Cytolytic mechanisms and expression of activation-regulating receptors on effector-type CD8+CD45RA+CD27- human T cells

Circulating CD8+ T cells with a CD45RA+CD27- phenotype resemble cytolytic effector cells because they express various cytolytic mediators and are able to execute cytotoxicity without prior stimulation in vitro. We here demonstrate that CD8+CD45RA+CD27- T cells can use both granule exocytosis and Fas/Fas ligand pathways to induce apoptosis in target cells. The availability of these cytolytic mechanisms in circulating T cells suggests that the activity of these cells must be carefully controlled to prevent unwanted tissue damage. For this reason, we analyzed the expression of surface receptors that either enhance or inhibit T cell function. Compared with memory-type cells, effector cells were found to express normal levels of CD3epsilon and TCRzeta and relatively high levels of CD8. CTLA-4 was absent from freshly isolated effector cells, whereas a limited number of unstimulated memory cells expressed this molecule. In line with recent findings on CD8+CD28- T cells, CD45RA+CD27- T cells were unique in the abundant expression of NK cell-inhibitory receptors, both of Ig superfamily and C-type lectin classes. Binding of NK cell-inhibitory receptors to classical and nonclassical MHC class I molecules may inhibit the activation of the cytolytic machinery induced by either Ag receptor-specific or nonspecific signals in CD8+CD45RA+CD27- T cells.     

Lymphocyte activation in response to melanoma: interaction of NK-associated receptors and their ligands

In recent years, studies on the molecular and cellular mechanisms of immune responses against melanoma have contributed to a better understanding of how these tumours can be recognised by cytotoxic cells and the mechanisms they have developed to escape from innate and adaptive immunity. Lysis of melanoma cells by natural killer (NK) cells and cytolytic T cells is the result of a fine balance between signals transmitted by activating and inhibitory receptors. In addition to the T cell receptor, these were initially described as NK cell-associated receptors (NKRs) and were later also found on subsets of T lymphocytes, particularly effector-memory and terminally differentiated CD8 T cells. An increase of NKR(+)CD8(+) T cells has been found in melanoma patients, correlating with the expansion of differentiated effector CD8(+)CD28(null) CD27(null) T cells. NKRs can regulate the lysis of target cells expressing appropriate ligands. Activating receptors recognise ligands on tumours whereas inhibitory receptors are specific for MHC class I antigens and sense missing self. Altered expression of MHC class I antigens is frequently found on melanoma cells, preventing recognition by specific cytolytic T cells but favouring NK cell recognition. Changes in the expression of NKR-ligands in melanoma contribute in explaining the differences in the capacity of cytotoxic immune cells to control melanoma growth and dissemination.     

Stimulatory killer Ig-like receptors modulate T cell activation through DAP12-dependent and DAP12-independent mechanisms

Stimulatory killer Ig-like receptors (KIRs) are expressed by various lymphocytes, including NK cells and subsets of T cells. In NK cells, KIRs associate with the adapter molecule KARAP/DAP12, which confers the ability to function as an independent activation unit. The function of KIRs and killer cell activating receptor-associated protein (KARAP)/DAP12 in T cells is unclear. By flow cytometry, we demonstrated that CD4+CD28null T cells heterogeneously express KIRs and/or KARAP/DAP12. In clones that lacked expression of KARAP/DAP12, the stimulatory KIR KIR2DS2 signaled through the JNK pathway, but did not activate the ERK pathway. However, in the presence of KARAP/DAP12, stimulation through KIR2DS2 led to phosphorylation of both JNK and ERK. Transfection experiments confirmed that KIR2DS2-mediated ERK phosphorylation was dependent on KARAP/DAP12. The differential signaling of KIR2DS2 through association with alternative adapter molecules resulted in differential regulation of cellular activity. In clones that lacked expression of KARAP/DAP12, stimulation of KIR2DS2 did not induce cytotoxicity. However, KIR2DS2 did augment suboptimal TCR stimulation, leading to enhanced IFN-gamma production. In clones that expressed KARAP/DAP12, KIR2DS2 directly activated both cytotoxicity and IFN-gamma production without the need for TCR-derived signals. The function of stimulatory KIRs in T cells is determined by the expression of the appropriate adapter molecule. Expression of KARAP/DAP12 is sufficient to convert a costimulatory KIR into a stimulatory molecule. These differing functions mediated by alternative signaling pathways have implications for the pathogenesis of diseases such as rheumatoid arthritis and acute coronary syndromes, in which aberrant expression of KIRs on T cells is frequently observed.     

