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Hiv resistance

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Promettenti i risultati di un nuovo studio sulla resistenza al virus dell'HIV. Un team internazionale di ricercatori guidati dal Bruce Walker del Ragon Institute in Massachusetts, USA, ha infatti …

Promettenti i risultati di un nuovo studio sulla resistenza al virus dell'HIV. Un team internazionale di ricercatori guidati dal Bruce Walker del Ragon Institute in Massachusetts, USA, ha infatti scoperto come mai alcuni individui (circa 1 su 300) presentano la capacità innata di controllare l'HIV senza fare ricorso ai farmaci.


Lo studio, pubblicato su Nature Immunology, mostra come questi individui presentino un ceppo specifico di cellule immunitarie 'killer', molto efficaci contro il virus. "Ogni essere umano presenta delle cellule dette linfociti T citotossici (CTL).

Tuttavia, nonostante vengano prodotte in grandissime quantità durante un'infezione da HIV, queste non sono efficaci contro il virus; a meno che non appartengano a uno specifico ceppo che presenta un recettore in grado di riconoscere il virus" ha spiegato Walker.

"Finora, la produzione di un vaccino contro l'HIV è stata inefficace perchè si sono prodotte cellule T, ma del tipo sbagliato. Il prossimo passo è ora capire cosa c'è in questi recettori da renderli così efficaci. Ogni nuova scoperta di questo tipo ci porta un passo più vicini alla sconfitta dell'AIDS".

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  • 1. Articles TCR clonotypes modulate the protective effect of HLA class I molecules in HIV-1 infection Huabiao Chen1,2,7, Zaza M Ndhlovu1,2,7, Dongfang Liu1, Lindsay C Porter1, Justin W Fang1, Sam Darko3, Mark A Brockman1,4, Toshiyuki Miura1,5, Zabrina L Brumme1,4, Arne Schneidewind1,6, Alicja Piechocka-Trocha1,2, Kevin T Cesa1, Jennifer Sela1, Thai D Cung1, Ildiko Toth1, Florencia Pereyra1, Xu G Yu1, Daniel C Douek3, Daniel E Kaufmann1, Todd M Allen1 & Bruce D Walker1,2© 2012 Nature America, Inc. All rights reserved. The human leukocyte antigens HLA-B*27 and HLA-B*57 are associated with protection against progression of disease that results from infection with human immunodeficiency virus type 1 (HIV-1), yet most people with alleles encoding HLA-B*27 and HLA-B*57 are unable to control HIV-1. Here we found that HLA-B*27-restricted CD8 + T cells in people able to control infection with HIV-1 (controllers) and those who progress to disease after infection with HIV-1 (progressors) differed in their ability to inhibit viral replication through targeting of the immunodominant epitope of group-associated antigen (Gag) of HIV-1. This was associated with distinct T cell antigen receptor (TCR) clonotypes, characterized by superior control of HIV-1 replication in vitro, greater cross-reactivity to epitope variants and enhanced loading and delivery of perforin. We also observed clonotype-specific differences in antiviral efficacy for an immunodominant HLA-B*57-restricted response in controllers and progressors. Thus, the efficacy of such so-called ‘protective alleles’ is modulated by specific TCR clonotypes selected during natural infection, which provides a functional explanation for divergent HIV-1 outcomes. A subset of people infected with human immunodeficiency virus type 1 of perforin14, specific targeting of conserved viral regions15,16, immu- (HIV-1), called ‘elite controllers’ here, are distinguished by their abil- noregulatory mechanisms17–19, concurrent responses to multiple viral ity to maintain a state of apparently durable control of HIV-1 replica- epitopes restricted by different HLA alleles20, CD8+ T cell–associated tion without the need for antiviral therapy1,2. Viral control is linked mutations that impair viral fitness21,22 and escape from the immune to the expression of certain alleles encoding human leukocyte antigen response23. Many studies have also suggested that properties of the (HLA) class I molecules3–5, particularly HLA-B*57, HLA-B*27 and interaction among the T cell antigen receptor (TCR), viral peptide HLA-B*5801, which suggests an immunological basis related to the and major histocompatibility complex may be involved24,25. However, function of CD8+ T cells. A published genome-wide association study the extent to which any of these factors influences the antiviral effi- has indicated that the nature of the HLA–viral peptide interaction is cacy of the human immune response, as reflected by in vivo viral load, the main factor that modulates durable control of infection with HIV-1 remains unclear, in part because of a lack of direct comparison of viral in the absence of antiretroviral therapy6. However, the mechanistic epitope–specific CD8+ T cell responses in people able to control infec- basis for the association remains unclear, and it is also unclear why tion with HIV-1 (controllers) and those who progress to ­disease after most people with so-called ‘protective HLA alleles’ actually develop infection with HIV-1 (progressors), sequence diversity in epitopes of progressive disease. HIV-1 targeted by the immune system that leads to escape from the Many studies have attempted to define quantitative and qualita- immune response, and the potential confounding effect of the target- tive differences in CD8+ T cell responses that may be associated with ing of multiple epitopes via diverse HLA molecules. different outcomes in terms of control of viremia by the immune To address those limitations, we focused on HIV-1-infected people response. Simple quantitative measures have shown little correla- who express HLA-B*2705, which represents a situation in which the tion with viral control7,8, which suggests that qualitative features of immune response is mostly if not exclusively mediated by targeting CD8+ T cells may modulate efficacy. Factors that potentially modu- of a single epitope, KK10, of the group-associated antigen (Gag) pro- late protective HLA-associated CD8+ T cell responses include, among tein p24 (sequence, KRWIILGLNK; amino acids 263–272)23. From a others, polyfunctionality9, antigen sensitivity or functional avidity10,11, large, well-pedigreed cohort26, we specifically selected five control- proliferative capacity12, loading of lytic granules13, ex vivo expression lers and five progressors who express HLA-B*2705 for whom the 1Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Boston, Massachusetts, USA. 2Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. 3Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, Maryland, USA. 4Faculty of Health Sciences, Simon Fraser University, Burnaby, Canada. 5Institute of Medical Science, University of Tokyo, Tokyo, Japan. 6Department of Internal Medicine, University Hospital Regensburg, Regensburg, Germany. 7These authors contributed equally to this work. Correspondence should be addressed to B.D.W. (bwalker@partners.org). Received 15 March; accepted 11 May; published online 10 June 2012; doi:10.1038/ni.2342 nature immunology  aDVANCE ONLINE PUBLICATION
  • 2. Articles c ­ irculating viruses and cellular proviruses all had wild-type sequences dominance of single clonotypes in each person, we did not observe in the dominant KK10 epitope targeted by this molecule at the time ‘preferential’ use of a particular complementarity-determining of analysis. This allowed comparative assessment of adaptive CD8 + region 3 (CDR3) motif among the various KK10-specific CD8 + T cell responses in people in whom the dominant CD8+ T cell response T cell clonotypes (Table 1). Indeed, of the clonotypes identified, is to a single epitope of Gag in a setting in which the viral load and, by only two clonotypes were the same in two different people (subjects inference, the degree of CD8+ T cell–­mediated control and not HLA CTR22 and CR420 had the same clonotype (TCRBV27TCRBJ1-1) allele or sequence variation in the targeted viral epitope, were the main and CDR3 sequence (CASSGGRRAF); and subjects CTR22 and variables. We made a detailed analysis of the epitope-­specific CD8+ CR540 had the same clonotype (TCRBV21TCRBJ2-7) and CDR3 T cell responses and then extended that to include the dominant sequence (CASTNRGSEQY)), and only one subject (CR420) had a HLA-B*57-restricted epitope TW10 of Gag (sequence, TSTLQEQIGW; TCRBV4-3TCRBJ1-3 clonotype similar to that reported before in amino acids 240–49). Our data indicated that HLA-B*27- and HLA- people who express HLA-B*27 in whom viral loads vary between B*57-restricted CD8+ T cells targeting the same epitopes in elite 1,880 and 202,590 RNA copies per milliliter of plasma28,30,31. These controllers and progressors were distinctly different, on the basis of data indicated that KK10-specific CD8 + T cells were quantitatively potency and cross-reactivity of TCR recognition of HIV-1 and viral similar but demonstrated considerable heterogeneity in TCR use variants, which was in turn related to specific TCR clonotypes selected among people who targeted a genetically identical epitope through a during natural infection. genetically identical HLA allele, in whom we observed considerable differences in viral load. RESULTS Quantitative measures of KK10-specific T cells Functional characteristics of KK10-specific T cells Many reports have suggested qualitative features of CD8+ T cells© 2012 Nature America, Inc. All rights reserved. Published studies have shown that people who express HLA-B*2705 generate an immunodominant