CD85/LIR-1/ILT2 and CD152 (cytotoxic T lymphocyte antigen 4) inhibitory molecules down-regulate the cytolytic activity of human CD4+ T-cell clones specific for Mycobacterium tuberculosis

Abstract

Antigen-specific cytolytic CD4+ T lymphocytes control Mycobacterium tuberculosis infection by secreting cytokines and by killing macrophages that have phagocytosed the pathogen. However, lysis of the latter cells promotes microbial dissemination, and other macrophages engulf the released bacteria. Subsequently, CD4+ T-cell-mediated killing of macrophages goes on, and this persistent process may hamper control of infection, unless regulatory mechanisms maintain a subtle balance between lysis of macrophages by cytolytic CD4+ cells and activation of cytolytic CD4+ cells by infected macrophages. We asked whether inhibitory molecules expressed by CD4+ cytolytic T lymphocytes could play a role in such a balance. To this end, human CD4+ T-cell clones specific for M. tuberculosis were produced that displayed an autologous major histocompatibility complex class II-restricted lytic ability against purified protein derivative (PPD)-pulsed antigen-presenting cells. All T-cell clones expressed CD152 (cytotoxic T-lymphocyte antigen 4 [CTLA-4]) and CD85/leukocyte immunoglobulin-like receptor 1 (LIR-1)/immunoglobulin-like transcript 2 (ILT2) inhibitory receptors, but not CD94 and the killer inhibitory receptor (or killer immunoglobulin-like receptor [KIR]) p58.2. CD3-mediated activation of the clones was inhibited in a redirected killing assay in which CD152 and CD85/LIR-1/ILT2 were cross-linked. Specific antigen-mediated proliferation of the clones was also sharply reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked by specific monoclonal antibody (MAb) followed by goat anti-mouse antiserum. In contrast, blockade of the receptors by specific MAb only increased their proliferation. Production of interleukin 2 (IL-2) and gamma interferon (IFN-gamma) by the T-cell clones was also strongly reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked. The lytic activity of the T-cell clones against PPD-pulsed autologous monocytes or Epstein-Barr virus-activated B cells was increased by blockade and decreased by cross-linking of the receptors. These results indicate that CD152 and CD85/LIR-1/ILT2 play a role in the regulation of the antigen-specific activity of CD4+ cytolytic T lymphocytes against PPD-presenting cells.

FIG. 1

MHC class II restriction and antigen specificity of cytolytic CD4⁺ T-cell clones. The lytic activity of five cytolytic CD4⁺ T-cell clones specific for PPD from M. tuberculosis was assayed against monocytes (upper panel) or EBV-infected B cells (lower panel). Autologous target cells were untreated or previously cultured for 18 h with PPD (“PPD pulsed”). The targets were also tested in the presence of soluble anti-HLA class II MAbs, which impair presentation of PPD antigen by HLA class II molecules to cytolytic CD4⁺ T cells. Lysis of allogeneic PPD-pulsed target cells did not occur. Asterisks indicate differences that are statistically significant between cytolysis in the presence of PPD and PPD plus anti-HLA class II MAb culture conditions. P < 0.05.

FIG. 2

CD85/LIR-1/ILT2 and CD152 are expressed by M. tuberculosis-specific cytolytic CD4⁺ T-cell clones. CD85/LIR-1/ILT2 and CD152 surface and cytoplasmic immunofluorescence histograms of clones AM1 and FM1. CD85/LIR-1/ILT2 is detected by the M402 MAb on the surface and by the M402 (data not shown) and HP-F1 MAbs in the cytoplasm. CD152 shows similar behavior. Dotted lines represent negative controls obtained with isotype-matched irrelevant MAbs.

FIG. 3

CD85/LIR-1/ILT2 and CD152 regulate antigen (Ag)-specific proliferation of cytolytic CD4⁺ T-cell clones. T-cell clones were restimulated by autologous irradiated PBMCs pulsed with PPD, in the absence or presence of soluble MAbs, alone or cross-linked by GAM antiserum. Four days later, proliferation was measured by [³H]thymidine uptake. Controls were provided by cells cultured with autologous PBMCs not pulsed with PPD, as well as by cells restimulated by PPD-pulsed PBMCs in the presence of GAM antiserum alone or in the presence of two irrelevant MAbs used as controls (i.e., anti-CD8 MAb or anti-MHC class I MAb). Asterisks indicate values that are significantly different for PPD, PPD plus HP-F1, PPD plus HP-F1 plus GAM, PPD plus anti-CD152, and PPD plus anti-CD152 plus GAM culture conditions. P < 0.05. ▨, blocking MAb conditions; ░⃞, cross-linking of MAb specific for the inhibitory receptors.

FIG. 4

Cross-linked CD85/LIR-1/ILT2 and CD152 inhibit restimulation and induce delayed cell death. Flow cytometric DNA content analysis was performed on clone AM1 10 days after restimulation carried out in the absence or presence of HP-F1 or anti-CD152, soluble or cross-linked by GAM antiserum. The marker to the left of the G₁ peak (sub-G₁ region) accounts for apoptotic cells, and the marker to the right of the G₁ peak indicates S + G₂M cells expressed as a percentage of the viable cells (i.e., S+G₂M/G₁+ S + G₂M).

FIG. 5

CD85/LIR-1/ILT2 and CD152 regulate the antigen-specific cytotoxic activity of CD4⁺ T-cell clones. The function of cytolytic CD4⁺ T-cell clones against autologous antigen-presenting B-EBV cells or monocytes was assayed in a conventional 4-h ⁵¹Cr-release assay, carried out in the absence or presence of soluble MAbs, alone or cross-linked by GAM antiserum. Soluble HP-F1 or anti-CD152 MAb significantly increased specific lysis, while the same MAb cross-linked by GAM antiserum reduced lysis. Control samples were treated with GAM antiserum alone or two irrelevant MAbs (i.e., anti-CD8 or anti-MHC class I MAb). Asterisks indicate values that are significantly different for PPD, PPD plus HP-F1, PPD plus HP-F1 plus GAM, PPD plus anti-CD152, and PPD plus anti-CD152 plus GAM culture conditions. P < 0.05. ▨, blocking MAb conditions; ░⃞, cross-linking of MAb specific for inhibitory receptors.

FIG. 6

CD85/LIR-1/ILT2 and CD152 down-regulate IL-2 and IFN-γ production by cytolytic CD4⁺ T-cell clones. Twenty-four hours after restimulation of CD4⁺ T-cell clones with OKT3 and irradiated feeder PBMCs, the amount of IL-2 and IFN-γ in the culture medium was measured and expressed as picograms per milliliter. At the time of restimulation, HP-F1 and anti-CD152 were added to the cultures alone (blockade of receptors) or cross-linked by GAM antiserum (cross-linking of the receptors). A negative “restimulation control” was provided by T cells exposed to irradiated autologous feeder PBMCs only, in the absence of OKT3. Asterisks indicate values that are significantly different for anti-CD3, anti-CD3 plus HP-F1, anti-CD3 plus HP-F1 plus GAM, anti-CD3 plus anti-CD152, and anti-CD3 plus anti-CD152 plus GAM culture conditions. P < 0.05.