Multiple effector functions mediated by human immunodeficiency virus-specific CD4(+) T-cell clones

Abstract

Mounting evidence suggests that human immunodeficiency virus type 1 (HIV-1) Gag-specific T helper cells contribute to effective antiviral control, but their functional characteristics and the precise epitopes targeted by this response remain to be defined. In this study, we generated CD4(+) T-cell clones specific for Gag from HIV-1-infected persons with vigorous Gag-specific responses detectable in peripheral blood mononuclear cells. Multiple peptides containing T helper epitopes were identified, including a minimal peptide, VHAGPIAG (amino acids 218 to 226), in the cyclophilin binding domain of Gag. Peptide recognition by all clones examined induced cell proliferation, gamma interferon (IFN-gamma) secretion, and cytolytic activity. Cytolysis was abrogated by concanamycin A and EGTA but not brefeldin A or anti-Fas antibody, implying a perforin-mediated mechanism of cell lysis. Additionally, serine esterase release into the extracellular medium, a marker for cytolytic granules, was demonstrated in an antigen-specific, dose-dependent fashion. These data indicate that T helper cells can target multiple regions of the p24 Gag protein and suggest that cytolytic activity may be a component of the antiviral effect of these cells.

FIG. 1

(A) PBMC from subject CTS-01 were tested in a lymphocyte proliferation assay for responses to synthetic 22-amino-acid peptides spanning HIV-1 p24 protein sequence (peptides 1 to 23). These PBMC recognized several epitopes within p24, with the dominant response directed against peptide 9, sequence DRVHPVHAGPIAPGQMREPRGS. (B) The average proliferative response (net cpm) of PBMC from 10 HIV-1-seronegative control subjects to each of peptides 1 to 23 is shown.

FIG. 2

(A) TCR Vβ repertoire analysis: cDNA was derived from clone 16 from subject CTS-01 by RT-PCR as previously described (12). The cDNA was amplified using primer sets specific for different variable segments of the TCR Vβ chain (Vβ1 to Vβ24). Only the Vβ4-specific primer pair yielded a PCR product (lane 4). Each reaction also contained T-cell receptor α chain constant region 3′ and 5′ primers that produced a 100-bp fragment, included as a positive PCR control in each lane. Lanes 25 and 27 contained no Vβ-specific primers (negative control), whereas lane 26 contained a primer pair specific for the Vβ constant region (positive control). The DNA ladder shown at left contained 100-bp incremental fragments. (B) Flow cytometric analysis of clone 16 showed that the clone was 99.57% CD4⁺ and 0.34% CD8⁺.

FIG. 3

Ten p24-specific lines from donor CTS-01 were assayed for their ability to lyse B-LCL pulsed with p24 protein or peptide 9. A standard chromium release assay was performed, with results expressed as percent specific lysis. Negative controls included B cells pulsed with no antigen or with a control 22-amino-acid peptide derived from a portion of p24 not recognized by the subject. Lines were assayed at an E:T ratio of 100:1. Error bars were calculated by determining the standard deviation of assay results performed on at least two separate occasions.

FIG. 4

Fine mapping of the peptide-specific proliferative, cytotoxic, and Elispot responses of clone 16 from subject CTS-01. The peptide sequences are given on the left, followed by the graphs of (A) proliferation, (B) cytotoxicity, and (C) Elispot responses. Peptides that contained the minimal epitope are in bold. Elispot results were expressed as SFC/10⁶ clone cells added. Antigen concentration was 1 μg/ml for the proliferation and Elispot assays and 10 μg/ml for the cytotoxicity assays. In each assay the minimal epitope recognized was VHAGPIAPG.