response to the KK10 epitope of associated with viral control. One such measure is antigen sensitivity Gag p24; targeting of this epitope is critical to long-term control in (often called ‘functional avidity’)10,11,31, and HLA-B*27 is character- these people23,27. Although escape from this response leads to accel- ized by T cell responses of high sensitivity28. However, whether anti- erated disease progression, variable viral loads and rates of CD4+ gen sensitivity varies with viral load in people who express HLA-B*27 T cell decrease are already observed before escape occurs23. Given and contain virus with the wild-type epitope has not been determined, the importance of the KK10-specific CD8+ T cell response to dis- to our knowledge. We next assessed antigen sensitivity in each of our ease control, we reasoned that the identification of both controllers subjects by examining (by ELISPOT) IFN-γ responses at limiting con- and progressors with the wild-type KK10 sequence would afford the centrations of KK10. There was no difference between controllers and opportunity to define the characteristics of effective and ineffective progressors in the sensitizing dose of peptides needed to yield 50% CD8+ T cell responses independently of any confounding effects of maximal effector-cell triggering of IFN-γ production by peripheral escape from the immune response. blood mononuclear cells (PBMCs; Fig. 2a). These results, in which We recruited five elite controllers and five progressors for detailed all responses were detected in the presence of wild-type KK10 and studies; all expressed HLA-B*2705 and had autologous virus with therefore were not confounded by potentially cross-reactive responses the wild-type KK10 epitope in HIV-1 RNA obtained from plasma induced by substitutions in the epitope sequence, were consistent with and HIV-1 DNA obtained from cells (Table 1). We selected these published reports showing that most clones specific for KK10 have subjects from a larger population that included people in whom vari- similar antigen sensitivity10. ants in this epitope were present in vivo. Viral loads in the controllers We next examined polyfunctionality ex vivo, including the ability of were all 50 RNA copies per milliliter of plasma and ranged as low HLA-B*2705-restricted, KK10-specific CD8+ T cells to simultaneously as 0.2 RNA copies per milliliter of plasma in those for whom we did produce the effector cytokines and chemokines IFN-γ, interleukin 2 more sensitive testing. Progressors had viral loads that ranged from (IL-2), tumor necrosis factor and CCL4 (MIP-1β) and to release cyto- 4,073 to 22,094 RNA copies per milliliter of plasma (Table 1). We toxic factors by monitoring expression of the degranulation marker confirmed the dominance of the KK10-specific responses in these CD107a after stimulation with KK10. Published population studies subjects by fine mapping with HLA-restricted optimal peptides have shown that the ability of CD8+ T cells to produce four or five in enzyme-linked immunospot (ELISPOT) assays of interferon-γ cytokines and chemokines concurrently is associated with HIV-1 (IFN-γ; data not shown). We detected strong KK10-specific CD8+ controllers9, and although some epitope-specific responses have T cell responses, as defined by tetramer analysis, in the peripheral been evaluated in this manner10,28,32, to our knowledge this has not blood of both controllers and progressors (Fig. 1a). Comparison of been examined for HLA-B*27-restricted responses in people known controllers and progressors showed there was no significant difference to have the wild-type virus, in whom the inducing antigen is thus between these two groups in the proportion of KK10-specific cells in the same. When we did this analysis for the KK10 epitope (Fig. 2b), peripheral blood, as quantified by staining with a HLA-B*27–KK10 cells that secreted CCL4 (MIP-1β) dominated the response in both tetramer (Fig. 1b), or ELISPOT assay of IFN-γ (Fig. 1c), despite their progressors and controllers, consistent with published findings9. substantial differences in plasma viremia. Pairwise comparisons showed several subsets in the two groups with As the TCR is a key structure that defines antigen recognition, we significantly different functional profiles. For example, cells with dual next evaluated whether differences in TCR use might be associated expression of both IFN-γ and CCL4 (MIP-1β) were of significantly with different abilities to control viremia, due to TCR clonotypes greater frequency in controllers, whereas cells with dual expression with heterogeneous antiviral potential. We sorted cells positive for of both IFN-γ and tumor necrosis factor were of significantly greater the HLA-B*27–KK10 tetramer and sequenced their genes encoding frequency in progressors (Fig. 2b). However, although controllers the TCR β-chain variable (V) region (‘TCRBV’) and TCR β-chain showed enrichment for cells with expression of more than three joining (J) segment (’TCRBJ’). Consistent with the findings of other cytokines and chemokines, this did not reach statistical significance studies28,29, we found considerable diversity of clonotype recruit- (P = 0.1) and these cells made up only a small subset of the total KK10- ment in all KK10-specific CD8+ T cell populations, and despite the specific CD8+ T cell response. aDVANCE ONLINE PUBLICATION  nature immunology
  • 3. Articles Published studies have shown that chronic infection with HIV-1 in the controllers and progressors (Supplementary Fig. 1). Moreover, skews the maturation of HIV-1-specific CD8+ T cells toward devel- we observed that HIV-1-infected GXR cells (a green fluorescent opment into preterminally differentiated cells with poor cytotoxic protein (GFP) reporter T cell line, derived from the human lympho­ activity33. We stained peripheral blood cells for CD27 and CD45RA as blastoid CD4+ T cell line CEM, that fluoresces green after infection described34 to phenotypically distinguish four distinct subpopulations with HIV-1) expressing HLA-B*2705 (refs. 35,36) stimulated the pro- of KK10-specific cells. We detected similar proportions of KK10- liferation of KK10-specific cells to a similar degree in both groups specific cells with a central memory phenotype (KK10+CD27+CD45RA−), (Fig. 2c,d). Together these data indicated that the superior control of effector memory phenotype (KK10+CD27−CD45RA−) or terminally wild-type viremia in these elite controllers relative to that in chronic differentiated effector memory phenotype (KK10+CD27−CD45RA+) progressors expressing HLA-B*27, all of whom were ­treatment naive, Table 1  Clonal analysis of HLA-B*2705-restricted KK10-specific CD8+ T cell populations Subject and HLA KK10 Tet+ cells (%) pVL (copies/ml) CD4 count TCR β-chain and CDR3 Frequency EC CTR22 KRWIILGLNK 3.53 50 337 Vβ20-CSARDRTRANYGYT-J1.2 22/25 HLA-A*0201/0201 KRWIILGLNK Vβ27-CASSGGRRAF-J1.1 1/25 HLA-B*2705/5101 Vβ5.1-CASSSPDRTYGYT-J1.2 1/25 HLA-C*0102/1402 Vβ21-CASTNRGSEQY-J2.7 1/25 EC CTR40 KRWIILGLNK 1.56 50 1,415 Vβ7.2-CASSLSGRWSTDTQY-J2.3 11/21 HLA-A*0201/0201 KRWIILGLNK Vβ7.2-CASSLEGRYSNQPQH-J1.5 4/21 HLA-B*2705/5701 Vβ7.6-CASSLGTGKVEGYT-J1.2 1/21© 2012 Nature America, Inc. All rights reserved. HLA-C*0102/0602 Vβ7.9-CASSPEGPRAIEQF-J2.1 2/21 Vβ15-CATSRELTGGPSYEQY-J2.7 1/21 Vβ7.8-CASSQARASHLI-J1.4 1/21 Vβ9-CASSEDRDTEAF-J1.1 1/21 EC CTR203 KRWIILGLNK 9.99 1.1 411 Vβ25.1-CASSEADFEAF-J1.1 13/37 HLA-A*2601/6801 KRWIILGLNK Vβ18-CASSPGQFSHEQY-J2.7 11/37 HLA-B*0702/2705 Vβ27-CASSARTGELF-J2.2 2/37 HLA-C*0702/0202 Vβ20.1-CSARDGGEQY-J2.7 6/37 Vβ9-CASSPLGNSGNTIY-J1.3 2/37 Vβ7.9-CASSLDRLEQF-J2.1 3/37 EC FW56 KRWIILGLNK 7.08 0.2 937 Vβ4.3-CASRPGLASNEQF-J2.1 16/31 HLA-A*0201/0301 KRWIILGLNK Vβ6.5-CASRPGQGATEAF-J1.1 10/31 HLA-B*1501/2705 Vβ20.1-CSARDRGTREVADNYGYT-J1.2 3/31 HLA-C*0304/0102 Vβ15-CATSETGTTLEQY-J2.7 2/31 EC 13587 KRWIILGLNK 1.35 50 994 Vβ7.9-CASSRDSNEQF-J2.1 15/27 HLA-A*0206/3201 NA Vβ20.1-CSAREGLAGVLYEQY-J2.7 1/27 HLA-B*1524/2705 Vβ28-CASSSSGGAGDTQY-J2.3 1/27 HLA-C*0202/0304 Vβ20.1-CSAPTTEVAGSTDTQY-J2.3 1/27 Vβ5.4-CASSLTNLGEQY-J2.7 3/27 Vβ27-CASSRTTGELF-J2.2 6/27 CP CR540 KRWIILGLNK 6.85 17,300 505 Vβ2-CASSAGPGQYGNTIY-J1.3 16/34 HLA-A*0201/0201 KRWIILGLNK Vβ5.6-CASGGGTVYEQY-J2.7 8/34 HLA-B*2705/4402 Vβ21-CASTNRGSEQY-J2.7 6/34 HLA-C*0102/0501 Vβ4.3-CASSPGTNAYEQY-J2.7 4/34 CP CR420 KRWIILGLNK 2.93 13,900 582 Vβ20.1-CSAREGVEGYT-J1.2 21/30 HLA-A*0201/1101 KRWIILGLNK Vβ27-CASSGGRRAF-J1.1 6/30 HLA-B*2705/4402 Vβ4.3-CASSQGSGSGNTIY-J1.3 1/30 HLA-C*0102/0501 Vβ4.3-CASSQVLRGVYGYT-J1.2 1/30 Vβ5.4-CASSLLAGGTDTQY-J2.3 1/30 CP CR338 KRWIILGLNK 2.2 6,800 870 Vβ27-CASSPRTGELF-J2.2 16/29 HLA-A*0101/0201 KRWIILGLNK Vβ27-CASSQRTGELF-J2.2 3/29 HLA-B*0702/2705 Vβ27-CASSRATGELF-J2.2 4/29 HLA-C*0102/0702 Vβ5.1-CASSLEGGANL-J1.2 1/29 Vβ6.4-CASSVVRGNPNEQF-J2.1 5/29 CP 8222 KRWIILGLNK 1.9 4,073 269 Vβ27-CASSGSNLEAF-J1.1 21/30 HLA-A*0201/2902 KRWIILGLNK Vβ7.9-CASSPLGVRAYEQY-J2.7 3/30 HLA-B*2705/4403 Vβ4.3-CASSPGTSTYEQY-J2.7 6/30 HLA-C*0102/1601 CP FEN33 KRWIILGLNK 6.4 22,094 603 Vβ7.9-CASSLAGGDSYEQY-J2.7 21/23 HLA-A*0201/1101 KRWIILGLNK Vβ27-CASSGGVFYGYT-J1.2 1/23 HLA-B*2705/5101 Vβ19-CATLGGFPDGYT-J1.2 1/23 HLA-C*0102/0303 Clonal analysis includes HLA subtypes, presented below subject identifier (far left); KK10 epitope sequence of the virus in plasma (top) and provirus in PBMCs (bottom); frequency of tetramer-positive (Tet+) cells among total CD8+ T cells; plasma viral load (pVL), in RNA copies per milliliter of plasma; CD4+ T cells (CD4 count) per microliter of plasma (cells/µl); TCR β-chain variable region (Vβ), CDR3 sequence and β-chain joining region (J); and frequency of clonotype among KK10-specific CD8+ T cell populations (far right). P = 0.4206, CD4 count, controllers versus progressors (Mann-Whitney test). Data are from one experiment (time point) per subject. nature immunology  aDVANCE ONLINE PUBLICATION
