specific response defined by using unstimulated peripheral blood mononuclear cells and cloned effector cells. J Immunol 1:1-1.. Juelg, B., E. Moodely, Y. Qi, K. Bishop, S. Reddy, P. J. Goulder, R. Detels, T. Ndung'u, B. D. Walker, and M. Carrington. 0. Presented at the Conference on Retroviruses and Oportunistic Infections, San Francisco, CA.. Kalams, S. A., S. P. Buchbinder, E. S. Rosenberg, J. M. Billingsley, D. S. Colbert, N. G. Jones, A. K. Shea, A. K. Trocha, and B. D. Walker. 1. Association between Downloaded from on September 1, 01 by guest

28 virus-specific cytotoxic T-lymphocyte and helper responses in human immunodeficiency virus type 1 infection. J Virol :1-0.. Kalams, S. A., and B. D. Walker. 1. The critical need for CD help in maintaining effective cytotoxic T lymphocyte responses [comment]. J Exp Med 1: Kaufmann, D. E., P. M. Bailey, J. Sidney, B. Wagner, P. J. Norris, M. N. Johnston, L. A. Cosimi, M. M. Addo, M. Lichterfeld, M. Altfeld, N. Frahm, C. Brander, A. Sette, B. D. Walker, and E. S. Rosenberg. 00. Comprehensive analysis of human immunodeficiency virus type 1-specific CD responses reveals marked immunodominance of gag and nef and the presence of broadly recognized peptides. J Virol :-. 1. Keet, I. P. M., J. Tang, M. R. Klein, S. LeBlanc, C. Enger, C. Rivers, R. J. Apple, D. Mann, J. J. Goedert, F. Miedema, and R. A. Kaslow. 1. Consistent associations of HLA class I and II and transporter gene products with progression of human immunodeficiency virus type 1 infection in homosexual men. Journal of Infectious Diseases :-0.. Klein, M. R., C. A. van Baalen, A. M. Holwerda, S. R. Kerkhof Garde, R. J. Bende, I. P. Keet, J. K. Eeftinck-Schattenkerk, A. D. Osterhaus, H. Schuitemaker, and F. Miedema. 1. Kinetics of Gag-specific cytotoxic T lymphocyte responses during the clinical course of HIV-1 infection: a longitudinal analysis of rapid progressors and longterm asymptomatics. J Exp Med :-.. Koup, R. A., J. T. Safrit, Y. Cao, C. A. Andrews, G. McLeod, W. Borkowsky, C. Farthing, and D. D. Ho. 1. Temporal association of cellular immune responses with Downloaded from on September 1, 01 by guest

29 the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol :0-.. Lalvani, A., T. Dong, G. Ogg, A. A. Patham, H. Newell, A. V. Hill, A. J. McMichael, and S. Rowland-Jones. 1. Optimization of a peptide-based protocol employing IL- for in vitro restimulation of human cytotoxic T lymphocyte precursors. J Immunol Methods :-.. Louwagie, J., F. E. McCutchan, M. Peeters, T. P. Brennan, E. Sanders-Buell, G. A. Eddy, G. van der Groen, K. Fransen, G. M. Gershy-Damet, R. Deleys, and et al.. Phylogenetic analysis of gag genes from 0 international HIV-1 isolates provides evidence for multiple genotypes. AIDS :-0.. Malhotra, U., S. Holte, S. Dutta, M. M. Berrey, E. Delpit, D. M. Koelle, A. Sette, L. Corey, and M. J. McElrath Role for HLA class II molecules in HIV-1 suppression and cellular immunity following antiretroviral treatment. J Clin Invest :0-1.. Martinez-Picado, J., J. G. Prado, E. E. Fry, K. Pfafferott, A. Leslie, S. Chetty, C. Thobakgale, I. Honeyborne, H. Crawford, P. Matthews, T. Pillay, C. Rousseau, J. I. Mullins, C. Brander, B. D. Walker, D. I. Stuart, P. Kiepiela, and P. Goulder. 00. Fitness cost of escape mutations in p Gag in association with control of human immunodeficiency virus type 1. J Virol 0:1-.. Matloubian, M., R. J. Concepcion, and R. Ahmed. 1. CD+ T cells are required to sustain CD+ cytotoxic T-cell responses during chronic viral infection. J Virol :0-. Downloaded from on September 1, 01 by guest

