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TIM-3 has been considered as a target in cancer immunotherapy. In T cells, inhibitory as well as activating functions have been ascribed to this molecule. Its role may therefore depend on the state of T cells and on the presence of interaction partners capable to perform functional pairing. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) has been proposed to bind TIM-3 and to regulate its function. Using a T cell reporter platform we confirmed CEACAM1-mediated inhibition, but CEACAM1 did not functionally engage TIM-3. TIM-3 and CEACAM1 coexpression was limited to a small subset of activated T cells. Moreover, results obtained in extensive binding studies were not in support of an interaction between TIM-3 and CEACAM1. Cytoplasmic sequences derived from TIM-3 induced inhibitory signaling in our human T cell reporter system. Our results indicate that TIM-3 functions are independent of CEACAM1 and that this receptor has the capability to promote inhibitory signaling pathways in human T cells., (© 2020 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mast cells are innate immune cells usually residing in peripheral tissues, where they are likely to activate T-cell responses. Similar to other myeloid immune cells, mast cells can function as antigen-presenting cells. However, little is known about the capacity of human mast cells to costimulate CD4(+) T cells. Here, we studied the T-cell stimulatory potential of human mast cells. Peripheral blood derived mast cells were generated and cocultured with isolated CD4(+) T cells. In the presence of T-cell receptor triggering using anti-CD3, mast cells promoted strong proliferation of T cells, which was two- to fivefold stronger than the "T-cell promoting capacity" of monocytes. The interplay between mast cells and T cells was dependent on cell-cell contact, suggesting that costimulatory molecules on the mast cell surface are responsible for the effect. However, in contrast to monocytes, the T-cell costimulation by mast cells was independent of the classical costimulatory molecule CD28, or that of OX40L, ICOSL, or LIGHT. Our data show that mast cells can costimulate human CD4(+) T cells to induce strong T-cell proliferation, but that therapies aiming at disrupting the interaction of CD28 and B7 molecules do not inhibit mast cell mediated T-cell activation., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intrinsic immunosuppression is a major obstacle for successful cancer therapy. The mechanisms for the induction and regulation of immunosuppression in humans are ill defined. A microenvironmental component that might prevent antitumor immunity is the presence of dying tumor cells, which are abundant following conventional cancer ablation methods such as chemo- or radiotherapy. Shedding of apoptotic debris and/or secretion of factors to the tumor bed or draining lymph nodes thus might have a profound impact on professional phagocytes, such as DCs, and subsequent priming of lymphocytes. Here, we exposed human DCs to supernatants of live, apoptotic, or necrotic human breast cancer cells and cocultured them with autologous T cells. Priming with apoptotic debris prevented DCs from establishing cytotoxicity toward live human tumor cells by inducing a Treg-cell population, defined by coexpression of CD39 and CD69. Immunosuppression via Treg cells was transferable and required the release of sphingosine-1-phosphate (S1P) from apoptotic cells, acting via S1P receptor 4 on DCs to induce IL-27 secretion. We propose that CD69 expression on CD39(+) Treg cells enables them to interact with CD73-expressing CD8(+) T cells to generate adenosine, thereby suppressing cytotoxicity. These findings aid the understanding of how dying tumor cells limit antitumor immunity., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CD4(+) Foxp3(+) regulatory T (Treg) cells are required for the maintenance of self-tolerance, as demonstrated by profound autoimmunity in mice and humans with inactivating Foxp3 mutations. Recent studies demonstrate that Treg cells are anatomically compartmentalized within secondary lymphoid organs based on their TCR repertoire and specific organ-protective function; however, whether this reflects differential homing or in situ selection is not known. Here, using Foxp3-GFP reporter mice, we have examined the ability of polyclonal Treg cells from cervical LNs to return to their site-of-origin following adoptive transfer to nonlymphopenic congenic recipients. We find that bulk cervical LN Treg cells do not home directly to cervical LNs but rather accumulate site specifically over time following transfer. Site-specific enrichment is both more rapid and more pronounced among a population of recently activated (CD69(+) ) Treg cells. These data suggest that compartmentalization of Treg cells within secondary lymphoid organs may be governed by antigen recognition and implicate CD69 as a potential marker of recently activated Treg cells recognizing locally expressed antigens., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Expression by DCs of co-inhibitory molecules such as programmed death ligand-1 (PD-L1/B7-H1/CD274), a member of the B7 superfamily, is crucial for the downregulation of T-cell responses and the maintenance of immune homeostasis. Exposure of immature DCs to danger-associated molecular patterns (DAMPS) or pathogen-associated molecular patterns (PAMPs) generally results in their maturation and acquisition of immunostimulatory function. However, exposure of DCs to TLR ligands early during their differentiation can inhibit further differentiation and confer tolerogenic properties on these APCs. A report in this issue of The European Journal of Immunology reveals that early inhibition of human DC differentiation from blood monocytes by TLR agonists is associated with a tolerogenic phenotype and Treg generation. The tolerogenic function of these APCs is dependent on MAPK-induced IL-6 and IL-10 production, which drives STAT-3-mediated PD-L1 expression. These observations link IL-10 and IL-6 to PD-L1 expression, providing a new dimension to the anti-inflammatory properties of these cytokines. These findings also have implications for understanding the inherent function of DCs in non-lymphoid tissues such as the liver and lung, where they are exposed to PAMPs that are found constitutively in the local microenvironment., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