  • 4. Articles Figure 1  Quantification of KK10-specific CD8+ a b c T cell responses. (a) Flow cytometry of CD8+ EC FW56 CP CR540 11 * 5 * SFCs 103 (per 106 PBMCs) (VL = 0.2) (VL = 17,300) 10 T cells from elite controller (EC) FW56 and 9 5 4 Tet CD8 cells (%) 10 6.85 chronic progressor (CP) CR540, assessed by 7.08 8 10 4 7 staining with HLA class I tetramers. Numbers 6 3 adjacent to outlined areas indicate percent 3 + 10 5 4 2 CD8+ T cells specific for the HLA-B*27–KK10 10 2 3 + CD8 0 tetramer (KK10 tet). (b) Frequency of cells 2 3 4 5 2 1 0 10 10 10 10 1 positive for the HLA-B*27–KK10 tetramer KK10 tet 0 0 among bulk CD8+ T cells from controllers EC CP EC CP (n = 5) and progressors (n = 5). *P = 0.7531 (Mann-Whitney test). (c) Responses of KK10-specific CD8+ T cells among PBMCs from controllers (n = 5) and progressors (n = 5), analyzed ex vivo by ELISPOT assay of IFN-γ after stimulation with KK10 peptide and calculated as spot-forming cells (SFC) per 1 × 10 6 PBMCs. *P = 0.7383 (Mann- Whitney test). Each symbol represents an individual subject; small horizontal lines indicate the mean. Data are representative of two experiments. was not reflected in the quantitative measurement of KK10-specific resulted in three to four logs less production of p24 antigen at day 7 CD8+ T cells or qualitative assessment of the functional avidity, in culture, whereas viral inhibition was over 90% less after the addi- cytokine secretion, proliferative capacity or differentiation pheno- tion of cell samples from which KK10-specific CD8+ T cells were types of these cells. depleted (Fig. 3a). This confirmed that the main immune control was mediated by the KK10-specific response in each of these subjects Neutralization of virus by KK10-specific T cells and showed that all of the HLA-B*27+ controllers were able to limit viral replication in vitro.© 2012 Nature America, Inc. All rights reserved. Having shown that the measures of responses to the immunodomi- nant KK10 Gag epitope noted above did not differentiate control- We next sought to assess the antiviral function of CD8+ T cells from lers from progressors, we next evaluated the functional ability of the progressors. However, outgrowth of autologous virus in HIV-1 responses to this epitope to inhibit HIV-1 replication in vitro37. We progressors complicated the virus-inhibition assay when we used limited our initial analysis to the elite controllers, in whom outgrowth autologous CD4+ T cells as target cells (data not shown). We therefore of autologous virus in CD4+ T cells is delayed considerably38, which sought to confirm the utility of an assay based on the use of GXR cells allowed us to use controlled inocula of exogenous HIV-1 isolates encoding HLA-B*2705 as target cells35,36. When we used those target to infect these cells and measure the ability of defined numbers of cells together with bulk CD8+ T cells from controllers, we noted con- CD8+ T cells to inhibit viral replication. We included in this analysis siderable inhibition of replication; moreover, there was nearly complete all five HLA-B*2705+ elite controllers demonstrated to have wild- loss of viral inhibition after depletion of KK10-specific cells from bulk type KK-10 epitope sequences in virus in their plasma and provirus CD8+ T cell populations (Fig. 3b). In contrast, we observed no viral in their PBMCs (Table 1) and assessed the antiviral ability of bulk inhibition by bulk CD8+ T cells from HIV-1− people in the presence of CD8+ T cells and CD8+ T cell samples depleted of KK10-specific cells HIV-1-infected autologous CD4+ T cells or HLA-B*2705-expressing to inhibit viral replication in autologous CD4+ T cells by measuring GXR cells. In addition, we observed no inhibition by bulk CD8+ the production of p24 antigen in the supernatant over 7 d (ref. 35). T cells from controllers in the presence of HLA-B*2705− GXR cells The addition of bulk CD8+ T cells to HIV-1-infected CD4+ T cells after infection with the same virus (data not shown). a b c EC FW56 CP CR540 50 10 5 38.4 61.6 31.9 68.1 * Cytokine-secreting KK10+ cells (%) 40 EC 4 10 30 CP 3 100 20 10 CTR22 * 2 CTR40 10 * IFN-γ production (%) CTR203 Tet 75 0 0 0 0 0 FW56 10 * 2 3 4 5 13587 0 10 10 10 10 50 CR338 CFSE CR420 25 CR540 5 d 50 * Proliferation (%) FEN33 8222 40 0 0 5 4 3 2 1 1 2 3 4 5 6 7 IFN-γ 30 10 10 10 10 10 10 10 Peptide concentration (pM) TNF 20 IL-2 CD107a 10 CCL4 EC CP Figure 2  Functional characteristics of KK10-specific CD8 + T cells. (a) Functional avidity of KK10-specific CD8 + T cells from various subjects (identifiers in key), assessed by peptide ‘titration’ of PBMCs in an ELISPOT assay of IFN-γ. P = 0.4678 (Mann-Whitney test). (b) Expression of IFN-γ, tumor necrosis factor (TNF), IL-2, CD107a and CCL4 (MIP-1β) by KK10-specific CD8 + T cells from HLA-B*2705 + elite controllers (n = 5) and chronic progressors (n = 5) after stimulation of PBMCs with KK10, presented as the proportion (bar height) of subpopulations of KK10-specific cells expressing various combinations (below graph) of effector molecules (numbers in colored rectangles along horizontal axis indicate number of effector molecules assessed in group of bars encompassed by rectangle).*P 0.05 (Mann-Whitney test). (c) Proliferation of KK10-specific CD8 + T cells at day 7 after stimulation of bulk CD8 + T cells from a controller (FW56) and a progressor (CR540) with HIV-1-infected, HLA-B*2705- expressing GXR cells, assessed by staining with the cytosolic dye CFSE (proliferation) and with the HLA-B*27–KK10 tetramer (KK10-specific cells). Numbers in quadrants indicate percent CFSE lo (proliferating) cells (top left) and CFSE hi (nonproliferating) cells (top right). (d) Proliferative capacity of KK10-specific CD8 + T cells from controllers (n = 5) and progressors (n = 5), measured by flow cytometry of CFSE intensity. Each symbol represents an individual subject; small horizontal lines indicate the mean. *P = 0.2222 (Mann-Whitney test). Data are representative of two experiments (error bars (b), s.d.). aDVANCE ONLINE PUBLICATION  nature immunology
  • 5. Articles Figure 3  Neutralization of virus ex vivo by KK10-specific CD8 + T cells. (a) Production of p24 antigen by autologous CD4 + T cells a CD4+ only CD4+ + virus + bulk CD8+ + KK10-depleted CD8+ EC FW56 106 left uninfected (CD4 + only; negative control) or infected with wild- type HIV-1 NL4-3 and assessed alone (CD4 + + virus; positive control) 106 10 5 p24 (pg/ml) or in the presence of bulk CD8 + T cells (+ bulk CD8 +) or CD8+ T cell 105 104 p24 (pg/ml) populations depleted of KK10-specific cells (+ KK10-depleted CD8 +), 104 3 10 presented for controller FW56 over 7 d (left) and for all controllers 103 (n = 5) and an HIV − subject at day 7 (right). (b) Viral replication in 102 102 HLA-B*2705-expressing GXR cells left uninfected (GXRB27 only) or 1 10 101 infected with HIV-1 and cultured alone (GXRB27 + virus) or in the 2 3 4 5 6 7 – 40 3 56 22 7 20 IV 58 TR FW TR presence of bulk or depleted CD8 + T cells (as in a), analyzed by flow H Time after infection (d) TR 13 C C C cytometry and presented as GFP + cells (infected GXR cells with viral replication) in controller FW56 over 7 d (left) and for all controllers b GXRB27 only GXRB27 + virus + bulk CD8+ + KK10-depleted CD8+ 20 (n = 5) and an HIV − subject at day 7 (right). (c) Viral replication in 20 EC FW56 HLA-B*2705-expressing GXR cells left uninfected or infected and 15 GFP+ cells (%) GFP+ cells (%) 15 cultured alone (as in b) or in the presence of bulk CD8 + T cells (as 10 in b), analyzed by flow cytometry and presented as GFP + cells from 10 1.5 progressor FEN33 over 7 d (left) and for all progressors (n = 5) and an 1.0 HIV− subject at day 7 (right). P 0.0001 (Mann-Whitney test). Data are 5 0.5 representative of two experiments. 0 0 2 3 4 5 6 7 7 – 58 22 40 3 56 20 IV Having shown that this assay provided evidence of active viral neu- 13 TR TR FW Time after infection (d) H TR C C C tralization by ex vivo KK10-specific CD8+ T cells in HLA-B*2705+ elite© 2012 Nature America, Inc. All rights reserved. controllers and that this assay was sensitive to KK10 epitope specificity c GXRB27 only GXRB27 + virus + bulk CD8+ CP FEN33 25 and HLA-B*2705 expression, we next evaluated CD8+ T cells from the 25 20 progressors. Infected GXR cells expressing HLA-B*2705 were inhi­ GFP+ cells (%) GFP+ cells (%) 20 bited by the addition of CD8+ T cells from progressors (Fig. 3c), but 15 15 to a much smaller degree than had been noted with cells from con- 10 10 trollers. We obtained this result despite the finding of no quantitative 5 differences in KK10-specific cells in controllers and progressors, as 5 shown by tetramer staining and ELISPOT assay for IFN-γ (Fig. 1). 