TCR clonotypes modulate the protective effect of HLA class I molecules in HIV-1 infection - PubMed (original) (raw)

. 2012 Jun 10;13(7):691-700.

doi: 10.1038/ni.2342.

Zaza M Ndhlovu, Dongfang Liu, Lindsay C Porter, Justin W Fang, Sam Darko, Mark A Brockman, Toshiyuki Miura, Zabrina L Brumme, Arne Schneidewind, Alicja Piechocka-Trocha, Kevin T Cesa, Jennifer Sela, Thai D Cung, Ildiko Toth, Florencia Pereyra, Xu G Yu, Daniel C Douek, Daniel E Kaufmann, Todd M Allen, Bruce D Walker

Affiliations

TCR clonotypes modulate the protective effect of HLA class I molecules in HIV-1 infection

Huabiao Chen et al. Nat Immunol. 2012.

Abstract

The human leukocyte antigens HLA-B27 and HLA-B57 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-B27 and HLA-B57 are unable to control HIV-1. Here we found that HLA-B27-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-B57-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.

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Figures

Fig. 1

Fig. 1. Quantification of KK10-specific CD8+ T cell responses

(a) Scatterplot of the percentage of HLA-B*27-KK10-specific CD8+ T cells in a controller (FW56) and a progressor (CR540) determined by flow cytometry and staining with HLA class I tetramers. (b) No significant difference between the controllers (n = 5) and progressors (n = 5) in terms of the percentage of KK10 tetramer-positive cells in bulk CD8+ T cells. (c) KK10-specific CD8+ T cell responses in PBMC were assessed directly ex vivo in an IFN-γ ELISPOT assay following KK10 peptide stimulation. There were no significant differences in response magnitude (calculated as spot forming cells (SFC) per million PBMC) between the controllers (n = 5) and progressors (n = 5). Statistical comparisons were made using the Mann-Whitney test.

Fig. 2

Fig. 2. Functional characteristics of KK10-specific CD8+ T cells

(a) Functional avidity of KK10-specific CD8+ T cells as measured by peptide titration of PBMC in the IFN-γ ELISPOT assay. (b) Expression of IFN-γ, TNF, IL-2, MIP-1β and CD107a were measured in KK10-specific CD8+ T cells from the five HLA-B*2705-positive controllers (EC) and five progressors (CP) following PBMC stimulation with KK10 peptide. The bars represent proportion of subpopulations of KK10-specific cells expressing different combinations of effector functions. The y-axis shows the mean percentage of all cells displaying a particular combination. Statistical comparisons were made using the Mann-Whitney test; * denotes p < 0.05. (c) Scatterplot of proliferation of tetramer-positive KK10-specific CD8+ T cells, as shown by CFSE-low cells, at day 7 following stimulation of bulk CD8+ T cells from a controller (FW56) and a progressor (CR540) with virally infected HLA-B*2705-encoding GXR cells. (d) No significant difference was observed in proliferative capacity of KK10-specific CD8+ T cells as measured by CFSE intensity by flow cytometry between the controllers (n = 5) and progressors (n = 5).

Fig. 3

Fig. 3. Virus neutralization by ex vivo KK10-specific CD8+ T cells

(a) The production of p24 antigen by autologous CD4+ T cells infected with NL4-3 wild-type virus were evaluated in the presence of bulk CD8+ T cells or KK10-specific cell-depleted CD8+ T cells over 7 days for all 5 controllers. Data are shown over 7 days for a representative subject (FW56, left) and for all 5 controllers at day 7 (right). Uninfected CD4+ T cells and virally infected CD4+ T cells were used as negative and positive controls, respectively. (b) The virus inhibition assay was performed using the HLA-B*2705-encoding GXR cell line and GFP reporter assay. The reporter cells were cultured in the presence of bulk CD8+ T cells or KK10-specific cell-depleted CD8+ T cells over 7 days. The proportions of GFP-positive cells were analyzed by flow cytometry as shown for an representative subject over 7 days (FW56, left) and for all 5 controllers at day 7 (right). (c) Virus replication was evaluated in HLA-B*2705-encoding GFP reporter GXR cells in the presence of bulk CD8+ T cells over 7 days for all 5 progressors. The proportion of GFP-positive cells was analyzed by flow cytometry as shown for an example over 7 days (FEN33, left) and for all 5 progressors at day 7 (right).