P2X7 receptor is an adenosine triphosphate (ATP)-gated ion channel within the multiprotein inflammasome complex. Until now, little is known about regulation of P2X7 effector functions in macrophages. In this study, we show that nucleoside triphosphate diphosphohydrolase 1 (NTPDase1)/CD39 is the dominant ectonucleotidase expressed by murine peritoneal macrophages and that it regulates P2X7-dependent responses in these cells. Macrophages isolated from NTPDase1-null mice (Entpd1(-/-)) were devoid of all ADPase and most ATPase activities when compared with WT macrophages (Entpd1(+/+)). Entpd1(-/-) macrophages exposed to millimolar concentrations of ATP were more susceptible to cell death, released more IL-1beta and IL-18 after TLR2 or TLR4 priming, and incorporated the fluorescent dye Yo-Pro-1 more efficiently (suggestive of increased pore formation) than Entpd1(+/+) cells. Consistent with these observations, NTPDase1 regulated P2X7-associated IL-1beta release after synthesis, and this process occurred independently of, and prior to, cytokine maturation by caspase-1. NTPDase1 also inhibited IL-1beta release in vivo in the air pouch inflammatory model. Exudates of LPS-injected Entpd1(-/-) mice had significantly higher IL-1beta levels when compared with Entpd1(+/+) mice. Altogether, our studies suggest that NTPDase1/CD39 plays a key role in the control of P2X7-dependent macrophage responses.

The hallmark of chronic viral infections is a progressive exhaustion of antigen-specific CD8(+) T cells that leads to persisting viral replication. It is generally believed that exhaustion is a consequence of the accumulation of multiple inhibitory receptors on CD8(+) T cells that makes them dysfunctional. Here, we show that during human chronic HIV-1 infection, a CD8(+) T-cell positive costimulatory pathway mediated by DNAX-activating molecule-1 is also disrupted. Thus, DNAX-activating molecule-1 downregulation on CD8(+) T cells aggravates the impairment of CTL effector function in chronic HIV-1 infection.