0 0 2 3 4 5 6 7 – We next extended the studies reported above to determine the abil- 0 8 33 0 22 54 33 42 IV N 82 Time after infection (d) H R R R FE C C C ity of KK10-specific CD8+ T cell responses in controllers and pro- gressors to recognize viral variants known to arise in vivo that are able to escape the immune response36. Computational studies have capa­city in response to HIV-1-infected, HLA-B*2705-expressing suggested that protective HLA alleles are associated with enhanced GXR cells (Fig. 2c). We consistently observed the superiority of cross-reactivity25, but this has not been evaluated, to our knowledge, HIV-1-specific CD8 + T cells from elite controllers in antiviral in the context of a single allele encoding HLA class I and single viral efficacy against wild-type HIV-1 NL4-3 and viral variants when epitope in a comparison of controllers and progressors. For this, we extended these detailed studies to all the elite controllers and we analyzed by flow cytometry the recognition of wild-type HIV-1 chronic progressors (Fig. 4b). In addition, bulk CD8+ T cells from and viral variants by KK10-specific CD8+ T cells among bulk CD8+ HIV-1 − people (which included HLA-B*2705 + donors) did not T cells from controllers and progressors. We evaluated the proportion inhibit viral replication in HLA-B*2705-expressing GXR cells of GFP+ cells after infecting HLA-B*2705-expressing GXR cells. We (Fig. 4b). We obtained similar results in terms of killing efficacy again used the GXR cell assay system rather than autologous CD4+ against the same HIV-1-infected, HLA-B*2705-expressing GXR T cells to overcome the problem of outgrowth of autologous virus cells by standard chromium-release assays of cells from the control- from the chronic progressors and avoid potential variability of CD4+ lers and progressors (Fig. 4c). T cell responses among study subjects. Together these data indicated a clear distinction between elite We assessed the recognition of HIV-1 strain NL4-3 with the wild- controllers and chronic progressors in HLA-B*2705-restricted CD8+ type KK10 epitope, as well as HIV-1 NL4-3 variants with mutant T cells targeting the KK10 epitope based on the potency and KK10 sequences (Table 2), by KK10-specific CD8 + T cells from elite controllers and chronic progressors (results for HLA-B*2705 + Table 2  HLA-B*2705-restricted KK10 and HLA-B*57-restricted elite controller FW56 and chronic progressor CR540, Fig. 4a). We TW10 sequences found essentially complete inhibition of the replication of wild-type Viral isolate Epitope sequence HIV-1 NL4-3 and broad recognition of viral variants by KK10- KK10 wild-type KRWIILGLNK specific CD8+ T cells from controller FW56, as well as considerable L6M KRWIIMGLNK inhibition of the typical early L6M mutant virus, which we did not R2T KTWIILGLNK R2TL6M KTWIIMGLNK detect in this subject. In contrast, inhibition of both of these targets R2Q KQWIILGLNK by CD8+ T cells from progressor CR540 was present but minimal. R2QL6M KQWIIMGLNK Although peak infection with the other mutant viruses was less TW10 wild-type TSTLQEQIGW than that noted with wild-type virus or the L6M mutant virus, T242N TSNLQEQIGW G248A TSTLQEQIAW CD8+ T cells from controller FW56 inhibited all variants, whereas G248D TSTLQEQIDW CD8+ T cells from progressor CR540 were ineffective, even though Sequences of the HLA-B*2705-restricted KK10 epitope and HLA-B*57-restricted both subjects had similar proportions of KK10-­specific effector TW10 epitope of wild-type HIV-1 NL4-3 and its variants (mutant residues bolded). cells, as quantified by tetramer staining (Table 1), and proliferative Derived from the Los Alamos Immunology Database. nature immunology  aDVANCE ONLINE PUBLICATION
  • 6. Articles a 25 WT b EC CP c 70 WT L6M L6M 100 * – 60 R2T HIV R2T 20 * R2TL6M R2TL6M 50 R2Q Specific lysis (%) R2Q 75 GFP cells (%) R2QL6M R2QL6M 15 * Inhibition (%) 40 * * 50 * 30 + 10 20 5 25 10 0 0 0 + + + No CD8 cells FW56 CD8 cells CR540 CD8 cells WT L6M R2T R2TL6M R2Q R2QL6M CTR203 FW56 CR540 FEN33 Figure 4  Recognition of viral variants by KK10-specific CD8 + T cells. (a) Replication of wild-type HIV-1 NL4-3 (WT) and various HIV-1 NL4-3 variants (Table 2 and key) ex vivo in HLA-B*2705-expressing GXR cells alone (No CD8 + cells) or in the presence of CD8 + T cells isolated from a controller (FW56 CD8 + cells) or a progressor (CR540 CD8 + cells) at an effector cell/target cell ratio of 1:1, presented (as in Fig. 3b) as GFP+ cells at day 7 of culture. (b) Inhibition of the replication of wild-type HIV-1 NL4-3 and various HIV-1 NL4-3 variants (horizontal axis) ex vivo by CD8+ T cells from controllers (n = 5), progressors (n = 5) and HIV-1 − people (n = 12 total, including n = 4 HLA-B*2705 + donors). *P 0.0001 (Mann-Whitney test). (c) Lysis of live, HIV-infected (GFP +), HLA-B*2705-expressing GXR cells (sorted as viable infected cells by flow cytometry after infection for 5 d by virus in key) ex vivo by CD8+ T cells from controllers (CTR203 and FW56) and progressors (CR540 and FEN33) at an effector cell/target cell ratio of 10:1, assessed by a 4-hour chromium-release assay. Data are representative of two experiments. these people, we cloned the dominant clonotypes identified in vivo,© 2012 Nature America, Inc. All rights reserved. cross-reactivity of the recognition of cells infected with wild-type HIV-1 and HIV-1 containing naturally arising substitutions in the KK10 and in three of the five, we were able to generate multiple clones. epitope. In addition, we found that the CDR3 sequences of KK10- We assessed the ability of the clonotypic KK10-specific CD8 + specific clonotypes were significantly closer to germline sequences T cells to kill HIV-infected target cells in a standard chromium- in controllers than in progressors (P = 0.001; Supplementary Fig. 2), release assay and assessed their ability to inhibit viral replication, which would confer a greater ability to recognize epitope variants39. again using the HLA-B*2705-expressing GXR cells. For controller These functional data were also consistent with computational mod- CTR203, we were able to establish clones representing five of the six eling showing that thymic selection in the context of protective HLA clonotypes detected in peripheral blood. We observed considerable alleles is more likely to generate a cross-reactive CD8 + T cell rep- variation in the ability of these clonotypes to recognize HIV-1 and ertoire that targets mutant viral epitopes, thereby contributing to viral variants (Fig. 5) that ranged from broad recognition of the improved control of a highly variable pathogen25. variants by the two most dominant CTR203 clonotypes to weak and narrow recognition by all three subdominant CTR203 TCR vari- Antiviral efficacy of KK10-specific clonotypes ants. Extending this to clones from five subjects, we found that the The data reported above indicated that there were differences most effective clonotypes were the immunodominant in vivo clono- between controllers and progressors in the potency and cross- types from the controllers, including the two codominant responses reactivity of recognition of wild-type HIV-1 and viral variants in in subject CTR203, the two codominant responses in subject FW56 the KK10-specific CD8+ T cell responses, which suggested that and the one dominant response in subject CTR40. These were sig- the fine specificity of the TCR might be modulating these effects. nificantly more potent at viral recognition than were the immuno- Given that we had observed different clonotypes in the tetramer- dominant clones from the progressors. We observed codominant positive populations in these subjects (Table 1), we next sought to determine clonotypic antiviral efficacy. We therefore cloned KK10- 100 KK10 WT L6M R2T R2TL6M specific CD8+ T cells by limiting dilution from the sorted KK10 tetramer–positive cells from three elite controllers (CTR203, FW56 CTR203 FW56 CTR40 CR540 CR420 75 and CTR40) and two chronic progressors (CR540 and CR420) and then determined clonotypes by TCR sequencing (Table 3). In each of Specific lysis 50 Table 3  Clonotypes of HLA-B*2705-restricted KK10-specific 25 CD8+ T cell clones Clone Vβ CDR3 J region 0 CTR203 S-C003 25.1 CASSEADFEAF 1.1 Freq 13/37 11/37 2/37 6/37 3/37 16/31 10/31 3/31 11/21 8/34 16/34 6/34 21/30 Clone S-C003 S-T001 L015 S-C007 S-T002 B3 B5 B6 H08 002 013 015 C05 CTR203 S-T001 18 CASSPGQFSHEQY 2.7 1 .7 2 .2 1 .1 .1 3 7 .7 .2 7 3 CTR203 L015 27 CASSARTGELF 2.2 Clonotype 1. 1. 2. 2. 2. 2. 1. J2 /1 J2 J1 J2 J2 /J /J /J /J /J /J J .1 1/ 3/ 5/ / / 2/ .1 .1 9 2 6 18 27 21 20 . 7. 4. 6. 7. 5. CTR203 S-C007 20.1 CSARDGGEQY 2.7 BV 25 20 20 BV BV BV BV BV BV BV BV BV BV BV BV CTR203 S-T002 7.9 CASSLDRLEQF 2.1 FW56 B3 4.3 CASRPGLASNEQF 2.1 Figure 5  Differences in the antiviral efficacy of clonotypes specific for FW56 B5 6.5 CASRPGQGATEAF 1.1 HLA-B*27–KK10. Lysis of HIV-1-infected, HLA-B*2705-expressing FW56 B6 20.1 CSARDRGTREVADNYGYT 1.2 GXR cells (as in Fig. 4c) by KK10-specific clonotypes from controllers CTR40 H08 7.2 CASSLSGRWSTDTQY 2.3 (CTR203, FW56 and CTR40) and progressors (CR540 and CR420), CR540 002 5.6 CASGGGTVYEQY 2.7 assessed by chromium-release assay at an effector cell/target cell ratio CR540 013 2 CASSAGPGQYGNTIY 1.3 of 1:1. Freq, frequency of clonotype among KK10-specific CD8 + T cell CR540 015 21 CASTNRGSEQY 2.7 populations. P = 0.005, dominant clonotypes from controllers versus CR420 C05 20.1 CSAREGVEGYT 1.2 those from progressors (Mann-Whitney test). Data are representative of TCR β-chain variable region, CDR3 sequence and joining region (as in Table 1). two experiments (error bars, s.d.). aDVANCE ONLINE PUBLICATION  nature immunology