CD4+CD28null T cells in autoimmune disease: pathogenic features and decreased susceptibility to immunoregulation

To determine the role of expanded CD4(+)CD28(null) T cells in multiple sclerosis and rheumatoid arthritis pathology, these cells were phenotypically characterized and their Ag reactivity was studied. FACS analysis confirmed that CD4(+)CD28(null) T cells are terminally differentiated effector memory cells. In addition, they express phenotypic markers that indicate their capacity to infiltrate into tissues and cause tissue damage. Whereas no reactivity to the candidate autoantigens myelin basic protein and collagen type II was observed within the CD4(+)CD28(null) T cell subset, CMV reactivity was prominent in four of four HC, four of four rheumatoid arthritis patients, and three of four multiple sclerosis patients. The level of the CMV-induced proliferative response was found to be related to the clonal diversity of the response. Interestingly, our results illustrate that CD4(+)CD28(null) T cells are not susceptible to the suppressive actions of CD4(+)CD25(+) regulatory T cells. In conclusion, this study provides several indications for a role of CD4(+)CD28(null) T cells in autoimmune pathology. CD4(+)CD28(null) T cells display pathogenic features, fill up immunological space, and are less susceptible to regulatory mechanisms. However, based on their low reactivity to the autoantigens tested in this study, CD4(+)CD28(null) T cells most likely do not play a direct autoaggressive role in autoimmune disease.     

Skewed distribution of proinflammatory CD4+CD28null T cells in rheumatoid arthritis

Expanded populations of CD4+ T cells lacking the co-stimulatory molecule CD28 (CD4+CD28null T cells) have been reported in several inflammatory disorders. In rheumatoid arthritis, increased frequencies of CD4+CD28null T cells in peripheral blood have previously been associated with extra-articular manifestations and human cytomegalovirus (HCMV) infection, but their presence in and contribution to joint manifestations is not clear. In the present article we investigated the distribution of CD4+CD28null T cells in the synovial membrane, synovial fluid and peripheral blood of RA patients, and analysed the association with erosive disease and anti-citrullinated protein antibodies. CD4+CD28null T cells were infrequent in the synovial membrane and synovial fluid, despite significant frequencies in the circulation. Strikingly, the dominant TCR-Vbeta subsets of CD4+CD28null T cells in peripheral blood were often absent in synovial fluid. CD4+CD28null T cells in blood and synovial fluid showed specificity for HCMV antigens, and their presence was clearly associated with HCMV seropositivity but not with anti-citrullinated protein antibodies in the serum or synovial fluid, nor with erosive disease. Together these data imply a primary role for CD4+CD28null T cells in manifestations elsewhere than in the joints of patients with HCMV-seropositive rheumatoid arthritis.     

NK cell inhibitory receptors prevent tyrosine phosphorylation of the activation receptor 2B4 (CD244)

2B4 is an NK cell activation receptor that can provide a co-stimulatory signal to other activation receptors and whose mode of signal transduction is still unknown. We show that cross-linking of 2B4 on NK cells results in its rapid tyrosine phosphorylation, implying that this initial step in 2B4 signaling does not require coligation of other receptors. Ligation of 2B4 in the context of an NK cell-target cell interaction leads to 2B4 tyrosine phosphorylation, target cell lysis, and IFN-gamma release. Coligation of 2B4 with the inhibitory receptors killer cell Ig-like receptor (KIR)2DL1 or CD94/NKG2 completely blocks NK cell activation. The rapid tyrosine phosphorylation of 2B4 observed upon contact of NK cells with sensitive target cells is abrogated when KIR2DL1 or CD94/NKG2 are engaged by their cognate MHC class I ligand on resistant target cells. These results demonstrate that NK inhibitory receptors can interfere with a step as proximal as phosphorylation of an activation receptor.     