2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM/CD150), is expressed on all NK cells, a subpopulation of T cells, monocytes and basophils. Human NK cells express two isoforms of 2B4, h2B4-A and h2B4-B that differ in a small portion of the extracellular domain. In the present investigation, we have studied the functions of h2B4-A and h2B4-B. Our study demonstrated that these two isoforms differ in their binding affinity for CD48, which results in differential cytotoxic activity as well as intracellular calcium release by NK cells upon target cell recognition. Analysis of the predicted 3-D structure of the two isoforms showed conformational differences that could account for their differences in binding affinity to CD48. h2B4-A was able to mediate natural cytotoxicity against CD48-expressing K562 target cells and induce intracellular calcium release, whereas h2B4-B showed no effects. NK-92MI, U937, THP-1, KU812, primary monocytes, basophils and NK cells showed expression of both h2B4-A and h2B4-B whereas YT and IL-2-activated NK cells did not show any h2B4-B expression. Stimulation of NK cells through 2B4 resulted in decreased mRNA levels of both h2B4-A and h2B4-B indicating that down-regulation of 2B4 isoforms may be an important factor in controlling NK cell activation during immune responses.

CTLA-4 is constitutively expressed by CD4(+)CD25(+)Foxp3(+) Treg but its precise role in Treg function is not clear. Although blockade of CTLA-4 interferes with Treg function, studies using CTLA-4-deficient Treg have failed to reveal an essential requirement for CTLA-4 in Treg suppression in vivo. Conditional deletion of CTLA-4 in Foxp3(+) T cells disrupts immune homeostasis in vivo but the immune processes disrupted by CTLA-4 deletion have not been determined. We demonstrate that Treg expression of CTLA-4 is essential for Treg control of lymphopenia-induced CD4 T-cell expansion. Despite IL-10 expression, CTLA-4-deficient Treg were unable to control the expansion of CD4(+) target cells in a lymphopenic environment. Moreover, unlike their WT counterparts, CTLA-4-deficient Treg failed to inhibit cytokine production associated with homeostatic expansion and were unable to prevent colitis. Thus, while Treg developing in the absence of CTLA-4 appear to acquire some compensatory suppressive mechanisms in vitro, we identify a non-redundant role for CTLA-4 in Treg function in vivo.

Regulatory T cells exert their function through the modulation of both T and B cell responses. Our previous studies demonstrated that IL-10-producing Treg (Tr1) can induce B cells to secrete IgG4 in a cell-contact-dependent manner. The benefit of such non-inflammatory B-cell responses is apparent in the hyporesponsive state of patients with helminth infections such as Onchocerciasis. Here, we investigated the mechanisms involved to induce IgG4, within B:Tr-cell co-cultures, using IL-10-producing tetanus-toxoid-specific regulatory T cell lines and clones (Tr-TCC) from human PBMC. During the generation process, we found that increasing Foxp3 levels in regulatory T cell lines correlated with their ability to induce IgG4 in B cells. Using Tr-TCC, we found that blocking glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR) molecules selectively prevented IgG4 production as did neutralizing Ab to glucocorticoid-induced tumour necrosis factor receptor-related protein ligand (GITR-L), IL-10 and TGF-beta. Furthermore, the prevention of IgG4 induction by anti-GITR Ab was reversed by excess rIL-10 but not rTGF-beta. In contrast, anti-ICOS and anti-CTLA-4 Abs had no effect. When compared with Tr-TCC, freshly isolated CD4+CD25+ T cells, but not effector T cell populations, induced low levels of IgG4, which were also blocked by anti-GITR and anti-GITR-L Ab. Thus, the mechanism of IgG4 induction by regulatory cells involves GITR-GITR-L interactions, IL-10 and TGF-beta.