  • 7. Articles Figure 6  Differences in the antiviral efficacy of clonotypes specific for 100 TW10 WT T242N G248A G248D HLA-B*57–TW10. Lysis of HIV-1-infected, HLA-B*2705-expressing GXR EC CTR53 EC CR462 CP CR555 cells (as in Fig. 4c) by CD8+ T cell clones specific for HLA-B*5701– TW10, generated from HLA-B*57+ elite controllers (CTR53 and CR462) 75 and a chronic progressor (CR555), assessed by chromium-release assay Specific lysis (%) at an effector cell/target cell ratio of 1:1. Data are representative of two experiments (error bars, s.d.). 50 clonotype TCRBV4-3 in controller FW56, which showed efficient recognition of wild-type virus and had less robust activity than 25 codominant FW56 clonotype TCRBV6-5 had against the L6M vari- ant and was unable to recognize any of the other variants, at a low frequency in progressors CR540, CR420 and 8222 in the context of 0 Clone 001 004 005 001 003 010 012 021 different sequences of CDR3 or joining segments. Clonotype J1 1 .1 1 3 3 2 .5 1. 2. 1. 2. 2. J2 J2 Subdominant clonotypes from the elite controllers included 1/ /J /J /J /J /J 5. 1/ 1/ .3 28 15 .1 19 BV 9. 9. 10 20 BV BV BV BV BV CRBV27TCRBJ2-2, TCRBV20-1TCRBJ2-7 and TCRBV20-1TCRBJ1- BV BV 2, all of which were associated with inferior recognition of wild-type virus and the L6M variant and showed the least efficacy against the other HIV-1-infected, HLA-B*5701-expressing GXR cells (Fig. 6). We gene­ viral variants. However, these less-effective clonotypes were dominantly rated HLA-B*5701-restricted TW10-specific CD8+ T cell clones by selected by progressors CR338, 8222 and CR420. They were rearranged limiting dilution of cells sorted from HLA-B*57+ elite controllers© 2012 Nature America, Inc. All rights reserved. with variant CDR3 or joining segments but were likewise less efficient (CTR53 and CR462) and a chronic progressor (CR555) through the at recognizing HIV-1-infected cells. Moreover, none of the subdomi- use of an HLA-B*57–TW10 tetramer and then clonotypically assessed nant clonotypes, whether from controllers or progressors, were able to these by TCR sequencing. Again, we observed that variant clonotypes efficiently recognize HIV-1-infected cells or to inhibit viral replication. had differences in antiviral efficacy and that, overall, clones from the We obtained consistent results in terms of the ability of individual clono- elite controllers were more potent and cross-reactive in recognition types to inhibit viral ­replication (Supplementary Fig. 3). Together these of HIV-1 and viral variants than were clones from the progressor data indicated that ­differences in the antiviral efficacy of KK10-specific (Fig. 6). Together these data indicated that the difference between the CD8+ T cells in HIV-1-infected people were defined by the dominance of controllers and progressors of the same epitope-specific CD8+ T cell clonotypes that conferred distinct antiviral potential on CD8+ T cells. responses in the recognition of HIV-1 and viral variants was related to distinct TCR clonotypes selected during natural infection. Antiviral efficacy of TW10-specific clonotypes We further examined the effect of individual TCR clonotypes on anti- Lytic granule loading and delivery by clonotypes viral efficacy with HLA-B*5701-restricted CD8 + T cell clones spe- The data reported above indicated clonotype-specific differences cific for the epitope TW10 of Gag in a chromium-release assay with in antiviral function, which offered the opportunity to define the a b c * DIC + GFP F-actin Perforin Merge * EC CTR203 CP CR450 * 100 Intensity per T cell Perforin-expression KK10+ cells (%) 10 (10 AU) 75 CTR203 5 S-C003 T GXR 5 50 0 S-C003 S-C007 013 015 CTR203 CR540 25 d Intensity per GXR cell 20 (10 AU) CTR203 0 15 Freg S-C007 GXR 4 11 7 2/ 7 37 37 37 2 1 34 34 34 10 /3 /3 6/ 3/ 8/ 6/ 6/ 13 T S- 003 01 S- 15 S- 007 02 3 5 Clone 5 00 01 01 T0 L0 T0 C C S- S-C003 S-C007 013 015 CTR203 CR540 Figure 7  Differences in the loading and delivery of perforin by clonotypes. (a) Perforin expression by KK10-specific (KK10+) cells after 3 d of culture with HIV-1-infected HLA-B*2705-expressing GXR cells, assessed by flow cytometry and presented as frequency after subtraction of background frequency obtained by incubation with uninfected target cells. P 0.0001, dominant clonotypes from controllers versus subdominant clonotypes from the controllers and all clonotypes from progressors (Mann-Whitney test). (b) Microcopy of the release of perforin from effector cells of the dominant clonotype S-C003 (top) and subdominant clonotype S-C007 (bottom) from controller CTR203 after 30 min of incubation with HIV-1-infected HLA-B*2705- encoding GXR cells (green; target cells), presented as differential interference contrast images (DIC + GFP; left) and as z-series confocal microscopy with projected serial confocal sections through conjugations between cells, by staining of F-actin with phalloidin–Alexa Fluor 647 (red) and of perforin with primary antibody to perforin, followed by Alexa Fluor 568–conjugated secondary monoclonal antibody (purple). Far right, merged overlays. Scale bars, 3.0 µm. (c) Intensity of perforin staining in T cells of dominant clonotypes (S-C003 from controller CTR203, and 013 from progressor CR540) and subdominant clonotypes (S-C007 from CTR203, and 015 from CR540) after exposure to HIV-1-infected GXR cells. *P 0.0001 (Mann-Whitney test). (d) Intensity of perforin staining in GXR cells after exposure to the clonotypes in c. Each symbol (c,d) represents an individual cell; small horizontal lines indicate the mean (and s.e.m.). Data are representative of two experiments. nature immunology  aDVANCE ONLINE PUBLICATION
  • 8. Articles mechanisms that account for these phenotypes. We next determined studies done here of a small cohort of well-pedigreed control- the effect of TCR clonotype on the loading of lytic granules after lers and progressors expressing protective alleles, none of those recognition of HIV-1-infected target cells and the ability of ­clonotype- reported associations reached statistical significance. In contrast, specific T cells to deliver perforin to infected target cells. We mea­ the ability of both bulk CD8+ T cells as well as epitope-specific TCR sured by flow cytometry the expression of perforin and granzyme B clonotypes to inhibit viral replication, cross-recognize viral variants by various clonotypes12,13. After culture for 3 d with KK10-specific, and upregulate perforin and granzyme B, which are probably the HLA-B*2705-expressing GXR cells infected with wild-type HIV-1, most important in vivo functions of these cells, was highly signifi- dominant clonotypes from controllers had efficient expression of cant. Overall, our study has linked the antiviral efficacy of the two perforin. In contrast, subdominant clonotypes from controllers and most protective HLA class I molecules to CD8+ T cell clonotypes all the clonotypes from progressors were significantly less efficient at selected during natural infection with HIV-1 and has demonstrated expressing perforin than were dominant clonotypes from controllers that TCR rearrangement modulated the effect of protective alleles (Fig. 7a). We obtained similar results for the expression of granzyme B on disease outcome. (P 0.0001; Supplementary Fig. 4). Our study is distinct from other reports of TCR clonotype use We also examined by confocal microscopy effector cell–target by KK10-specific CD8+ T cells in people who express HLA-B*27 in cell conjugation and the loading and delivery of granules40. After that we selected the subjects by ‘extremes’ of viral load. As noted incubation for 30 min with HIV-infected, HLA-B*2705-expressing in other published studies28,29, we observed considerable diversity GXR cells, more perforin was polarized to synapses and released in clonotype recruitment and CDR3 motifs in KK10-specific CD8+ into target cells for the inhibitory clonotypes than for the non­ T cell populations, as well as a dominance of clonotypes in progressors inhibitory clonotypes (Fig. 7b). Extended quantitative analysis of who were unable to cross-recognize the L6M mutant. The mutation© 2012 Nature America, Inc. All rights reserved. perforin loading and delivery showed that significantly less per- that results in this mutant is known to occur early during the course of forin was delivered to synapses (Fig. 7c) and released into target HIV-1 infection with little or no effect on peptide processing27, bind- cells (Fig. 7d) by subdominant clonotypes from controllers and all ing of peptide to HLA-B*27 (ref. 27) recognition by the TCR41 or viral the clonotypes from progressors than by dominant clonotypes from fitness36, but it is an important intermediate mutation on the path controllers. As a control, we confirmed that endogenous perforin to complete escape of the immune response. Although we detected was undetectable in HIV-1-infected GXR cells (Supplementary such ineffective responses at the clonal level in both controllers Fig. 5). We observed similar basal amounts of perforin in inhibi- and progressors in our cohort, HLA-B*27+ elite controllers had domi- tory and noninhibitory clonotypes without stimulation of target cells nant clonotypes that targeted not only the L6M mutant but also other (P = 0.96; Supplementary Fig. 6), which indicated that the func- mutants, including substitution at position 2 of KK10 alone or in tionally effective clones rapidly upregulated perforin loading after combination with the L6M substitution, which diminishes