Fig. 4

Fig. 4. Recognition of viral variants by KK10-specific CD8+ T cells

(a) Inhibition of replication of NL4-3 wild-type virus and the designated NL4-3 variants was evaluated in HLA-B*2705-encoding GFP reporter GXR cells in the presence of ex vivo CD8+ T cells isolated from a controller (FW56) and a progressor (CR540) at an effector/target cell ratio of 1:1. Virus replication was calculated as the proportion of GFP-positive cells by flow cytometry at day 7 in culture. (b) Summary of data from pools of 5 controllers, 5 progressors, and 12 HIV-1 negative individuals demonstrating different antiviral efficacy for ex vivo CD8+ T cells from these groups. Significance was tested with a Mann-Whitney test; * denotes p < 0.0001. (c) The ability of ex vivo CD8+ T cells from controllers (CTR203 and FW56) and progressors (CR540 and FEN33) to kill live virally infected HLA-B*2705-encoding GFP reporter GXR cells was tested in the standard 4-h chromium release assay at an effector/target cell ratio of 10:1. Viable virally infected (GFP-positive) GXR cells were sorted by a FACS Aria cell-sorting instrument after infection for 5 days and used as target cells.

Fig. 5

Fig. 5. Differential antiviral efficacy of B*27-KK10 specific clonotypes

The ability of KK10-specific clonotypes to recognize NL4-3 wild-type and variant viruses was tested in the standard 4-h chromium release assay with virally infected HLA-B*2705-encoding GFP reporter GXR cells at an effector/target cell ratio of 1:1. Viable infected (GFP-positive) GXR cells were sorted by a FACS Aria cell-sorting instrument after infection for 5 days and used as target cells. Data are shown for three controllers (CTR203, FW56 and CTR40) and two progressors (CR540 and CR420).

Fig. 6

Fig. 6. Differential antiviral efficacy of B*57-TW10 specific clonotypes

The ability of HLA-B*5701 TW10-specific CD8+ T cell clones generated from HLA-B*57 positive elite controllers (CTR53, CR462) and a chronic progressor (CR555) to recognize NL4-3 wild-type and variant viruses was tested in the standard 4-h chromium release assay with virally infected HLA-B*5701-encoding GFP reporter GXR cells at an effector/target cell ratio of 1:1. Viable infected (GFP-positive) GXR cells were sorted by a FACS Aria cell-sorting instrument after infection for 5 days and used as target cells.

Fig. 7

Fig. 7. Differential perforin loading and delivery of clonotypes

(a) Perforin expression of clonotypes was examined by flow cytometry after 3 days of culture with virally infected and uninfected HLA-B*2705-expressing GFP reporter GXR cells. Values indicate percentages of perforin secreting KK10-specific cells upon culture with virally infected target cells after subtracting background from effector cells incubated with uninfected target cells. (b) Differential interference contrast (DIC) images of effector cells with GFP-fluorescing target cells (DIC+GFP) are shown on the Left. Confocal microscope z-series were obtained. Projected serial confocal sections through conjugation between effector cells and HLA-B*2705-encoding GFP reporter GXR cells are shown (Green). F-actin was stained with phalloidin-Alexa Fluor 647 (Red). Perforin was stained with anti-perforin primary Ab followed by Alexa Fluor 568-conjugated secondary mAb (Purple). Merged overlays are on the Right. Clones were imaged 30 min after incubation with HIV-1 infected GXR cells. The dominant clonotype S-C003 (top) and subdominant clonotype S-C007 (bottom) from controller CTR203 are indicated. Scale bars are 3.0 μm. (c) Total intensity of perforin per T cell from representative dominant clonotypes (S-C003 and 013) and subdominant clonotypes (S-C007 and 015) from controller CTR203 and progressor CR540 are shown following exposure to HIV-1 infected GXR cells. (d) Intensity of perforin staining in GXR target cells following exposure to the clonotypes shown in (c). Data for (c) and (d) were obtained from two independent experiments. Error bars indicate the standard error of the mean (SEM).

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