Triggering of murine NK cells by CD40 and CD86 (B7-2)

NK cell-mediated cytotoxicity is regulated by both triggering and inhibitory signals. The interaction between MHC class I molecules expressed on target cells and specific MHC class I-binding receptors expressed by NK cells generally leads to inhibition of lysis. We have shown recently that CD80 (B7-1) in mice and CD40 in humans trigger NK cell-mediated cytotoxicity in vitro. In the present study, we show that murine CD40 and CD86 (B7-2) trigger murine NK cell-mediated cytotoxicity in vitro when expressed on tumor cells. Preincubation of the transfected cell lines with anti-CD40 F(ab')2 fragments or cytolytic T lymphocyte-associated Ag-4-Ig (CTLA-4-Ig) before the cytotoxic assay abolished the triggering effect. Furthermore, radiolabeled CD40- and B7-2-expressing cells were rapidly eliminated in vivo in an NK cell-dependent manner. NK cells from CD40 ligand (CD40L)-/- or CD28-/- mice were triggered by tumor cells transfected with CD40 and B7-2, respectively, and these transfectants were rapidly eliminated in vivo when inoculated into CD40L-/- and CD28-/- mice. This suggests that the CD40 and B7-2 molecules can interact with receptors on NK cells other than CD40L and CD28, respectively, and that these may account for some of the reactivities observed in the present study. Collectively, these data demonstrate that 1) costimulatory molecules, other than B7-1, can modulate NK cell responses in vitro, 2) they can also affect NK cell-dependent responses in vivo, and 3) parts of these reactions are independent of CD28 and CD40L.     

Human T-cell leukemia virus type 1 (HTLV-1) p12I down-modulates ICAM-1 and -2 and reduces adherence of natural killer cells, thereby protecting HTLV-1-infected primary CD4+ T cells from autologous natural killer cell-mediated cytotoxicity despite the reduction of major histocompatibility complex class I molecules on infected cells

Although natural killer (NK) cell-mediated control of viral infections is well documented, very little is known about the ability of NK cells to restrain human T-cell leukemia virus type 1 (HTLV-1) infection. In the current study we show that NK cells are unable to kill HTLV-1-infected primary CD4+ T cells. Exposure of NK cells to interleukin-2 (IL-2) resulted in only a marginal increase in their ability to kill HTLV-1-infected primary CD4+ T cells. This inability of NK cells to kill HTLV-1-infected CD4+ T cells occurred despite the down-modulation of major histocompatibility complex (MHC) class I molecules, one of the ligands for the major NK cell inhibitory receptor, by HTLV-1 p12(I) on CD4+ T cells. One reason for this diminished ability of NK cells to kill HTLV-1-infected cells was the decreased ability of NK cells to adhere to HTLV-1-infected cells because of HTLV-1 p12(I)-mediated down-modulation of intercellular adhesion molecule 1 (ICAM-1) and ICAM-2. We also found that HTLV-1-infected CD4+ T cells did not express ligands for NK cell activating receptors, NCR and NKG2D, although they did express ligands for NK cell coactivating receptors, NTB-A and 2B4. Thus, despite HTLV-1-mediated down-modulation of MHC-I molecules, HTLV-1-infected primary CD4+ T cells avoids NK cell destruction by modulating ICAM expression and shunning the expression of ligands for activating receptors.     

Formation of the killer Ig-like receptor repertoire on CD4+CD28null T cells

Killer Ig-like receptors (KIRs) are expressed on CD4(+)CD28(null) T cells, a highly oligoclonal subset of T cells that is expanded in patients with rheumatoid arthritis. It is unclear at what stage of development these T cells acquire KIR expression. To determine whether KIR expression is a consequence of clonal expansion and replicative senescence, multiple CD4(+)CD28(null) T cell clones expressing the in vivo dominant TCR beta-chain sequences were identified in three patients and analyzed for their KIR gene expression pattern. Based on sharing of TCR sequences, the clones were grouped into five clone families. The repertoire of KIRs was diverse, even within each clone family; however, the gene expression was not random. Three particular receptors, KIR2DS2, KIR2DL2, and KIR3DL2, had significant differences in gene expression frequencies between the clone families. These data suggest that KIRs are successively acquired after TCR rearrangement, with each clone family developing a dominant expression pattern. The patterns did not segregate with the individual from whom the clones were derived, indicating that peripheral selection in the host environment was not a major shaping force. Several models were examined using a computer algorithm that was designed to simulate the expression of KIRs at various times during T cell proliferation. The computer simulations favored a model in which KIR gene expression is inducible for a limited time during the initial stages of clonal expansion.     