the bind- the recognition of cognate antigens. These data indicated that TCR ing of peptide to HLA-B*27 and impairs viral replication36. Thus, clonotypes associated with enhanced ­ability to inhibit HIV-1 replica- the TCR clonotypes in controllers comprised effective and ineffec- tion did so by rapidly upregulating lytic ­granules at immunological tive clonotypes, whereas those in progressors were limited to less- synapses after engagement of a target cell and delivery of these effective clonotypes. granules into the infected cell. In contrast to some other published reports42,43, we found no sig- nificant difference between the two groups in the use of ‘public’ clono- DISCUSSION types (defined as those that expressed identical TCR β-chain amino Alleles encoding certain HLA-B types are associated with enhanced acid sequences and recurred in many people), despite significant dif- control of viremia in HIV-1-infected people3–5, and this effect maps ferences in plasma viremia and the fact that ‘public’ clonotypes do not to specific host amino acids in the HLA-B peptide-binding groove6. seem to dominate among controllers29. However, controllers used However, most people with such so-called ‘protective alleles’, such TCR β-chain clonotypes with sequences encoding CDR3 that were as those encoding HLA HLA-B*27 and HLA-B*57, experience pro- significantly more ‘germline-like’ than those used by the progressors. gressive infection26. To address the basis of these differences in out- The lower number of nucleotide additions in the germline-like genes come, we compared CD8+ T cell responses to immunodominant viral encoding CDR3 is a hallmark of clonotypes found at high frequency epitopes in treatment-naive elite controllers and chronic progressors. in naive and memory T cell pools and also shared by many people44. To limit the number of potential confounding variables, we stud- The mechanism underlying this advantage bestowed by germline- ied only subjects with wild-type sequences of the respective T cell like genes encoding CDR3 may be related to higher precursor fre- epitopes in the virus in their plasma and provirus in their PBMCs quency and/or greater ability to recognize mutational variants of the and with the same HLA class I–restricting allele. In this setting, in epitope39,42. Furthermore, the most effective clonotypes, in terms which contemporaneous escape from the immune response is not a of viral inhibition and cytotoxic recognition of wild-type and vari- confounding issue, we found that CD8+ T cell responses by HIV-1 ant viruses, were dominantly selected in vivo by the controllers but controllers were more potent at inhibiting HIV-1 replication than were either absent or subdominantly selected in KK10-specific CD8+ were those of progressors that targeted the same epitopes and were T cell populations of the progressors. In contrast, the clonotypes better able to ‘cross-recognize’ HIV-1 viral variants that typically arise associated with inferior recognition of wild-type virus and the least in vivo. Moreover, these effects were associated with a unique ability of efficacy against the viral variants were dominantly selected by the the dominant TCR clonotypes to upregulate perforin and granzyme B, progressors but subdominantly selected by the controllers. which provides a mechanistic explanation for the divergent disease Although our study has clearly shown that the TCR modulated outcomes in people with protective HLA alleles. the protective effect of HLA molecules, it has many limitations. Our Many characteristics of CD8+ T cells have been reported to be HLA-B*2705 studies were limited to only five controllers and five associated with enhanced control of viremia, including differences progressors, and many published reported associations with viral in polyfunctionality, proliferative capacity and functional avidity of control did not reach statistical significance in this small study group. KK10-specific CD8+ T cells. In the carefully controlled comparative Nevertheless, even with these small numbers, the results showing that aDVANCE ONLINE PUBLICATION  nature immunology
  • 9. Articles TCR clonotypes modulated the protective effect of HLA-B were of D.E.K. did the imaging experiments; T.D.C. and X.G.Y. helped with TCR high statistical significance in demonstrating greater cytotoxic killing sequencing; F.P., A.P.-T. and I.T. provided clinical samples; and H.C., Z.M.N. and B.D.W. wrote the paper and all authors contributed to revisions. and greater cross-reactivity by the dominant clonotypes in controllers than by those in progressors. We were not able to generate CD8+ T cell COMPETING FINANCIAL INTERESTS clones that represented all detectable TCR clonotypes in all people, The authors declare no competing financial interests. but in one subject we were able to test five of six clonotypes in vivo that represented 90% of the detectable TCR diversity in that subject. Published online at http://www.nature.com/doifinder/10.1038/ni.2342. Moreover, all controllers evaluated at the clonal level had dominant Reprints and permissions information is available online at http://www.nature.com/ TCR clonotypes that were highly effective, whereas these were absent reprints/index.html. in both dominant and subdominant clones established in the progres- sors. Of note, the important role of TCR clonotypes in distinguishing 1. Migueles, S.A. Connors, M. Long-term nonprogressive disease among untreated HIV-infected individuals. J. Am. Med. Assoc. 304, 194–201 (2010). viral control from lack of viral control was probably mediated by direct 2. Deeks, S.G. Walker, B.D. Human immunodeficiency virus controllers: mechanisms cytotoxicity of HIV-1-infected cells, with perforin upregulation noted of durable virus control in the absence of antiretroviral therapy. Immunity 27, within 30 min of recognition of the cognate epitope and not requiring 406–416 (2007). 3. Kaslow, R.A. et al. Influence of combinations of human major histocompatibility proliferation of CD8+ T cells. The data presented here contrast with the complex genes on the course of HIV-1 infection. Nat. Med. 2, 405–411 (1996). well-documented differences between nonprogressors and progres- 4. Migueles, S.A. et al. HLA B*5701 is highly associated with restriction of virus replication in a subgroup of HIV-infected long term nonprogressors. Proc. Natl. sors in the proliferative capacity of CD8+ T cells12, probably because Acad. Sci. USA 97, 2709–2714 (2000). of the way proliferation was measured. In our study here, we used 5. Carrington, M. O’Brien, S.J. The influence of HLA genotype on AIDS. Annu. Rev. HIV-1-infected GXR cells expressing HLA-B*2705 as stimulator cells Med. 54, 535–551 (2003). 6. Pereyra, F. et al. The major genetic determinants of HIV-1 control affect HLA class in culture with bulk CD8+ T cells and observed similar proliferative© 2012 Nature America, Inc. All rights reserved. I peptide presentation. Science 330, 1551–1557 (2010). capacity for HLA-B*27+ KK10-specific CD8+ T cells from controllers 7. Betts, M.R. et al. Analysis of total human immunodeficiency virus (HIV)-specific and progressors. This could be explained by the similar abundance of CD4+ and CD8+ T-cell responses: relationship to viral load in untreated HIV infection. J. Virol. 75, 11983–11991 (2001). CD4+ T cells in the progressors and controllers, the properties of regu- 8. Addo, M.M. et al. Comprehensive epitope analysis of human immunodeficiency latory T cells in people who express alleles encoding protective HLA virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral molecules17 or compensation for in vivo impaired CD4+ helper T cell load. J. Virol. 77, 2081–2092 (2003). function in progressors by cytokines produced by the cell line used for 9. Betts, M.R. et al. HIV nonprogressors preferentially maintain highly functional stimulation. Finally, whether our results can be extrapolated to other HIV-specific CD8+ T cells. Blood 107, 4781–4789 (2006). 10. Almeida, J.R. et al. Antigen sensitivity is a major determinant of CD8+ T-cell protective alleles and other epitopes will require additional study. polyfunctionality and HIV-suppressive activity. Blood 113, 6351–6360 Together our data have indicated that TCR use modulates virus- (2009). inhibitory capacity and recognition of naturally arising HIV-1 vari- 11. Bennett, M.S., Ng, H.L., Dagarag, M., Ali, A. Yang, O.O. Epitope-dependent avidity thresholds for cytotoxic T-lymphocyte clearance of virus-infected cells. ants and thus modulates the effect of protective HLA alleles. Our data J. Virol. 81, 4973–4980 (2007). have suggested that TCR clonotypes that inhibit viral replication and 12. Migueles, S.A. et al. HIV-specific CD8+ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors. Nat. Immunol. 3, 1061–1068 confer cross-recognition of viral epitope variants that can eventually (2002). arise in vivo may be critical to long-term control of viremia. Efforts 13. Migueles, S.A. et al. Lytic granule loading of CD8+ T cells is required for HIV- to define the factors that contribute to junctional rearrangement of infected cell elimination associated with immune control. Immunity 29, 1009–1021 (2008). more effective TCRs may be of critical importance for the design of 14. Hersperger, A.R. et al. Perforin expression directly ex vivo by HIV-specific CD8 T cell vaccines and therapeutic strategies for highly variable patho- T-cells is a correlate of HIV elite control. PLoS Pathog. 