HIV-specific T cell cytotoxicity mediated by RANTES via the chemokine receptor CCR3

Abstract

CC chemokines produced by CD8(+) T cells are known to act as HIV-suppressive factors. We studied the possible role of these chemokines in HIV-1-specific killing of target cells. We found that the activity of cytotoxic T lymphocytes (CTLs) in CTL lines or freshly isolated peripheral blood mononuclear cells from HIV-1-infected individuals is markedly enhanced by RANTES (regulated on activation, normal T cell expressed and secreted) and virtually abolished by an antibody neutralizing RANTES or the RANTES receptor antagonist RANTES(9-68). Lysis was mediated by CD8(+) major histocompatibility complex class I-restricted T cells and was obtained with target cells expressing epitopes of the HIV-1LAI proteins Gag, Pol, Env, and Nef. The cytolytic activity observed in the presence or absence of added RANTES could be abolished by pretreatment of the CTLs with pertussis toxin, indicating that the effect is mediated by a G protein-coupled receptor. The chemokines monocyte chemotactic protein (MCP)-3, MCP-4, and eotaxin acted like RANTES, whereas macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MCP-1, and stromal cell-derived factor 1 were inactive, suggesting a role for the eotaxin receptor, CCR3, and ruling out the involvement of CCR1, CCR2, CCR5, and CXCR4. CTL activity was abrogated by an antibody that blocks CCR3, further indicating that specific lysis is triggered via this chemokine receptor. These observations reveal a novel mechanism for the induction of HIV-1-specific cytotoxicity that depends on RANTES acting via CCR3.

Figure 1

RANTES enhances HIV-1–specific CTL activity. (A) Transformed autologous B cells infected with recombinant vaccinia virus expressing Gag HIV-1 antigens were used as targets. Lysis by HIV-1–specific CTLs was determined in the presence of 25 nM RANTES (closed squares), 1 μg/ml anti-RANTES (open circles), or without additions (closed circles). No lysis was obtained in the presence or absence of 25 nM RANTES (open squares and closed triangles, respectively) when transformed autologous B cells infected with wild-type recombinant vaccinia virus were used as targets. Similar effects were obtained in eight experiments with CTLs from different HIV-1–infected donors. (B) Transformed autologous B cells preincubated with the synthetic peptide 476–484 corresponding to the Pol epitope of HIV-1_LAI were used as targets. Lysis by HIV-1–specific CTLs was determined in the presence of 25 nM RANTES (closed squares), 25 nM RANTES(9-68) (open squares), 1 μg/ml anti-RANTES (open circles), or without additions (closed circles). Data from one experiment. (C) Human plasmocytoid cells Hmy cotransformed with the HLA-A2 and the HIV-1_LAI nef genes were used as targets. Lysis by HIV-1–specific CTLs was determined in the presence of 25 nM RANTES (closed squares), 25 nM RANTES(9-68) (open squares), 1 μg/ml anti-RANTES (open circles), or without additions (closed circles). The data are representative for two experiments. (D) RANTES does not affect the lytic activity of NK cells. Lysis of K562 target cells by NK cells obtained from venous blood of healthy donors was determined in the presence of 25 nM RANTES (closed squares), 1 μg/ml anti-RANTES (open circles), or without additions (open squares). The data are representative for four experiments. (E) Effects of 25 nM RANTES, 1 μg/ml anti-RANTES and 25 nM RANTES(9−68) on the activity of HIV-1–specific CTLs from eight different donors. Transformed autologous B cells infected with recombinant vaccinia virus expressing Gag (squares), Pol (circles), or Env (triangles) were used as targets. Data are shown for an E/T ratio of 40:1. The straight lines connect the lytic activity values obtained in the absence (−) or presence (+) of the added reagent as indicated.

Figure 2

(A) Concentration dependence of the effect of RANTES on HIV-1–specific cytolysis. Transformed autologous B cells infected with recombinant vaccinia virus expressing Gag were used as targets. Lysis by HIV-1–specific CTLs was assessed at an E/T ratio of 120:1 in the presence of increasing concentrations of RANTES (closed circles), the antagonist RANTES(9-68) (open circles), or MIP-1α (closed triangles). The data are representative for two experiments. (B) Effect of B. pertussis toxin. The effector cells were pretreated with RANTES in the absence (closed circles) or the presence (open circles) of pertussis toxin, washed, and then mixed with target cells expressing HIV-1 Pol protein. The toxin concentration was 10, 100, 1,000, or 5,000 ng/ml (open circles, top to bottom). The data are representative for four experiments. (C) RANTES acts on the HIV-1–specific CTLs but not on the target cells. Lysis was assessed at a similar E/T ratio after pretreatment of the effector (white bars) or the target cells (hatched bars) with RANTES, MIP-1α (all 25 nM) or without additions. Pretreatment was performed for 1 h at 37°C followed by washing, immediately before the Cr-release assay. The data are representative for two experiments.

Figure 3

Lysis by HIV-1–specific CTLs is mediated by CCR3. Transformed autologous B cells infected with recombinant vaccinia virus expressing Gag were used as target cells. Lysis by HIV-1–specific CTL lines obtained from two individuals (A and B) was determined at an E/T ratio of 120:1 in the presence of different chemokines (all 25 nM), 25 nM RANTES(9-68), 1 μg/ml anti-RANTES, 1 μg/ml anti-CCR3, 1 μg/ml anti-CCR3 plus 25 nM RANTES, or without additions. Similar effects were obtained in eight experiments with CTLs from different HIV-1–infected donors.

Figure 4

(Top) RANTES enhances the CTL activity of freshly isolated PBMCs. The lytic activity of PBMCs obtained from two HIV-1– infected individuals (A and B) was assayed at an E/T ratio of 120:1 against transformed autologous B cells infected with recombinant vaccinia virus expressing Pol (A) and Gag (B) in the presence of 25 nM RANTES, 1 μg/ml anti-RANTES, 1 μg/ml anti-CCR3, 1 μg/ml anti-CCR3 plus 25 nM RANTES, or without additions. (Middle and bottom) RANTES activity is mediated by MHC-restricted CD8⁺ T cells. HIV-1–specific lysis by CTL lines derived from the PBMCs of the same patients (A and B) was determined against autologous and mismatched transformed B cells infected with recombinant vaccinia virus expressing Pol (A) and Gag (B) in the presence of 25 nM RANTES, 1 μg/ml anti-RANTES, 1 μg/ml anti-CD8, 1 μg/ml anti-CD8 plus 25 nM RANTES, or without additions.