Clonality and longevity of CD4+CD28null T cells are associated with defects in apoptotic pathways

CD4(+)CD28(null) T cells are oligoclonal lymphocytes rarely found in healthy individuals younger than 40 yr, but are found in high frequencies in elderly individuals and in patients with chronic inflammatory diseases. Contrary to paradigm, they are functionally active and persist over many years. Such clonogenic potential and longevity suggest altered responses to apoptosis-inducing signals. In this study, we show that CD4(+)CD28(null) T cells are protected from undergoing activation-induced cell death. Whereas CD28(+) T cells underwent Fas-mediated apoptosis upon cross-linking of CD3, CD28(null) T cells were highly resistant. CD28(null) T cells were found to progress through the cell cycle, and cells at all stages of the cell cycle were resistant to apoptosis, unlike their CD28(+) counterparts. Neither the activation-induced up-regulation of the IL-2R alpha-chain (CD25) nor the addition of exogenous IL-2 renders them susceptible to Fas-mediated apoptosis. These properties of CD28(null) T cells were related to high levels of Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein, an inhibitor of Fas signaling that is normally degraded in T cells following activation in the presence of IL-2. Consistent with previous data showing protection of CD28(null) cells from spontaneous cell death, the present studies unequivocally show dysregulation of apoptotic pathways in CD4(+)CD28(null) T cells that favor their clonal outgrowth and maintenance in vivo.     

2B4 (CD244) signaling via chimeric receptors costimulates tumor-antigen specific proliferation and in vitro expansion of human T cells

Regulatory NK cell receptors can contribute to antigen-specific adaptive immune responses by modulating T cell receptor (TCR)-induced T cell activation. We investigated the potential of the NK cell receptor 2B4 (CD244) to enhance tumor antigen-induced activation of human T cells. 2B4 is a member of the CD2 receptor subfamily with both activating and inhibitory functions in NK cells. In T cells, its expression is positively associated with the acquisition of a cytolytic effector memory phenotype. Recombinant chimeric receptors that link extracellular single-chain Fv fragments specific for the tumor-associated surface antigens CD19 and G_D2 to the signaling domains of human 2B4 and/or TCRζ were expressed in non-specifically activated peripheral blood T cells by retroviral gene transfer. While 2B4 signaling alone failed to induce T cell effector functions or proliferation, it significantly augmented the antigen-specific activation responses induced by TCRζ. 2B4 costimulation did not affect the predominant effector memory phenotype of expanding T cells, nor did it increase the proportion of T cells with regulatory phenotype (CD4+CD25^hiFoxP3+). These data support a costimulatory role for 2B4 in human T cell subpopulations. As an amplifier of TCR-mediated signals, 2B4 may provide a powerful new tool for immunotherapy of cancer, promoting sustained activation and proliferation of gene-modified antitumor T cells.     

The repertoire of CD4+ CD28- T cells in rheumatoid arthritis.

BACKGROUND: While oligoclonality of circulating CD4- CD8 and of CD8+ T cells is not uncommon, clonal dominance within the CD4 compartment is not frequently found in healthy individuals. In contrast, the majority of patients with rheumatoid arthritis (RA) have clonally expanded CD4+ T cell populations. Previous studies have demonstrated that these clonogenic CD4+ T cells do not express the CD28 molecule. To examine the correlation between CD28 expression and clonal proliferation, we have analyzed the T cell receptor (TCR) diversity of CD4+ CD28- T cells in normal individuals and in RA patients. MATERIAL AND METHODS: The size of the peripheral blood CD4+ CD28- compartment was determined in 30 healthy individuals and 30 RA patients by two-color FACS analysis. In 10 RA patients and five controls with more than 2.5% CD4+ CD28- T cells, TCR BV gene segment usage was analyzed with 19 BV-specific antibodies. Oligoclonality was assessed in sorted CD4+ CD28+ and CD28- T cells using TCR BV-BC-specific polymerase chain reaction and size fractionation. Clonal dominance was confirmed by direct sequencing. RESULTS: The CD4+ CD28- T cell compartment was expanded to more than 2.5% in 70% of the RA patients and 30% of the normal individuals. Compared with the CD4+ CD28+ T cells, the TCR BV gene segment usage among CD4+ CD28- cells was grossly skewed with the dominance of single BV elements. Molecular TCR analysis provided evidence for oligoclonality in 17 of 21 expanded BV elements. In two unrelated RA patients who shared both HLA-DRB1 alleles, the TCR beta-chain sequences of dominant clonotypes were highly conserved. CONCLUSIONS: Oligoclonality is a characteristic feature of CD4+ CD28- T cells which are expanded in some healthy individuals and in the majority of RA patients. The lack of CD28 expression is a common denominator of CD4+, CD8+, and CD4- CD8- T cells prone to develop clonal dominance. The limited TCR diversity of clonal CD4+ CD28- populations in RA patients suggests that these T cells recognize a limited spectrum of antigens. The fact that the majority of individuals with marked expansions and oligoclonality of CD4+ CD28- T cells are RA patients suggests a role for these unusual lymphocytes in the pathogenetic events leading to RA.     