Impaired myeloid dendritic cells (mDC) fail to elicit host antiviral immune responses, leading to disease progression in HIV-1 infection. However, mechanisms underlying mDC suppression remain elusive. In this study, we found that the T-cell co-stimulatory molecule programmed death-1 ligand-1 (B7-H1) is significantly up-regulated on peripheral mDC in HIV-1-infected typical progressors and AIDS patients, but is maintained at a relatively low level in long-term non-progressors. Successful immune reconstitution after highly active antiretroviral therapy, indicated by full suppression of HIV-1 replication and substantial increases of CD4 T-cell counts, correlated with a decrease in B7-H1 expression. Importantly, we also found that X4 HIV-1 isolates directly induced B7-H1 expression on mDC in vitro, while adding antiviral agents hampered this B7-H1 up-regulation. Blockade of B7-H1 in vitro strongly enhanced mDC-mediated allostimulatory capacity and IL-12 production. In contrast, B7-H1 ligation with soluble programmed death-1 (PD-1) reduced mDC maturation and IL-12 production but increased mDC apoptosis and IL-10 production. Thus, B7-H1 up-regulation may inhibit mDC-mediated immune response, thereby facilitating viral persistence and disease progression in HIV-1-infected patients. This study provides new evidence that B7-H1 inhibitory signaling may reversely mediate functional impairment of mDC in HIV-1 infection, which further supports the notion that B7-H1 blockade represents a novel therapeutic approach to this disease.

The magnitude and quality of T cell responses generated when Ag is targeted to receptors on DC is influenced by both the specific receptor targeted and its distribution among DC subsets. Here we examine the targeting of the model Ag OVA to potential DC targets, including CD11c, CD205, MHC class II, CD40, TLR2 and FcgammaRII/III, using a panel of (Fab' x OVA) conjugates. In vitro studies identified CD11c, CD205 and MHC class II as superior and comparably effective immunotargets for the delivery of OVA to APC for presentation to T cells. In vivo studies, however, showed a marked advantage of targeting Ag to CD11c for both CD4 (OT-II) and CD8 (OT-I) responses, with robust stimulation after a single, low dose (equivalent to 0.5 microg OVA); in contrast, (anti-CD205 x OVA) and (anti-MHC class II x OVA) resulted in markedly less proliferation of both OT-I and OT-II cells. Biodistribution and immunohistochemical studies suggest that the exceptional ability of CD11c to capture Ag in lymphoid tissues may, at least partially, explain its ability to promote T cell responses. These results suggest that targeting antigen via CD11c offers a previously unappreciated strategy for vaccine development which, unlike most targets, delivers robust responses of both CD4 and CD8 T cells.

Myeloid cells bear Fc receptors (FcR) that mediate inflammatory signaling through the ITAM-containing FcRgamma adaptor. They express FcRgamma-associated FcalphaRI, which modulate either activating or inhibitory signaling depending on the type of ligand interaction. The role of FcalphaRIgamma in disease progression remains unknown, notably in IgA nephropathy (IgAN), one of major causes of end-stage renal disease, in which large amounts of circulating IgA-immune complexes (IC) may mediate receptor activation. To analyze the involvement of FcalphaRI activation in glomerulonephritis (GN), we generated Tg mice expressing a mutated, signaling-incompetent, human FcalphaRI(R209L) that cannot associate with FcRgamma. Like FcalphaRI(wt)-Tg mice, they developed mesangial IgA deposits but not macrophage infiltration. FcalphaRI activation in FcalphaRI(wt), but not in FcalphaRI(R209L), Tg mice resulted in marked inflammation with severe proteinuria and leukocyte infiltration in spontaneous IgAN or anti-glomerular basement membrane Ab-induced GN models. Receptor triggering of syngenically transferred FcalphaRI(wt) Tg macrophages into non-Tg animals induced their recruitment into injured kidneys during GN development. FcalphaRI(wt) cross-linking on macrophages activated MAP kinases and production of TNF-alpha and MCP-1. Moreover, IgA-IC from IgAN patients activated FcalphaRI and induced TNF-alpha production. Thus, FcalphaRI activation mediates GN progression by initiating a cytokine/chemokine cascade that promotes leukocyte recruitment and kidney damage.

