Protective genotypes in HIV infection reflect superior function of KIR3DS1+ over KIR3DL1+ CD8+ T cells

Abstract

Certain human class I histocompatibility-linked leukocyte antigen (HLA)/killer cell immunoglobulin-like receptor (KIR) genotypic combinations confer more favourable prognoses upon exposure to human immunodeficiency virus (HIV). These combinations influence natural killer (NK) cell function, thereby implicating NK cells in protection from HIV infection or disease progression. Because CD8(+) T cells restrict HIV replication, depend upon HLA class I antigen presentation and can also express KIR molecules, we investigated how these HLA/KIR combinations relate to the phenotype and function of CD8(+) T cells from uninfected controls and individuals with chronic HIV infection. CD8(+) T cells from KIR3DL1 and KIR3DS1 homozygous individuals, and expressing the corresponding KIR, were enumerated and phenotyped for CD127, CD57 and CD45RA expression. Ex vivo and in vitro responsiveness to antigen-specific and polyclonal stimulation was compared between KIR-expressing and non-expressing CD8(+) T cells by interferon-γ production. There were higher numbers and fractions of KIR3DL1-expressing CD8(+) T cells in HIV-infected individuals independent of HLA-Bw4 co-expression, whereas expansion of KIR3DS1-expressing CD8(+) T cells reflected HLA-Bw4*80I co-expression. KIR3DL1(+) and S1(+) CD8(+) T cells were predominantly CD127(-)CD57(+)CD45RA(+). KIR3DL1-expressing CD8(+) T cells were insensitive to ex vivo stimulation with peptides from HIV or common viruses, but responded to anti-CD3 and recovered responsiveness to common viruses in vitro. Ex vivo non-responsiveness of KIR3DL1-expressing CD8(+) T cells was also independent of HLA-Bw4. KIR3DS1-expressing T cells responded normally to ex vivo antigenic stimulation, illustrating functional superiority over KIR3DL1(+) CD8(+) T cells.

Figure 1

Distributions of percentage CD8⁺ T cells expressing KIR3DL1 or KIR3DS1 in different HIV-infected and uninfected subgroups. The percentage of CD8⁺ T cells expressing KIR3DL1 was compared between HIV-infected and uninfected KIR3DL1 homozygous subjects (a) and between subgroups of the HIV-infected individuals distinguished by HLA-Bw4*80I, Bw4*80T or Bw6 status (b). The percentage of CD8⁺ T cells expressing KIR3DS1 was compared between KIR3DS1 homozygous subjects expressing Bw4*80I or not with different symbols used to distinguish HIV-infected (□) from uninfected (○) individuals (c).

Figure 2

Representative examples of analysis of CD45RA, CD57 and CD127 expression on KIR3DL1⁺ (a) or KIR3DS1⁺ (b) CD8⁺ T cells from KIR3DL1 or KIR3DS1 homozygous subjects by flow cytometry. Gating for analysis was on CD3⁺CD8⁺ lymphocytes with DX9-FITC or DX9-PE identifying KIR3DL1⁺ cells and Z27-PE identifying KIR3DS1⁺ cells.

Figure 3

Distribution of KIR3DL1/S1⁺CD8⁺ T cell phenotypes. The percentage of KIR3DL1⁺ and KIR3DS1⁺ CD8⁺ T cells expressing CD45RA, CD57 and CD127 in groups of HIV-infected KIR3DL1 or KIR3DS1 homozygous individuals was compared by flow cytometry.

Figure 4

Representative ex vivo antigen-specific responses of KIR3DL1⁺CD8⁺ T cells from KIR3DL1 homozygous individuals co-expressing HLA-Bw4. Freshly-isolated PBMC from an uninfected (a) and HIV-infected individual (b) were incubated with overlapping peptides from CMV pp65 (a) or HIV Gag (b) as described in the methods section and production of intracellular IFN-γ was assessed by flow cytometry with gating on CD3⁺CD8⁺ lymphocytes. Unstimulated controls are shown in the left hand panel of each set.

Figure 5

Representative ex vivo antigen-specific responses of KIR3DL1⁺CD8⁺ T cells from HLA-Bw6 subjects and representative ex vivo response to P815/anti-CD3 stimulation. Freshly-isolated PBMC from three KIR3DL1 homozygous HIV-infected individuals defined as HLA-Bw6 (a, b, c) and one uninfected KIR3DL1 homozygous individual co-expressing HLA-Bw4 were incubated with overlapping peptides from CMV pp65 (a, c) or HIV Gag (b) as described in the methods. Freshly-isolated PBMC from an uninfected KIR3DL1 homozygous individual who co-expressed HLA-Bw4 were incubated with P815 cells and anti-CD3 as described in the methods section (d). Intracellular IFN-γ production was assessed by flow cytometry with gating on CD3⁺CD8⁺ (a, b, c) or CD8⁺ lymphocytes (d). Unstimulated controls, including PBMC incubated with P815 cells without anti-CD3 (d) are shown in the left hand panel of each set.

Figure 6

Representative ex vivo antigen-specific responses of KIR3DS1⁺CD8⁺ T cells. Gating was on CD3⁺KIR3DS1⁺ cells as in (a) with CD8⁺ cells producing IFN-γ shown in the upper right hand quadrants of the subsequent plots. Freshly-isolated PBMC from a KIR3DS1 homozygous individual were unstimulated (b) or incubated with overlapping peptides from CMV pp65 (c) or HIV Gag (d).

Figure 7

Representative secondary antigen-specific responses of KIR3DL1⁺CD8⁺ T cells following in vitro stimulation and expansion. Freshly-isolated PBMC from 2 HLA-Bw4⁺ HIV-infected individuals were cultured for 7 days with specific HLA-A2-restricted flu (a) or HIV-Gag peptides (b), restimulated with peptide-pulsed autologous BLCL as described in the methods section, and tested for antigen-specific IFN-γ production by intracellular flow cytometry with gating on CD3⁺CD8⁺ lymphocytes. Cells expressing KIR3DL1 were positively selected from freshly isolated PBMC of an uninfected HLA-Bw4 individual, incubated with a specific HLA-A2-restricted CMV peptide for 7 days, restimulated with peptide-pulsed autologous BLCL as described in the methods section, and tested for antigen-specific IFN-γ production by intracellular flow cytometry with gating on CD3⁺CD8⁺ lymphocytes (c).