6, e1000917 gens such as HIV-1. (2010). 15. Dahirel, V. et al. Coordinate linkage of HIV evolution reveals regions of immunological vulnerability. Proc. Natl. Acad. Sci. USA 108, 11530–11535 (2011). Methods 16. Kiepiela, P. et al. CD8+ T-cell responses to different HIV proteins have discordant associations with viral load. Nat. Med. 13, 46–53 (2007). Methods and any associated references are available in the online 17. Elahi, S. et al. Protective HIV-specific CD8+ T cells evade Treg cell suppression. version of the paper. Nat. Med. 17, 989–995 (2011). 18. Kaufmann, D.E. et al. Upregulation of CTLA-4 by HIV-specific CD4+ T cells correlates with disease progression and defines a reversible immune dysfunction. Note: Supplementary information is available in the online version of the paper. Nat. Immunol. 8, 1246–1254 (2007). 19. Day, C.L. et al. PD-1 expression on HIV-specific T cells is associated with T-cell Acknowledgments exhaustion and disease progression. Nature 443, 350–354 (2006). 20. Pérez, C.L. et al. Broadly immunogenic HLA class I supertype-restricted elite CTL We thank J. Wong (Massachusetts General Hospital) for monoclonal antibody 12F6 epitopes recognized in a diverse population infected with different HIV-1 subtypes. to CD3 and monoclonal antibody CD3:8 bispecific for CD3 and CD8; and all study J. Immunol. 180, 5092–5100 (2008). participants for their contributions. Supported by the Harvard University Center 21. Miura, T. et al. HLA-B57/B*5801 human immunodeficiency virus type 1 elite for AIDS Research (5 P30 AI060354-04), the Bill and Melinda Gates Foundation controllers select for rare Gag variants associated with reduced viral replication capacity (B.D.W. and D.C.D.), the Doris Duke Charitable Foundation (B.D.W.), the US and strong cytotoxic T-lymphotye recognition. J. Virol. 83, 2743–2755 (2009). National Institutes of Health (AI030914 to B.D.W. and AI074415 to T.M.A.), 22. Lassen, K.G. et al. Elite suppressor derived HIV-1 envelope glycoproteins exhibit the Howard Hughes Medical Institute (B.D.W.), the Mark and Lisa Schwartz reduced entry efficiency and kinetics. PLoS Pathog. 5, e1000377 (2009). Foundation (B.D.W.), the Intramural Research Program and the Office of AIDS 23. Goulder, P.J. et al. Late escape from an immunodominant cytotoxic T-lymphocyte response associated with progression to AIDS. Nat. Med. 3, 212–217 (1997). Research of the US National Institutes of Health (D.C.D. and S.D.), the Canadian 24. Lee, K.-H. et al. The immunological synapse balances T cell receptor signaling and Institutes for Health Research (New Investigator Award to Z.L.B.) and the Canada degradation. Science 302, 1218–1222 (2003). Research Chair in Viral Pathogenesis and Immunity (M.A.B.). 25. Kosmrlj, A. et al. Effects of thymic selection of the T-cell repertoire on HLA class I-associated control of HIV infection. Nature 465, 350–354 (2010). AUTHOR CONTRIBUTIONS 26. Pereyra, F. et al. Genetic and immunologic heterogeneity among persons who control HIV infection in the absence of therapy. J. Infect. Dis. 197, 563–571 (2008). H.C. was responsible for the overall conduct of the study, under the supervision 27. Goulder, P.J. et al. Evolution and transmission of stable CTL escape mutations in of B.D.W.; H.C., Z.M.N. and B.D.W. contributed to the experimental design; H.C., HIV infection. Nature 412, 334–338 (2001). Z.M.N., L.C.P. and J.W.F. did the experiments and analyzed the data; S.D. and 28. Almeida, J.R. et al. Superior control of HIV-1 replication by CD8+ T cells is reflected D.C.D. did germline analyses; T.M., Z.L.B., K.T.C. and J.S. did virus sequencing; by their avidity, polyfunctionality, and clonal turnover. J. Exp. Med. 204, 2473– M.A.B., A.S. and T.M.A. constructed HIV-1 variants and GXR cell lines; D.L. and 2485 (2007). nature immunology  aDVANCE ONLINE PUBLICATION
  • 10. Articles 29. Mendoza, D. et al. HLA B*5701+ long-term nonprogressors/elite controllers are not 37. Chen, H. et al. Differential neutralization of human immunodeficiency virus (HIV) distinguished from progressors by the clonal composition of HIV-specific CD8+ replication in autologous CD4 T cells by HIV-specific cytotoxic T lymphocytes. T-cells. J. Virol. 86, 4014–4018 (2012). J. Virol. 83, 3138–3149 (2009). 30. van Bockel, D.J. et al. Persistent survival of prevalent clonotypes within an 38. Julg, B. et al. Infrequent recovery of HIV from but robust exogenous infection of immunodominant HIV Gag-specific CD8+ T cell response. J. Immunol. 186, activated CD4+ T cells in HIV elite controllers. Clin. Infect. Dis. 51, 233–238 359–371 (2011). (2010). 31. Iglesias, M.C. et al. Escape from highly effective public CD8+ T-cell clonotypes by 39. Miles, J.J., Douek, D.C. Price, D.A. Bias in the αβ T-cell repertoire: implications HIV. Blood 118, 2138–2149 (2011). for disease pathogenesis and vaccination. Immunol. Cell Biol. 89, 375–387 32. Day, C.L. et al. Proliferative capacity of epitope-specific CD8 T-cell responses is (2011). inversely related to viral load in chronic human immunodeficiency virus type 1 40. Liu, D. et al. Integrin-dependent organization and bidirectional vesicular traffic at infection. J. Virol. 81, 434–438 (2007). cytotoxic immune synapses. Immunity 31, 99–109 (2009). 33. Champagne, P. et al. Skewed maturation of memory HIV-specific CD8 T lymphocytes. 41. Stewart-Jones, G.B.E. et al. Crystal structures and KIR3DL1 recognition of three Nature 410, 106–111 (2001). immunodominant viral peptides complexed to HLA-B*2705. Eur. J. Immunol. 35, 34. Hamann, D.r. et al. Phenotypic and functional separation of memory and effector 341–351 (2005). human CD8+ T cells. J. Exp. Med. 186, 1407–1418 (1997). 42. Price, D.A. et al. Public clonotype usage identifies protective Gag-specific CD8+ 35. Brockman, M.A., Tanzi, G.O., Walker, B.D. Allen, T.M. Use of a novel GFP reporter T cell responses in SIV infection. J. Exp. Med. 206, 923–936 (2009). cell line to examine replication capacity of CXCR4- and CCR5-tropic HIV-1 by flow 43. Dong, T. et al. HIV-specific cytotoxic T cells from long-term survivors select a unique cytometry. J. Virol. Methods 131, 134–142 (2006). T cell receptor. J. Exp. Med. 200, 1547–1557 (2004). 36. Schneidewind, A. et al. Structural and functional constraints limit options for cytotoxic 44. Venturi, V. et al. A mechanism for TCR sharing between T cell subsets and T-lymphocyte escape in the immunodominant HLA-B27-restricted epitope in human individuals revealed by pyrosequencing. J. Immunol. 186, 4285–4294 immunodeficiency virus type 1 capsid. J. Virol. 82, 5594–5605 (2008). (2011).© 2012 Nature America, Inc. All rights reserved. 10 aDVANCE ONLINE PUBLICATION  nature immunology
  • 11. ONLINE METHODS (M5E2), anti–human CD19 (HIB19) and anti–human CD56 (B159). Samples Study subjects. PBMCs and plasma samples from HIV-1-infected people and were acquired on an LSRFortessa (BD Biosciences) and data were analyzed HIV-1− people were used in this study according to protocols approved by with FlowJo software (version 9.0.2). the Institutional Review Board of the Massachusetts General Hospital. Elite controllers were defined as having a concentration of HIV-1 RNA below the Surface and intracellular staining. Cells (2 × 106) were stimulated for limit of detection for the assay used (for example, 75 RNA copies per ml by 1 h at 37 °C and 5% CO2 with KK10 at a final concentration of 20 ng/ml. branched DNA assay, or 50 copies by ultrasenstive PCR), without antiretro- Phycoerythrin-indodicarbocyanine–conjugated anti-CD107a (H4A3; BD) viral therapy. Treatment-naive chronic progressors in the study had a median was also added at the beginning of the stimulation period for analysis of viral load of 12,833 copies per ml (range, 4,073–22,094 copies per ml). CD4+ degranulation. After 1 h, 10 ng/ml of brefeldin A (B7651; Sigma) was added T cell counts, viral loads and HLA types were determined as described26 and the cells were incubated for another 5 h. After stimulation, surfaces of (characteristics of study subjects, Table 1). cells were stained with the following antibodies: V500–anti-CD8 (RPA- T8; BD), allophycocyanin-indotricarbocyanine–anti-CD27 (MT271; BD) Viruses and synthetic peptides. The chemokine receptor CXCR4-tropic and Qdot 605–anti-CD45RA (H100; eBioscience). Cells were then fixed HIV-1 laboratory strain NL4-3 was obtained from the AIDS Research and and permeabilized with Cytofix/Cytoperm solution (554722; BD) and stained Reference Reagent Program, Division of AIDS, National Institute of Allergy with phycoerythrin-indotricarbocyanine–anti-IFN-γ (B27; BD), fluorescein and Infectious Diseases, US National Institutes of Health. HIV-1 laboratory i ­ sothiocyanate–anti-IL-2 (5344.111; BD), Alexa Fluor 700–anti-TNF (Mab11; strain NL4-3 was also modified to express one or more mutations in the gene BD), phycoerythrin–anti-MIP-1β (D21-1351; BD) and Qdot 655–anti-CD3 encoding Gag p24 as described36,45. Peptides corresponding to described opti- (S4.1; Invitrogen). Cells were acquired on an LSRFortessa (BD Biosciences) mal HIV-1 epitopes and their variants were synthesized at the Massachusetts and data were analyzed with FlowJo software (TreeStar). General Hospital Peptide Core Facility on an automated peptide synthesizer by fluorenylmethoxycarbonyl technology. Proliferation assay. Primary CD8+ T cells were isolated from PBMCs by nega-© 2012 Nature America, Inc. All rights