Recruitment of activation receptors at inhibitory NK cell immune synapses

Natural killer (NK) cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR) family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.     

Coordinated expression of Ig-like inhibitory MHC class I receptors and acquisition of cytotoxic function in human CD8+ T cells

MHC class I-specific inhibitory receptors are expressed by a subset of memory-phenotype CD8(+) T cells. Similar to NK cells, MHC class I-specific inhibitory receptors might subserve on T cells an important negative control that participates to the prevention of autologous damage. We analyzed here human CD8(+) T cells that express the Ig-like MHC class I-specific inhibitory receptors: killer cell Ig-like receptor (KIR) and CD85j. The cell surface expression of Ig-like inhibitory MHC class I receptors was found to correlate with an advanced stage of CD8(+) T cell maturation as evidenced by the reduced proliferative potential of KIR(+) and CD85j(+) T cells associated with their high intracytoplasmic perforin content. This concomitant regulation might represent a safety mechanism to control potentially harmful cytolytic CD8(+) T cells, by raising their activation threshold. Yet, KIR(+) and CD85j(+) T cells present distinct features. KIR(+)CD8(+) T cells are poor IFN-gamma producers upon TCR engagement. In addition, KIR are barely detectable at the surface of virus-specific T cells during the course of CMV or HIV-1 infection. By contrast, CD85j(+)CD8(+) T cells produce IFN-gamma upon TCR triggering, and represent a large fraction of virus-specific T cells. Thus, the cell surface expression of Ig-like inhibitory MHC class I receptors is associated with T cell engagement into various stages of the cytolytic differentiation pathway, and the cell surface expression of CD85j or KIR witnesses to the history of qualitatively and/or quantitatively distinct T cell activation events.     

Association of human NK cell surface receptors NKR-P1 and CD94 with Src-family protein kinases

 Human natural killer (NK) cells express on their surface several members of the C-type lectin family such as NKR-P1, CD94, and NKG2 that are probably involved in recognition of target cells and delivery of signals modulating NK cell cytotoxicity. To elucidate the mechanisms involved in signaling via these receptors, we solubilized in vitro cultured human NK cells by a mild detergent, Brij-58, immunoprecipitated molecular complexes containing the NKR-P1 or CD94 molecules, respectively, by specific monoclonal antibodies, and performed in vitro kinase assays on the immunoprecipitates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and phospho-amino acid analysis revealed the presence of in vitro tyrosine phosphorylated proteins that were subsequently identified by re-precipitation (and/or by western blotting) as the respective C-type lectin molecules and Src family kinases Lck, Lyn, and Fyn. The NKR-P1 and the CD94-containing complexes were independent of each other and both very large, as judged by Sepharose 4B gel chromatography. Crosslinking of NKR-P1 on the cell surface induced transient in vivo tyrosine phosphorylation of cellular protein substrates. These results indicate involvement of the associated Src-family kinases in signaling via the NKR-P1 and CD94 receptors. Received: 4 February 1997 / Revised: 28 February 1997     

Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells.     

自然杀伤细胞受体的研究进展

自然杀伤细胞（ＮＫ细胞）可表达两类功能相悖的识别受体,即活化受体（ＫＡＲ）和抑制受体（ＫＩＲ）。ＫＩＲ能识别自身细胞上的ＭＨＣⅠ类分子与自身或外来肽形成２的复合物,所产生的抑制信号可阴断ＫＡＲ的活化,以此抑制ＮＫ细胞的细胞毒作用。如果靶细胞失去ＫＩＲ所识别的配体,ＮＫ细胞即可通过ＫＡＲ对靶细胞进行攻击。本文将介绍此类受体的结构及基识别与信号转导机制的研究进展。