Hiv resistance

803 views

Published on

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".

Published in: Health & Medicine, Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
803
On SlideShare
0
From Embeds
0
Number of Embeds
2
Actions
Shares
0
Downloads
6
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Hiv resistance

  1. 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. 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. 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. 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. 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

×