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Human natural killer cells: Origin, clonality, specificity, and receptors

Publisher Summary This chapter focuses on human natural killer (NK) cells. NK cells are considered as effector cells that lyse tumor cells or virally infected cells via nonspecific mechanisms. Both their cytolytic effect and the spectrum of their antitumor activity are greatly increased after culture of NK cells in interleukin- 2 (IL-2). IL-2-activated NK cells represent the most potent effector cells among the lymphokine-activated killer (LAK) cells. Several of the generally accepted theories concerning NK cells have been challenged by evidence based on a variety of new experimental approaches and technological breakthroughs. NK cells may originate from precursors present in early fetal liver and even in immature postnatal thymocytes. They specifically recognize class I molecules. Different from T cells, binding of NK cells to class I molecules appears to result in a “negative signal” leading to target cell protection. Many tumor cells that are susceptible to lysis mediated by NK/LAK cells express normal amounts of surface class I antigens. This phenomenon is explained in that (1) the tumor cells express class I antigens that do not function as specific protective elements for the allogeneic NK cells most frequently used in the cytolytic assays and (2) the tumor cells may lack the expression of single class I alleles functioning as protective elements. The ability of activated NK cells to lyse efficiently a wider spectrum of tumor cells can be interpreted as the expression of appropriate activation receptors on these cells as a consequence of cell activation.

Allorecognition by NK cells: nonself or no self?

The issue of antigen recognition by NK cells is complex, fascinating and, as yet, unresolved. This article reviews recent research on the repertoire of human NK cell clones for the recognition of different allogeneic cells, and summarizes the studies, most of which have been performed in mice, that implicate the MHC in NK cell recognition. It goes on to provide a common conceptual framework within which these different systems may be understood.

The CD85/LIR-1/ILT2 inhibitory receptor is expressed by all human T lymphocytes and down-regulates their functions

The inhibitory molecule CD85/LIR-1/ILT2 has been detected previously on the surface of a small proportion of T lymphocytes. In this study, evidence is provided that, although only a fraction of CD3+ cells are stained by mAb specific for CD85/LIR-1/ILT2 on their surface, this inhibitory receptor is present in the cytoplasm of all T lymphocytes, and that it is detectable on the surface of all T cell clones by the M402 mAb. Biochemical analyses further demonstrate that CD85/LIR-1/ILT2 is present in all T clones analyzed, and that the protein is tyrosine-phosphorylated. Expression of mRNA coding for CD85/LIR-1/ILT2 has been assessed by RT-PCR. Notably, in the NKL cell line and in one T cell clone, amplification of the messenger required 30 cycles only, whereas, in other T cell clones, an amplification product was detected by increasing the number of cycles. CD85/LIR-1/ILT2 inhibits CD3/TCR-mediated activation in both CD4+ and CD8+ clones, and it down-regulates Ag recognition by CD8+ cells in a clonally distributed fashion. Addition of anti-ILT2 HP-F1 mAb in the cytolytic assay enhances target cell lysis mediated by Ag-specific CTL. This could be due to interference of the mAb with receptor/ligand interactions. In contrast, HP-F1 mAb cross-linking triggers inhibitory signals that reduce cytotoxicity. CD85/LIR-1/ILT2 also controls responses to recall Ags and, in low responders, its engagement sharply increases T cell proliferation. The inhibitory function of the molecule is also confirmed by its ability to reduce CD3/TCR-induced intracellular Ca2+ mobilization.

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-β. In contrast, the production of cytokines that support T cell expansion and function (e.g., IL-2, IFN-γ, 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-γ, 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-γ, 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-β production.

CD1d is expressed on B‐chronic lymphocytic leukemia cells and mediates α‐galactosylceramide presentation to natural killer T lymphocytes

Generation of immune responses against B cell chronic lymphocytic leukemia (B‐CLL) has been the aim of several studies that have demonstrated a poor antigen presenting ability of B‐CLL cells and an inconsistent emergence of T cells capable of killing efficiently the leukemic cells. CD1d is a restriction element structurally related to the major histocompatibility complex (MHC) and capable of presenting lipid antigens to CD1d‐restricted T cells (also defined as natural killer‐T [NKT] cells). The synthetic lipid α‐galactosylceramide (α‐GalCer) has been characterized as a potent stimulator of CD1d‐restricted T cells. We have investigated the expression of CD1d on B‐CLL cells. CD1d was detected by flow cytometric analyses on leukemic cells of all B‐CLL cases studied (n = 38) and was expressed at higher density on cells carrying unmutated immunoglobulin variable region (IgV) genes. In addition, CD1d on B‐CLL cells mediated the presentation of α‐GalCer to CD1d‐restricted T cells, which in turn induced B‐CLL cell death. At variance with another study (Metelitsa et al., Leukemia 2003;17:1068–77), no correlation between expression levels of CD1d and susceptibility to NKT cell lysis was observed. Proliferation and production of interferon‐γ (IFN‐γ) and tumor necrosis factor‐α (TNF‐α) by CD1d‐restricted T cells, in the presence of B‐CLL cells loaded with α‐GalCer, were also observed. Our study demonstrates that B‐CLL cells express a monomorphic restriction element that is functionally capable of antigen presentation and can be useful to design novel B‐CLL immunotherapies.