The transmembrane glycoprotein CD83 has been described as a specific maturation marker for dendritic cells and several lines of evidence suggest that CD83 regulates thymic T cell maturation as well as peripheral T cell activation. Here we show for the first time that CD83 is involved also in the regulation of B cell function. CD83 is up-regulated on activated B cells in vivo, specifically in the draining lymph nodes of Leishmania major-infected mice. The ubiquitous transgenic (Tg) expression of CD83 interferes with Leishmania-specific T cell-dependent and with T cell-independent antibody production. This defect is restricted to the B cell population since the antigen-specific T cell response of CD83Tg mice to L. major infection is unchanged. The defective immunoglobulin (Ig) response is due to Tg expression of CD83 on the B cells because wild-type B cells display normal antigen-specific responses in CD83Tg hosts and CD83Tg B cells do not respond to immunization in a mixed wild-type/CD83Tg bone marrow chimera. Finally, the treatment of non-Tg C57BL/6 mice with anti-CD83 mAb induces a dramatic increase in the antigen-specific IgG response to immunization, thus demonstrating a regulatory role for naturally induced CD83 on wild-type B cells.

Natural IFN-producing cells (NIPC), also called plasmacytoid dendritic cells, represent an essential component of the innate immune defense against infection. Despite this, not much is known about the pathways involved in their activation by non-enveloped viruses. The present study demonstrates that the non-enveloped foot-and-mouth disease virus (FMDV) cannot stimulate IFN-alpha responses in NIPC, unless complexed with FMDV-specific immunoglobulins. Stimulation of NIPC with such immune complexes employs FcgammaRII ligation, leading to strong secretion of IFN-alpha. In contrast to the stimulation of NIPC by many enveloped viruses, FMDV induction of IFN-alpha production requires live virus. It is necessary for the virus to initiate its replicative cycle. Moreover, it is an abortive replication, as witnessed by the decrease of dsRNA levels and viral titers with time post infection. Sensitivity of the NIPC stimulation to wortmannin and chloroquin, but not leupeptin, indicates an essential role for the pre-lysosomal stage endosomal compartment. In conclusion, the present study demonstrates that immune complexes provide the means for a non-interferogenic virus to induce IFN-alpha responses by NIPC. This indicates an important link between NIPC and antibodies in immune responses against non-enveloped viruses such as FMDV.

The B7 family member programmed death ligand 2 (PD-L2) has been implicated in both positive and negative regulation of T cell activity. In this study, we demonstrate that on human T cells, PD-L2 acts only as a negative regulator of T cell activity, inhibiting proliferation, IL-2 production, and IFN-gamma production via its interaction with programmed death-1 (PD-1). This study also shows a novel role for PD-1 in inhibiting beta1 and beta2 integrin-mediated adhesion. PD-L2 inhibition of T cell function involves modulation of the phosphoinositide 3-OH kinase (PI 3-K)/AKT and extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathways, with PD-L2 inhibiting anti-CD3-induced AKT phosphorylation within minutes and ERK phosphorylation after hours. Analysis of phosphatase activity of Src homology 2 domain-containing tyrosine phosphatase (SHP)-1 and SHP-2 in response to anti-CD3 mAb or anti-CD3 mAb + PD-L2 stimulation revealed that while SHP-1 phosphatase activity is not affected by stimulation, SHP-2 phosphatase activity is significantly increased by anti-CD3 mAb + PD-L2 stimulation. Anti-CD3 mAb + PD-L2 stimulation also increased the level of SHP-2 associated with the PD-1 receptor. These results suggest that catalytically active SHP-2 associated with the PD-1 receptor is involved in modulating T cell function.

