Inhibitory receptors CD85j, LAIR-1, and CD152 down-regulate immunoglobulin and cytokine production by human B lymphocytes.

Class switching consists in the substitution of the heavy-chain constant region of immunoglobulin M (IgM) with that of IgG, IgA, or IgE. This enables antibodies to acquire new effector functions that are crucial to combat invading pathogens. Class switching usually requires engagement of CD40 on B cells by CD40 ligand (CD40L) on antigen-activated CD4(+) T cells and the production of cytokines. The process must be regulated tightly because abnormal IgG and IgA production favors the onset of autoimmunity, whereas increased switching to IgE leads to atopy. These inflammatory disorders can be triggered or exacerbated by costimulatory signals. Although thoroughly investigated on T cells, the roles of the inhibitory receptors CD85j, LAIR-1, and CD152 on B-cell functions have not been fully elucidated. In this study we show that cross-linking of the B-cell inhibitory receptors by specific monoclonal antibodies inhibits IgG and IgE production, reduces the percentage of IgG- and IgE-expressing B cells, and down-regulates interleukin 8 (IL-8), IL-10, and tumor necrosis factor alpha production. These effects were demonstrated using different B-cell stimulatory pathways (recall antigens, CD40L-transfected cells plus IL-4, and lipopolysaccharide plus IL-4). It thus appears that CD85j, LAIR-1, and CD152 play a central role for the control of IL-4-driven isotype switching.

CD85j (leukocyte Ig-like receptor-1/Ig-like transcript 2) inhibits human osteoclast-associated receptor-mediated activation of human dendritic cells.

Immature dendritic cells (DCs) derived from freshly isolated human monocytes were used to evaluate the effect of the inhibiting receptor CD85j (leukocyte Ig-like receptor-1/ILT2) on activation induced by cross-linking of the human osteoclast-associated receptor (hOSCAR). CD85j and hOSCAR were expressed consistently at the same density on monocytes and on monocyte-derived DCs (both immature and mature). Cross-linking of hOSCAR, which activates via the FcR-associated gamma-chain, induced Ca(2+) flux in DCs. Concomitant cross-linking of anti-CD85j mAb abolished this early activation event. Likewise, CD85j stimulation strongly reduced IL-8 and IL-12 production by hOSCAR-activated DCs. Inhibition of DCs via CD85j also impaired their ability to enhance Ag-specific T cell proliferation induced by hOSCAR. Finally, because hOSCAR prevents apoptosis of DCs in the absence of growth/survival factors, CD85j cross-linking was able to counteract completely this antiapoptotic effect and to reduce Bcl-2 expression enhanced by hOSCAR stimulation. Thus, CD85j is an inhibiting receptor that is functional in human DCs.

Specific recognition of the viral protein UL18 by CD85j/LIR-1/ILT2 on CD8+ T cells mediates the non-MHC-restricted lysis of human cytomegalovirus-infected cells.

Immune evasion mechanisms of human CMV are known; however, the immune control of infection remains poorly elucidated. We show that interaction between the viral protein UL18 on infected cells and the invariant receptor CD85j/LIR-1/ILT2 expressed on CTL is relevant for the control of infection. Resting and activated CD8(+) T cells lysed UL18 expressing cells, whereas cells infected with CMV defective for UL18 were not killed. Lysis was not dependent on CD8(+) T cell Ag specificity, MHC-unrestricted and specifically blocked by anti-CD85j and anti-UL18 mAb. Moreover, soluble recombinant UL18Fc immunoprecipitated CD85j from T cells. Activation is mediated by CD85j and its pathway is unrelated to CD3/TCR engagement. UL18 is detected in immunocompromised patients with productive infection and the mechanism used in vivo by human CMV to ensure survival of the immunocompetent host may be mediated by activation signals delivered by infected cells to T lymphocytes via UL18/CD85j interactions.

Death of T cell precursors in the human thymus: a role for CD38.

Thymic T cell maturation depends on interactions between thymocytes and cells of epithelial and hematopoietic lineages that control a selective process whereby developing T cells with inappropriate or self-reactive receptors die. Molecules involved in this process are the TCR expressed on thymocytes together with the CD3 complex and MHC-peptide on accessory cells. However, other molecules may favor or prevent death of thymocytes, thus playing a role in selection. CD38 is expressed by the majority of human thymocytes, mainly at the double-positive (DP) stage. In contrast, CD38 is not found on subcapsular double-negative (DN) thymocytes and on a proportion of medullary single-positive (SP) thymocytes. CD38 enhances death of thymocytes when it is cross-linked by goat anti-mouse (GAM) antiserum or by one of its ligands, CD31, expressed by thymic epithelial cells or transfected into murine fibroblasts (L cells). As most thymocytes are at an intermediate (DP) stage of development, it is likely that these cells are most vulnerable to death mediated via MHC-peptide-TCR interactions that is increased by CD38 cross-linking. DN and SP thymocytes are refractory to CD38-induced apoptosis. Accessory molecules, e.g. CD38, are expressed during thymic cell maturation and their presence is relevant for the survival or death of DP T cells in the course of selection. Based on our data, CD38 enhances thymocyte death by interacting with CD31 expressed by accessory cells. In addition, CD28 expression on developing thymocytes also appears to play a role for their selection and it synergizes with CD38 to induce apoptosis of DP thymocytes.