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.

Inhibitory Receptors CD85j, LAIR-1, and CD152 Down-Regulate Immunoglobulin and Cytokine Production by Human B Lymphocytes

ABSTRACT 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.

Molecular and Cellular Analysis of Human T Lymphocytes Expressing γδ T-Cell Receptor

A minor subset of T lymphocytes expresses a CD3-associated TCR composed of gamma and delta chains. The majority of TCR gamma/delta+ cells lack surface CD4 and CD8 antigen and do not react with WT31 mAb. These negative criteria were utilized in early studies to identify TCR gamma/delta+ cells. More recently, mAb to TCR gamma/delta, selected in different laboratories, have permitted the direct identification of TCR gamma/delta+ cells and their subsets. TCR gamma/delta molecules were found to be heterogeneous in size and charge mobility. Two major forms of TCR gamma/delta could be identified that are characterized by the presence or absence of an inter-chain disulphide bond. Biochemical analysis originally suggested that a precise correlation existed between reactivity with BB3 or delta TCS1/A13 mAb and expression of a disulphide (C gamma 1-encoded) or non-disulphide linked (C gamma 2-encoded) form of TCR gamma/delta. However, more recent studies have indicated that these mAb react with the molecular product of V delta 2 or V delta 1, respectively, mAb directed to one or another form of TCR gamma/delta activate the functional program of the cell, leading to intracellular Ca++ mobilization, lymphokine production and triggering of the lytic machinery. Analysis of the target molecules for TCR gamma/delta-mediated recognition revealed that at least some TCR gamma/delta+ cells are capable of specific responses to (allo)antigen and that polymorphic determinants of class I molecules can be recognized (as shown by the specific lysis of P815 cells transfected with HLA-24 allele). Unlike TCR alpha/beta+ cells, TCR gamma/delta+ cells are homogeneously composed of cytolytic precursors, as shown by the analysis of a large panel of clones in both lectin-dependent and redirected killing assays. In spite of their LGL morphology, freshly isolated TCR gamma/delta+ cells do not lyse NK-sensitive targets but do so after exposure to rIL-2. A modest cytolytic activity, however, could be induced also in fresh cells by anti-TCR/CD3 mAb in a redirected killing assay. Analysis of the distribution of the subsets expressing different TCR gamma/delta types showed that BB3+ cells are prevalent in the peripheral blood and virtually absent in the thymus; in contrast, A13+ (delta TCS1+) cells represent the majority of TCR gamma/delta+ thymocytes. Electron microscopic analysis of fresh TCR gamma/delta+ cells showed an extended cytoplasm containing numerous electron-dense granules identifiable as primary lysosomes. Upon stimulation with IL-2, TCR gamma/delta+ cells, similar to other LAK cells, display an increase in their cytoplasmic granules together with a redistribution of cytoskeletal structures.(ABSTRACT TRUNCATED AT 400 WORDS)

CD1d expression on B-precursor acute lymphoblastic leukemia subsets with poor prognosis.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Although therapeutical advances have been achieved, some ALL subgroups still fare poorly. CD1d is a monomorphic molecule that provides a suitable target for immunotherapy in view of the characterization of a glycolipid, α-galactosylceramide (α-GalCer), capable of being presented to CD1d-restricted T cells with cytotoxic potential. We investigated CD1d expression in 80 pediatric B-cell precursor (BCP) ALL cases defined according to immunophenotype, cytogenetic features and age at onset. CD1d was detected on ALL cells in 15% of the patients. CD1d+ ALLs were significantly associated with infant leukemia, pro-B phenotype and mixed-lineage leukemia (MLL)/AF4 gene rearrangement. Accordingly, overall survival of patients with CD1d+ ALL was significantly shorter. CD1d+ leukemic blasts were able to present α-GalCer via CD1d to cytotoxic CD1d-restricted T cells, which induced apoptosis of ALL cells that was inhibited by mAb to CD1d. CD1d+ blasts loaded with α-GalCer elicited cytokine secretion by CD1d-restricted T cells. Analysis of bone marrow (BM) cells derived from normal donors revealed that CD19+/CD1d+ cells were mostly mature B lymphocytes. However, a minority of BCPs expressed CD1d. Thus, expression of CD1d in ALL cases heralds an adverse prognosis but may provide a therapeutic tool.

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 controlMycobacterium 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-γ) 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.