30 Migueles, S. A., M. S. Sabbaghian, W. L. Shupert, M. P. Bettinotti, F. M. Marincola, L. Martino, C. W. Hallahan, S. M. Selig, D. Schwartz, J. Sullivan, and M. Connors HLA B*01 is highly associated with restriction of virus replication in a subgroup of HIV-infected long term nonprogressors. Proc Natl Acad Sci U S A : Miura, T., M. A. Brockman, A. Schneidewind, M. Lobritz, F. Pereyra, A. Rathod, B. L. Block, Z. L. Brumme, C. J. Brumme, B. Baker, A. C. Rothchild, B. Li, A. Trocha, E. Cutrell, N. Frahm, C. Brander, I. Toth, E. J. Arts, T. M. Allen, and B. D. Walker. 00. HLA-B/B*01 human immunodeficiency virus type 1 elite controllers select for rare gag variants associated with reduced viral replication capacity and strong cytotoxic T-lymphocyte [corrected] recognition. J Virol :-. 1. Nakamura, Y., M. Kameoka, M. Tobiume, M. Kaya, K. Ohki, T. Yamada, and K. Ikuta. 1. A chain section containing epitopes for cytotoxic T, B and helper T cells within a highly conserved region found in the human immunodeficiency virus type 1 Gag protein. Vaccine 1:-.. Nishino, Y., K. Ohki, T. Kimura, S. Morikawa, T. Mikami, and K. Ikuta.. Major core proteins, ps, of human, simian, and feline immunodeficiency viruses are partly expressed on the surface of the virus-infected cells. Vaccine :-.. Ogg, G. S., X. Jin, S. Bonhoeffer, P. R. Dunbar, M. A. Nowak, S. Monard, J. P. Segal, Y. Cao, S. L. Rowland-Jones, V. Cerundolo, A. Hurley, M. Markowitz, D. D. Ho, D. F. Nixon, and A. J. McMichael. 1. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science :-.. Pereyra, F., M. M. Addo, D. E. Kaufmann, Y. Liu, T. Miura, A. Rathod, B. Baker, A. Trocha, R. Rosenberg, E. Mackey, P. Ueda, Z. Lu, D. Cohen, T. Wrin, C. J. Downloaded from on September 1, 01 by guest 0

31 Petropoulos, E. S. Rosenberg, and B. D. Walker. 00. Genetic and immunologic heterogeneity among persons who control HIV infection in the absence of therapy. J Infect Dis 1:-1.. Potter, S. J., C. Lacabaratz, O. Lambotte, S. Perez-Patrigeon, B. Vingert, M. Sinet, J. H. Colle, A. Urrutia, D. Scott-Algara, F. Boufassa, J. F. Delfraissy, J. Theze, A. Venet, and L. A. Chakrabarti. 00. Preserved central memory and activated effector memory CD+ T-cell subsets in human immunodeficiency virus controllers: an ANRS EP study. J Virol 1:0-1.. Rosenberg, E. S., J. M. Billingsley, A. M. Caliendo, S. L. Boswell, P. E. Sax, S. A. Kalams, and B. D. Walker. 1. Vigorous HIV-1-specific CD+ T cell responses associated with control of viremia. Science :1-0.. Schneidewind, A., M. A. Brockman, J. Sidney, Y. E. Wang, H. Chen, T. J. Suscovich, B. Li, R. I. Adam, R. L. Allgaier, B. R. Mothe, T. Kuntzen, C. Oniangue- Ndza, A. Trocha, X. G. Yu, C. Brander, A. Sette, B. D. Walker, and T. M. Allen. 00. Structural and functional constraints limit options for cytotoxic T-lymphocyte escape in the immunodominant HLA-B-restricted epitope in human immunodeficiency virus type 1 capsid. J Virol :-0.. Schneidewind, A., M. A. Brockman, R. Yang, R. I. Adam, B. Li, S. Le Gall, C. R. Rinaldo, S. L. Craggs, R. L. Allgaier, K. A. Power, T. Kuntzen, C. S. Tung, M. X. LaBute, S. M. Mueller, T. Harrer, A. J. McMichael, P. J. Goulder, C. Aiken, C. Brander, A. D. Kelleher, and T. M. Allen. 00. Escape from the dominant HLA-B- restricted cytotoxic T-lymphocyte response in Gag is associated with a dramatic reduction in human immunodeficiency virus type 1 replication. J Virol 1:-. Downloaded from on September 1, 01 by guest 1