FIG. 5

HLA restriction of CD4⁺ T-cell clone 16 from subject CTS-01. Antigen was presented to the clone by partially HLA matched B-LCL pulsed overnight with peptide 9. Negative controls included no antigen or peptide 23, a 22-amino-acid peptide from p24 not recognized by the subject's PBMC (data not shown). Only B-LCL which expressed the DQ7 allele elicited antigen-specific responses in proliferation, cytotoxicity, and IFN-γ Elispot assays. The top scale is for net cpm, and the bottom scale is for percent lysis and SFC/10⁶ cells. An E:T ratio of 10:1 was used for cytotoxicity assays.

FIG. 6

(A) Proliferative responses of each of six CD4⁺ T-cell clones derived from three individuals treated during acute HIV-1 infection are shown. The clones are labeled 1 to 6, with the first three derived from subject AC-01, the next two from subject AC-25, and the last from subject AC-36. Peptides 3 (amino acids 153 to 174), 4 (amino acids 163 to 184), 10 (amino acids 223 to 244), 13 (amino acids 253 to 274), 14 (amino acids 263 to 284), and 19 (amino acids 313 to 344) were recognized by one or more clones. (B) The six clones were tested for cytolytic ability. Autologous B-LCL were incubated with ⁵¹Cr and soluble p24 and used as targets in a 4-h CTL assay at an E:T ratio of 100:1. Spontaneous lysis ranged from 10 to 36%. Negative controls included B-LCL without added antigen. (C) The same six clones were assayed for their ability to kill B-LCL targets pulsed with the 22-amino-acid peptide within p24 recognized by each clone (see panel A). The assays were performed at an E:T ratio of 10:1, and negative controls included B-LCL without added antigen.

FIG. 7

(A) Dependence of proliferative, cytolytic, and IFN-γ secretion activity on peptide concentration. Autologous B cells were incubated with peptide 9 from p24 or with the control peptide 23 from p24 (not recognized) from 0 to 10 μg/ml. CD4⁺ T-cell clone responses from subject CTS-01 were measured in each of the three assays and expressed as stimulation index (SI), percent lysis (CTL), and SFC per 100. (B) Elaboration of serine esterase upon antigen-specific stimulation of clone 4 from subject AC-25. Autologous B cells were incubated with peptide 4 from 0 to 10 μg/ml prior to use as antigen-presenting cells. Serine esterase release was quantified by modification of the substrate BLT, and results were expressed as OD₄₀₅, with higher OD corresponding to greater granule secretion. The dotted line indicates the background release in response to the target cells without the peptide. (C) Abrogation of lysis mediated by clone 4 from subject AC-25 in the presence (○) and absence (●) of 2 mM Mg²⁺ and EGTA. Chelation of extracellular calcium caused abrogation of lysis over the range of peptide concentrations.

FIG. 8

(A) Effects of concanamycin A, brefeldin A, and anti-Fas antibody on cytolytic activity of the clone from subject CTS-01. T cells were incubated with various concentrations of concanamycin A, brefeldin A, or anti-Fas antibody for 2 h preceding a standard 4-h chromium release assay. The E:T ratio was 10:1, and B-cell targets were pulsed with 10 μg/ml of peptide 9 overnight prior to the assay. Similar results were obtained with clone 4 from subject AC-25. Spontaneous lysis ranged from 25 to 35%. (B) IFN-γ secretion after stimulation with peptide 4 was measured in clone 4 from subject AC-25 in the presence of various concentrations of concanamycin A (CMA). B-LCL were pulsed with peptide 4 and added at an E:T ratio of 100:1. The percentage of CD4⁺ T cells secreting IFN-γ is listed in the upper right of each dot plot. (C) Cytolysis was measured in clone 4 from subject AC-25 in the presence of various concentrations of concanamycin A and compared to IFN-γ production (from panel B). B-LCL were pulsed with peptide 4 and added at an E:T ratio of 10:1 for the CTL assay. While abrogation of IFN-γ secretion was seen at the highest concentration of concanamycin A, lysis was completely inhibited at concentrations of concanamycin A that had no effect on IFN-γ secretion. The left axis gives percent IFN-γ production and percent lysis.