Immunoglobulin Fcgamma receptors (FcgammaR) are comprised of a ligand-binding alpha-chain that sometimes associates with a cell signaling common gamma-chain. These receptors comprise an important family of effector molecules that link humoral and cell-mediated adaptive immunity and regulate innate immunity. Recent animal studies suggest that FcgammaR in general, and FcR alpha-chains in particular, are required for full development of experimental autoimmune encephalomyelitis (EAE). We show here that deletion of the gamma-chain renders mice resistant to EAE, whereas deletion of the alpha-chains of FcgammaRI, FcgammaRIIB and FcgammaRIII has no protective effect. Susceptibility to EAE is fully restored in common gamma-chain-/- mice into which wild-type splenocytes are adoptively transferred, but EAE is not restored in common gamma-chain-/- mice given wild-type splenocytes depleted of gammadelta T cells. These data indicate that although the common gamma-chain is required for full development of EAE in mice, this requirement is likely FcgammaR alpha-chain-independent. Expression of the common gamma-chain by gammadelta T cells, probably in conjunction with the T cell receptor/CD3 complex, is likely the key requirement for full development of EAE.

B cells can differentiate into antibody-secreting plasma cells, however the signals that control the entry into this pathway are not clearly understood. We have investigated the role of human CD72 in mature B cell differentiation. Human CD72 is preferentially expressed in naive B cells, but marginal levels of expression can be found in switched memory B cells. CD72 cross-linking promoted an increase in B cell activation and proliferation. Interestingly, expression of CD27, whose signal induces the differentiation of B cells into plasma cells, was down-modulated by CD72 stimulation. This CD72 signaling also induced tyrosine phosphorylation of various proteins such as Blk. Plasma cell differentiation and Ig syntheses were diminished by CD72 ligation in the presence of Staphylococcus aureus Cowan strain (SAC) plus IL-2 but not in the presence of CD40 signaling or CpG oligodeoxynucleotide. Our results show that CD72 signaling reduces the expression of X-box binding protein 1 in B cells stimulated with SAC plus IL-2, but the expression of PRDI-BF1 was unaffected. Taken together, these data demonstrate that CD72 is a key molecule in regulating mature B cell differentiation, particularly in preventing the differentiation of naive B cells into plasma cells, thus blocking the production of low-affinity antibodies.

The CD94/NKG2C killer lectin-like receptor (KLR) specific for HLA-E is coupled to the KARAP/DAP12 adapter in a subset of NK cells, triggering their effector functions. We have studied the distribution and function of this KLR in T lymphocytes. Like other NK cell receptors (NKR), CD94/NKG2C was predominantly expressed by a CD8(+) T cell subset, though TCRgammadelta(+) NKG2C(+) and rare CD4(+) NKG2C(+) cells were also detected in some individuals. Coculture with the 721.221 HLA class I-deficient lymphoma cell line transfected with HLA-E (.221-AEH) induced IL-2Ralpha expression in CD94/NKG2C+ NK cells and a minor subset of CD94/NKG2C(+) T cells, promoting their proliferation; moreover, a similar response was triggered upon selective engagement of CD94/NKG2C with a specific mAb. CD8(+) TCRalphabeta CD94/NKG2C(+) T cell clones, that displayed different combinations of KIR and CD85j receptors, expressed KARAP/DAP12 which was co-precipitated by an anti-CD94 mAb. Specific engagement of the KLR triggered cytotoxicity and cytokine production in CD94/NKG2C(+) T cell clones, inducing as well IL-2Ralpha expression and a proliferative response. Altogether these results support that CD94/NKG2C may constitute an alternative T cell activation pathway capable of driving the expansion and triggering the effector functions of a CTL subset.

NK cell receptors for MHC class I molecules (MHC-NKR) can be expressed by T cell subsets. The restricted repertoire and phenotypic characteristics of MHC-NKR(+) T cells indicate that expression of MHC-NKR is acquired upon antigenic challenge and might promote expansion of T cells. Previous studies performed on in vitro generated alphabeta T cell clones concluded that MHC-NKR expression was not a clonal attribute. Here, we examined a massive monoclonal expansion of a non-leukemic gammadelta T cell population found in the peripheral blood of a lung-transplanted patient who suffered from a cytomegalovirus infection. Despite their monoclonality, these T cells displayed a heterogeneous and stable in vivo Ig- and lectin-like MHC-NKR phenotype. Twenty percent of the cells displayed a CD94(+)NKG2A(+) phenotype, and 10% were labeled with an anti-CD158b1/b2/j monoclonal antibody. A CD158b/j(+) gammadelta T cell clone derived in vitro from patient's peripheral blood lymphocytes was shown to express the activating form CD158j (KIR2DS2), which once cross-linked stimulated the clone cytolytic function and costimulated the TCR-induced production of cytokines, independently of the killer-activating receptor-associated protein (KARAP). In conclusion, heterogeneity of MHC-NKR expression confers a functional intraclonal diversity that may participate to induction of specific gammadelta T cell effector functions or proliferation upon pathogen challenge.