32 Shacklett, B. L., J. W. Critchfield, and D. Lemongello. 00. Isolating mucosal lymphocytes from biopsy tissue for cellular immunology assays. Methods Mol Biol :-. 0. Shacklett, B. L., J. W. Critchfield, and D. Lemongello. 00. Quantifying HIV-1- specific CD (+) T-cell responses using ELISPOT and cytokine flow cytometry. Methods Mol Biol :-. 1. Shacklett, B. L., O. O. Yang, M. A. Hausner, J. Elliott, L. E. Hultin, C. Price, M. Fuerst, J. Matud, P. Hultin, C. A. Cox, J. Ibarrondo, J. T. Wong, D. F. Nixon, P. A. Anton, and B. D. Jamieson. 00. Optimization of methods to assess human mucosal T- cell responses to HIV infection and vaccination. Journal of Immunological Methods :1-1.. Southwood, S., J. Sidney, A. Kondo, M. F. del Guercio, E. Appella, S. Hoffman, R. T. Kubo, R. W. Chesnut, H. M. Grey, and A. Sette. 1. Several common HLA-DR types share largely overlapping peptide binding repertoires. J Immunol 10:-.. Thursz, M. R., D. Kwiatkowski, C. E. Allsopp, B. M. Greenwood, H. C. Thomas, and A. V. Hill. 1. Association between an MHC class II allele and clearance of hepatitis B virus in the Gambia. N Engl J Med :-.. Vingert, B., S. Perez-Patrigeon, P. Jeannin, O. Lambotte, F. Boufassa, F. Lemaitre, W. W. Kwok, I. Theodorou, J. F. Delfraissy, J. Theze, and L. A. Chakrabarti. 0. HIV Controller CD+ T Cells Respond to Minimal Amounts of Gag Antigen Due to High TCR Avidity. PLoS Pathog :e000.. Vyakarnam, A., D. Sidebottom, S. Murad, J. A. Underhill, P. J. Easterbrook, A. G. Dalgleish, and M. Peakman. 00. Possession of human leucocyte antigen DQ alleles Downloaded from on September 1, 01 by guest

33 1 and the rate of CD T-cell decline in human immunodeficiency virus-1 infection. Immunology :-.. Wilson, J. D., N. Imami, A. Watkins, J. Gill, P. Hay, B. Gazzard, M. Westby, and F. M. Gotch Loss of CD+ T cell proliferative ability but not loss of human immunodeficiency virus type 1 specificity equates with progression to disease. J Infect Dis :-.. Wong, J. T., A. A. Eylath, I. Ghobrial, and R. B. Colvin.. The mechanism of anti-cd monoclonal antibodies. Mediation of cytolysis by inter-t cell bridging. Transplantation 0:-.. Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M. Suresh, J. D. Altman, and R. Ahmed. 1. Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med 1:0-. Downloaded from on September 1, 01 by guest

34 FIGURE LEGENDS Figure 1. HIV Gag-specific CD+ T-cell responses in rectal mucosa (left, A thru C) and peripheral blood (right, B thru F). Panels A and D show the total percentage of CD+ T-cells capable of producing IFN, IL-, MIP-1, and/or TNF in response to HIV Gag stimulation in rectal mucosa (EC vs. HAART p=0.0) (A) and peripheral blood (D). Panels B and C show significant differences in CD+ T-cell cytokine/chemokine expression between groups in rectal mucosa. (B): IFN- (EC vs. NC p=0.01, EC vs. HAART p=0.0, VC vs. NC p=0.000, VC vs. HAART p=0.00); (C): MIP-1 EC vs. NC p=0.00, EC vs. HAART p=0.01, VC vs. NC p=0.0). Panels E and F show significant differences in CD+ T-cell cytokine expression between groups in peripheral blood. (E): IL- (EC vs. NC p=0.00, EC vs. HAART p=0.01, VC vs. NC p=0.00, VC vs. HAART p=0.001). (F): TNF VC vs. NC p=0.01, VC vs. HAART p=0.00). Horizontal bars represent the median response for each group. *p<0.0, **p<0.01, ***<p< Figure. HIV Gag-specific CD+ T-cell polyfunctional responses in rectal mucosa and peripheral blood across subject groups. CD+ T-cell polyfunctional responses in the rectal mucosa (A) and peripheral blood (B) of controllers, non-controllers, and HAART-suppressed individuals. Colors in the pie charts represent -function cells (black), -function cells (purple), -function cells (dark blue), and 1-function cells (light blue). Numbers in the pie charts are the median total CD+ T-cell response within each subject group. The bar charts below show the responses in controllers (blue), non-controllers (red), and HAART suppressed individuals (green) Downloaded from on September 1, 01 by guest

