Robust NK cell-mediated human immunodeficiency virus (HIV)-specific antibody-dependent responses in HIV-infected subjects

Abstract

Antibody-dependent cellular cytotoxicity (ADCC) is a potentially effective adaptive immune response to human immunodeficiency virus (HIV) infection. The study of ADCC responses has been hampered by the lack of simple methods to quantify these responses and map effective epitopes. We serendipitously observed that standard intracellular cytokine assays on fresh whole blood from a cohort of 26 HIV-infected subjects identified non-T lymphocytes expressing gamma interferon (IFN-gamma) in response to overlapping linear peptides spanning HIV-1 proteins. The effector cells were CD3(-) CD4(-) CD8(-) CD14(-) CD2(+) CD56(+/-) NK lymphocytes and degranulated granzyme B and perforin in response to antigen stimulation. Serum transfer assays demonstrated that the specific response was mediated by immunoglobulin G. Fresh blood samples from half of the HIV-infected cohort demonstrated robust HIV peptide-specific IFN-gamma expression by NK cells, predominately to Env, Pol, and Vpu HIV-1 proteins. Responses were readily mapped to define minimal epitopes utilizing this assay. Antibody-dependent, HIV-specific NK cell recognition, involving components of both innate and adaptive immune systems, represents a potentially effective immune response to induce by vaccination.

FIG. 1.

CD3-negative lymphocytes expressing IFN-γ in response to HIV peptides. Flow cytometric plots on gated lymphocytes from two subjects are shown. (A) Whole blood from HIV-infected subject 23 was stimulated with pools of HIV-1 consensus subtype B overlapping peptide pools in a 5-h ICS assay. Pol peptides were divided into two separate pools, and peptides for Rev, Tat, and Vpu and for Vif, Nef, and Vpr were combined. Gated lymphocytes were studied for expression of CD3 and intracellular IFN-γ. HIV-specific IFN-γ expression from non-CD3 lymphocytes in response to Pol and Env is detected. (B) Whole blood from HIV-infected subject 26 was similarly incubated with pools of overlapping 15-mer peptides from HIV-1 peptides for 5 h. Gated lymphocytes were studied for CD3 and intracellular IFN-γ expression as above. A large proportion of CD3-negative cells produced IFN-γ in response to the combined Rev, Tat, and Vpu peptide.

FIG. 2.

Epitope mapping: an HIV-specific non-T-lymphocyte response recognizes Vpu peptide. Whole blood from subject 26 was incubated with HIV peptides for 5 h, and gated lymphocytes were studied for the expression of CD3 and IFN-γ. (A) The recognition of pooled Tat, Rev, and Vpu peptide (shown in Fig. 1B) was specific for a Vpu pool of 19 peptides. (B) Individual peptides within the Vpu pool of 19 peptides were studied, with the response overlapping peptides 18 and 19. (C and D) The Vpu response was mapped to minimal epitopes. A 14-mer peptide had an equally strong response to larger peptides.

FIG. 3.

Cell markers on Vpu-specific IFN-γ-expressing lymphocytes. Whole blood from subject 26 was pulsed with the Vpu epitope. Gated lymphocytes are shown in all plots. The proportions of cells expressing IFN-γ are shown in the relevant quadrant. (A) IFN-γ-expressing lymphocytes all expressed CD2. CD3-negative gated lymphocytes are shown. (B) Only a proportion of the IFN-γ-expressing lymphocytes expressed CD56. CD2⁺ CD3⁻ gated lymphocytes are shown. Percentages in the upper-right and upper-left quadrants show the proportion of CD56⁺ and CD56⁻ cells that express IFN-γ, respectively. Overall ∼50% of IFN-γ-expressing cells are CD56⁺. (C and D) IFN-γ-expressing lymphocytes did not express intracellular TCRαβ or TCRγδ. Gated lymphocytes are shown. (E and F) There was no expression of the CD3 complex by IFN-γ-expressing lymphocytes, but intracellular expression of the CD3 epsilon subunit was detected. Gated lymphocytes are shown.

FIG. 4.

Vpu-specific expression of IFN-γ in subject 26 is mediated by autologous serum. (A) Ficoll-separated PBMCs or fresh blood washed twice to remove plasma from subject 26 was incubated with either fetal calf serum or autologous serum comprising 5% of the culture together with the Vpu peptide epitope in an ICS assay. (B) Whole blood from subject 26 was washed to remove plasma and incubated with autologous serum comprising 50%, 5%, or 0.5% of the culture together with the Vpu peptide epitope. (C) Autologous PBMCs and plasma from subject 26 were mixed with PBMCs or plasma from an HIV-negative donor.

FIG. 5.

IgG mediates the Vpu-specific IFN-γ expression. Whole blood from additional volunteers was incubated with plasma (or plasma components) from subject 26 in the presence of the Vpu epitope. The sources of the responder cells and plasma fraction are shown above each plot. (A) HIV-negative volunteer. All IFN-γ⁺ cells were CD56 positive. (B) When incubation was performed in the presence of an aliquot of Ig purified from the plasma of the HIV-infected subject, the response was preserved. (C) HIV-positive donor cells from subject 8 were incubated with plasma from subject 26. Only half of the IFN-γ-expressing cells expressed CD56. (D) IFN-γ⁺ cells did not express CD16. HIV-negative donor cells incubated with plasma from subject 26 are shown.

FIG. 6.

Serum from an additional HIV-positive subject mediates non-T-lymphocyte IFN-γ expression from multiple cell donors. Serum from HIV-positive subject 8, who responded to the Pol1 peptide pool, was incubated with either autologous PBMCs, with PBMCs from another HIV-positive subject (subject 26), or with PBMCs from an HIV-negative donor. Intracellular IFN-γ expression was examined in CD3-negative lymphocytes stained for either CD2 (top plots) or CD56 (bottom plots).

FIG. 7.

Cytolytic markers of NK cells in response to Vpu peptide. Serum from subject 26 was incubated with either control dimethyl sulfoxide or the HIV-1 Vpu peptide 19 epitope and HIV-negative donor PBMCs for 6 h. NK cells (CD3⁻ CD2⁺ CD56⁺ gated lymphocytes) were examined for surface expression of the degranulation marker CD107a and intracellular expression of the cytolytic effector molecules granzyme B and perforin. There was significant CD107a expression in response to Vpu, and the cells with highest expression of CD107a also lost intracellular granzyme B and perforin. Percentages of CD107a⁺ granzyme B-negative and CD107a⁺ perforin-negative cells are shown in the lower-right quadrants.