Surface density expression of the leukocyte-associated Ig-like receptor-1 is directly related to inhibition of human T-cell functions.

The relevance of inhibitory receptors that downregulate T-cell functions, such as CD152 (CTLA-4) and CD85j, have been extensively analyzed. This study will show that leukocyte-associated Ig-like receptor-1 (LAIR-1) acts as an inhibitory receptor for antigen-specific human effector T cells. To this end 28 CD8(+) and 22 CD4(+) T-cell clones were analyzed. LAIR-1 activity appears to be clonally distributed among T-cell clones and inhibition of T lymphocyte functions ranges from 4% to 49% in a redirected killing assay. This inhibitory function, although less efficient than that exerted by other inhibitory receptors expressed by T cells (i.e., CD152 and CD85j), downregulates the cytotoxic activity of CD8(+) T lymphocytes, both in a CD3-mediated and in an antigen-specific system. Furthermore, LAIR-1 inhibits the proliferative response of CD4(+) T lymphocytes to recall antigens and in CD3 stimulation. LAIR-1 also modulates cytokine production, downregulating IL-2 and IFN-gamma production. In contrast, LAIR-1 crosslinking induces secretion of transforming growth factor beta. This study will also demonstrate that a direct relationship exists between surface density expression of LAIR-1 molecules and their ability to modulate CD3-mediated activation of both CD8(+) and CD4(+) T-cell clones.

Apoptosis of squamous cells at different stages of carcinogenesis following 4-HPR treatment.

Squamous cell carcinoma (SCC) is the end product of a multistep process characterized by a progression from normal epithelial cells through metaplastic or dysplastic intraepithelial changes that evolve into invasive cancer. Since retinamides have shown promising in vivo anti-tumoral activity, we studied effects and effector mechanisms of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) on squamous cells at progressing stages of tumorigenesis. To this end, an in vitro model of squamous carcinogenesis consisting of normal human keratinocytes, human papilloma virus (HPV)-immortalized keratinocytes (UP) and tumorigenic HPV-immortalized/v-Ha-ras transfected keratinocytes (UPR) was used. 4-HPR treatment affected cell growth at doses higher than 1.5 microM. Flow cytometric measurements of DNA content and annexin V revealed that cell growth decrease was mainly due to apoptosis at 4-HPR concentrations of or below 15 microM, and necrosis at higher concentrations. The effects were similar in the three cell types of the in vitro model, as well as in three SCC cell lines, suggesting that sensitivity to 4-HPR is independent of the degree of squamous cell tumorigenesis in the in vitro model. We further investigated whether mitochondrial damage was involved in the course of 4-HPR-induced apoptosis. Treatment of squamous cells with the antioxidant L-ascorbic acid inhibited apoptosis, indicating that 4-HPR increases production of free radicals. Measures of mitochondrial membrane potentials showed that 4-HPR induced membrane permeability transition (MPT), and that MPT-inhibitors were able to reduce apoptosis. This indicates that MPT is involved in apoptosis signalling by 4-HPR. Finally, we studied the role of caspases. We found that caspases 8, 9 and 3 participate in 4-HPR-mediated apoptosis of squamous cells, and that MPT is an upstream event that regulates caspase activity. Caspase 8 was activated independently of the Fas-Fas ligand pathway.

Dual effect of CD85/leukocyte Ig-like receptor-1/Ig-like transcript 2 and CD152 (CTLA-4) on cytokine production by antigen-stimulated human T cells.

The functional outcome of a T cell response to Ag is the result of a balance between coactivation and inhibitory signals. In this study we have investigated the effects of the CD85/leukocyte Ig-like receptor (LIR)-1/Ig-like transcript (ILT) 2 and of CD152 (CTLA-4) inhibitory receptors on the modulation of cell-mediated immune responses to specific Ags, both at the effector and at the resting/memory cell level. Proliferation and cytokine production of CD4+ T lymphocytes stimulated by recall Ags have been evaluated. Cross-linking of CD85/LIR-1/ILT2 or CD152 molecules on cultured T cells using specific mAb and goat anti-mouse antiserum inhibits Ag-specific T cell proliferation. This inhibition is always paralleled by increased production of cytokines that down-regulate immune responses, e.g., IL-10 and TGF-beta. In contrast, the production of cytokines that support T cell expansion and function (e.g., IL-2, IFN-gamma, and IL-13) is significantly decreased. A long-term effect of CD85/LIR-1/ILT2 and of CD152 occurs during Ag-specific T cell activation and expansion. T cells, primed in the presence of anti-CD85/LIR-1/ILT2 and anti-CD152 blocking mAb (but in the absence of cross-linking), proliferate at higher rates and produce higher amounts of IL-2, IFN-gamma, and IL-13, in comparison with T cells stimulated with the Ag alone. We also show that the inhibitory receptors exert a similar effect during Ag activation of specific CD4+ effector T cells. Ag-specific polyclonal CD4+ T cell lines exhibit increased proliferation and IL-2, IFN-gamma, and IL-13 production when the CD85/LIR-1/ILT2 receptor is blocked by specific mAb. In contrast, cross-linking of this receptor down-regulates Ag-specific CD4+ T cell proliferation and increases IL-10 and TGF-beta production.