35 broken down into individual functional categories using inter-quartile ranges. Asterisks above the bar chart show significant differences (p<0.0; Wilcoxon Rank test) between controllers and non-controllers (red asterisks) or subjects on HAART (green asterisks) Figure. HLA class II alleles in study cohort. The frequency of controllers (blue bars) and non-controllers (red bars) in the current study who have HLA-DRB1* alone, HLA- DRBQ1*0 alone, the combined HLA-DRB1*/HLA-DQB1*0 haplotype, and those who have other class II HLA alleles not previously stated. Figure. Total HIV Gag-specific CD+ responses in the rectal mucosa of individuals with or without HLA-DRB1* and/or HLA-DQB1*0. Percentage of CD+ T-cells responding in any way (IFN, IL-, MIP-1, and/or TNF ) to HIV Gag stimulation in controllers with or without HLA-DRB1* and/or HLA-DQB1*0 and in non-controllers lacking these alleles. (Too few non-controllers possessed either of these alleles for a meaningful comparison). Horizontal bars represent the median response in each group. *p <0.0. Figure. Polyfunctional HIV Gag-specific CD+ T-cell responses in the rectal mucosa of controllers with or without HLA-DRB1* and/or HLA-DQB1*0. Colors in the pie charts represent -function cells (black), -function cells (purple), -function cells (dark blue), and 1- function cells (light blue). Numbers in the pie charts are the median total CD+ T-cell response within each group. The bar charts below show the responses in controllers with HLA-DQB1*0 alone (blue), the HLA-DRB1*/HLA-DQB1*0 haplotype (red), or without either allele (green) broken down into individual functional categories using inter-quartile ranges. Blue Downloaded from on September 1, 01 by guest

36 asterisk above the bar chart shows a significant difference (p<0.0) between those with the HLA- DRB1*/HLA-DQB1*0 haplotype and those with HLA-DQB1*0 alone Figure. Polyfunctional mucosal CD+ T-cell responses correlate with the magnitude and polyfunctionality of the CD+ T-cell response. Relationship between the magnitude of the total CD+ T-cell response vs. the magnitude of the total CD+ T-cell response (A), the frequency of polyfunctional CD+ T-cells vs. the magnitude of the total CD+ T-cell response (B), and the frequency of the polyfunctional CD+ T-cells vs. the frequency of the polyfunctional CD+ T-cell response (C) in rectal mucosa of controllers (blue), and non- controllers (red). Polyfunctional CD+ T-cells are defined as exhibiting or functional responses; polyfunctional CD+ T-cell are those which express, or functions. Linear regression lines and Spearman s r correlations were calculated using the combined controller and non-controller data set. Table 1: CD+ T-cell Epitope Mapping in four controllers with HLA-DRB1* and/or HLA-DQB1*0. Subject (Tissue a ) Class II HLA Gag p Gag p Gag p Gag p1 Nef Nef aa 1-1 aa 1- aa -0 aa - aa 1- aa 1-1 (PB) DRB1*/DQB1* (RM) DQB1* (RM) DQB1* (PB/RMC) DQB1* / 1 0 Downloaded from on September 1, 01 by guest 1 a PB = peripheral blood, RM = Rectal Mucosa 1 0 Values indicate magnitude of the CD+ IFN response to the given peptide in spot forming cells/million (SFC/million).

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