reserved. tive selection (Dynabeads; Invitrogen) with the proportion of CD3+ CD8+ Virus sequencing. Nested PCR for viral DNA or RNA was done as described46. T cells being 98%, as detected by flow cytometry. Cells were stained for PCR fragments were sequenced by population for the identification of regions 7 min at 37 °C with 0.35 µM CFSE (carboxyfluorescein diacetate succinimi- of sequence variation. All fragments were sequenced bidirectionally on an ABI dyl ester; Molecular Probes) and then were cultured for 7 d with medium 3730xl automated sequencer (Applied Biosystems). alone or with HIV-1-infected or uninfected HLA-B*27-encoding GXR cells in RPMI-1640 medium in the absence of IL-2. After being labeled with the ELISPOT assay. IFN-γ was measured by ELISPOT assay as described, with appropriate tetramer (Beckman Coulter), anti-CD8 (RPA-T8; BD) and anti- optimally defined epitopes and designated concentrations of peptide 8. The CD3 (UCHT1; BD), cells were fixed in 1% paraformaldehyde and analyzed density of input cells ranged from 1 × 104 cells per well to 1 × 105 cells per on an LSRII (BD Biosciences). well. For the quantification of specific spot-forming cells, the number of spots in the negative-control wells was subtracted from the number of spots in each Chromium release assay. HLA-B*27- or HLA-B*57-expressing GXR cells experimental well. Responses were considered positive if they had at least three (which contain a plasmid encoding GFP driven by the long terminal repeat times the mean number of spot-forming cells in the three negative-control of HIV-1) were constructed as described35,36 and were infected with wild- wells; positive responses also had to achieve a value of least 50 spot-forming type HIV-1 or viral variants at the appropriate multiplicity of infection. On cells per 1 × 106 PBMCs. The magnitude of the epitope-specific response is day 5 after infection, viable virus-infected cells were sorted on a FACSAria presented as spot-forming cells per 1 × 106 cells. (BD Biosciences) and labeled for 1 h at 37 °C with chromium. Bulk CD8 + T cells isolated from PBMCs by negative selection (Dynabeads; Invitrogen) Generation of CD8+ T cell clones. PBMCs were stained with fluorophore- or CD8+ T cell clones were then added at the appropriate effector cell/target conjugated HLA tetramer refolded with epitopic HIV-1 peptides (Beckman cell ratio, and a standard 4-hour chromium-release assay was done as Coulter) and fluorophore-labeled antibody to CD8 (anti-CD8; RPA-T8; BD) described49. Percent specific lysis was calculated as follows: [(mean experi- and anti-CD3 (UCHT1; BD). Tetramer-positive CD8+ cells were sorted on a mental c.p.m. − mean spontaneous c.p.m.) / (mean maximum c.p.m. − mean FACSAria (BD Biosciences) at 70 p.s.i., and single cells were placed into each spontaneous c.p.m.)] × 100. Spontaneous release or maximum release was well of 96-well plates, with irradiated allogeneic PBMCs and monoclonal anti- determined by incubation of labeled target cells with medium alone or 2% body 12F6 to CD3 (a gift from J. Wong) as a stimulus for T cell proliferation47. Triton X-100, respectively. Developing epitope-specific clones were further tested by ELISPOT assay of IFN-γ with optimal epitopes and with tetramer staining. Cloned CD8+ T cells Virus-inhibition assay. HLA-B*27- or HLA-B*57-expressing GXR cells were maintained by restimulation every 14–21 d with monoclonal anti-CD3 were infected for 4 h at 37 °C with the appropriate HIV-1 strain or viral vari- and irradiated allogeneic PBMCs in RPMI-1640 medium containing 50 U/ml ant at the specified multiplicity of infection, then were washed and cultured of recombinant IL-2, as described47. together with bulk CD8+ T cells isolated from PBMCs by negative selection (Dynabeads; Invitrogen) or CD8+ T cell clones at the appropriate effector Sequencing of TCR a- and b-chains. Tetramer-positive CD8+ cells were cell/target cell ratio. The ability to recognize HIV-1 and viral variants by CD8+ sorted from PBMCs or cloned CD8+ T cells and mRNA was extracted with T cells was analyzed by flow cytometry as the proportion of GFP+ cells over the RNeasy mini kit (Qiagen). Anchored RT-PCR was then done with a modi- 7 d in culture. To additionally address the relative antiviral efficacy of epitope- fied version of the SMART (switching mechanism at 5′ end of RNA transcript) specific CD8+ T cell responses, the ability of bulk CD8+ T cells and CD8+ procedure and a 3′-primer for the TCR α- or β-chain constant region (Cα or T cell populations depleted of epitope-specific cells to inhibit viral replica- Cβ) to obtain PCR products containing the Vα or Vβ chain in addition to the tion in autologous primary CD4+ T cells was measured by analysis of p24 CDR3, the Jα or Jβ region and the beginning of the Cα or Cβ region. RT-PCR production as described37. Primary CD4+ T cells were isolated from PBMCs and sequencing and analysis of genes encoding TCR α- and β-chain were by negative selection (Dynabeads; Invitrogen). Over 98% of these primary done as described48. cells coexpressed CD3 and CD4, as assessed by flow cytometry. Those CD4+ T cells were stimulated with monoclonal antibody bispecific for CD3 and CD8 Tetramer staining. Cells were first stained for 15 min with blue viability dye (CD3:8; a gift from J. Wong)50 and were infected for 4 h at 37 °C at day 3 with (L-23105; Molecular Probes, Invitrogen), then were stained for 30 min at room the appropriate HIV-1 isolates at a multiplicity of infection of 0.1, except as temperature with allophycocyanin- or phycoerythrin-conjugated MHC class I otherwise specified. Virus-infected cells were then washed and incubated in tetramer folded with KK10 peptide (Beckman Coulter). Surfaces of cells the presence or absence of effector cells at an effector cell/target cell ratio of were then stained with the following antibodies (all from BD Pharmingen): 1:1 in RPMI-1640 medium in addition of IL-2 at 50 U/ml. At regular inter- anti–human CD3 (UCHT1), anti–human CD8 (RPA-T8), anti–human CD14 vals, cultures were ‘fed’ by removal and replacement of one-half of the culture doi:10.1038/ni.2342 nature immunology
  • 12. supernatant with fresh medium. The supernatant removed was cryopreserved with the fluorescence images. Multitrack acquisition mode was used to avoid for subsequent quantification of p24 antigen by ELISA (Dupont). Viral inhibi- crosstalk between the different fluorophores. Images were analyzed with tion was calculated as follows: % inhibition = 100 × [1 − ([% GFP+ cells with Imaris software (Bitplane). effector cells] / [% GFP+ cells without effector cells])]. Statistical analyses. An unpaired t-test with Welch’s correction and Mann- Effector cell–target cell conjugation, granule loading and delivery. CD8+ Whitney tests were done with GraphPad Prism version 4.0a. All tests were two T cell clones were cultured for 30 min with HIV-1-infected or uninfected tailed, and P values of less than 0.05 were considered significant. HLA-B*27-expressing GXR cells in RPMI-1640 medium in the absence of IL-2. Perforin in CD8+ T cell clones was stained as described40. Cells were fixed for 15–30 min at room temperature with freshly prepared 4% para- 45. Miura, T. et al. Genetic characterization of human immunodeficiency virus type 1 formaldehyde and were washed three times with PBS. Cells were permeabi- in elite controllers: lack of gross genetic defects or common amino acid changes. lized for 30 min at room temperature in 0.5% Triton X-100 (Sigma) and 10% J. Virol. 82, 8422–8430 (2008). 46. Allen, T.M. et al. Selection, transmission, and reversion of an antigen-processing normal donkey serum (Jackson Immunoresearch) in PBS. Cells were stained cytotoxic T-lymphocyte escape mutation in human immunodeficiency virus type 1 for 60 min at room temperature with monoclonal mouse IgG2b antibody to infection. J. Virol. 78, 7069–7078 (2004). perforin (δG9; Pierce). Anti-perforin was diluted (1:333) with 0.05% Triton 47. Walker, B.D. et al. Long-term culture and fine specificity of human cytotoxic X-100 and 3% normal donkey serum in PBS. After three washes in PBS, cells T-lymphocyte clones reactive with human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 86, 9514–9518 (1989). were incubated for 1 h at room temperature with anti-perforin (A-20186; 48. Varadarajan, N. et al. A high-throughput single-cell analysis of human CD8+ T cell Invitrogen) in 0.05% Triton X-100 and 3% normal donkey serum in PBS. Anti- functions reveals discordance for cytokine secretion and cytolysis. J. Clin. Invest. perforin was conjugated to Alexa Fluor dyes (1:1,000 dilution). F-actin was 121, 4322–4331 (2011). stained with phalloidin–Alexa Fluor 647 (1:50 dilution; Invitrogen). Confocal 49. Yang, O.O. et al. Impacts of avidity and specificity on the antiviral efficiency of HIV-1-specific CTL. J. Immunol. 171, 3718–3724 (2003). images were collected on a Zeiss LSM510 Meta Confocal microscope with a 50. Wilson, C.C. et al. Ex vivo expansion of CD4 lymphocytes from human© 2012 Nature America, Inc. All rights reserved. plan apochromat 63× oil-immersion objective with a numerical aperture of immunodeficiency virus type 1-infected persons in the presence of combination 1.4. Differential interference contrast images were collected simultaneously antiretroviral agents. J. Infect. Dis. 172, 88–96 (1995). nature immunology doi:10.1038/ni.2342

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