Granulocytes form the first and fastest line of defense against pathogenic infections. Their survival is limited by apoptosis, a process that is critical for the resolution of inflammation. Pro-apoptotic and pro-inflammatory cytokines, as well as several receptors, can alter the lifespan of granulocytes. Here we report that the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, CD66a) is involved in the regulation of granulocyte survival. Until now CEACAM1 is described to control cell proliferation, cell migration, tumor growth, angiogenesis and diverse leukocyte functions. However, very little is known about its role in granulocytes. We found that CEACAM1 expression in resting rat granulocytes is significantly higher than in other leukocyte subtypes. Stimulation led to a strongly increased CEACAM1 cell surface expression and to release of soluble CEACAM1. DNA fragmentation assays and annexin V staining revealed that binding of CEACAM1-specific antibodies, Fab fragments and soluble CEACAM1-Fc constructs to cell surface-expressed CEACAM1 causes a delay of spontaneous and Fas ligand (CD95L)-induced apoptosis. Tyrosine phosphorylation of CEACAM1-L, its association with SHP-1, the activation of Erk1/2 and caspase-3 appeared to be crucial for the CEACAM1-mediated anti-apoptotic effect. These findings provide evidence that CEACAM1 influences the resolution of inflammation by prolonging the survival of rat granulocytes.

The role of membrane cofactor protein (MCP, CD46) on human T cell activation has been analyzed. Coligation of CD3 and CD46 in the presence of PMA or CD28 costimuli enhanced IL-2, IFN-gamma, or IL-10 secretion by CD4+ T lymphocytes. The effect of CD46 on IL-10 secretion did not require additional costimuli like anti-CD28 antibodies or phorbol esters. CD46 also enhanced IL-2 or IFN-gamma secretion by CD4+ blasts. In contrast, IL-5 secretion was inhibited upon CD46-CD3 coligation, in all the cells analyzed. These effects were independent of IL-12 and suggest that CD46 costimulation promotes a Th1-biased response in human CD4+ T lymphocytes. CD46 enhanced TCR/CD3-induced tyrosine phosphorylation of CD3zeta and ZAP-70, as well as the activation of the ERK, JNK, and p38, but did not modify intracellular calcium. The effect of specific inhibitors shows that enhanced ERK activation contributes to augmented IFN-gamma and lower IL-5 secretion and, consequently, to the Th1 bias. Cross-linking CD46 alone induced weak tyrosine phosphorylation of CD3zeta and ZAP-70. However, CD46 cross-linking by itself did not induce cell proliferation or lymphokine secretion, and pretreatment of CD4+ T lymphocytes with anti-CD46 antibodies did not significantly alter TCR/CD3 activation., (Copyright 2004 Wiley-VCH Verlag GmbH & Co.)

Interactions of natural killer (NK) cells with MHC class I proteins provide the main inhibitory signals controlling NK killing activity. It is therefore surprising to learn that TAP2-deficient patients suffer from autoimmune manifestations only occasionally in later stages of life. We have previously described that the CEACAM1-mediated inhibitory mechanism of NK cytotoxicity plays a major role in controlling NK autoreactivity in three newly identified TAP2-deficient siblings. This novel mechanism probably compensates for the lack of MHC class I-mediated inhibition. The CEACAM1 protein can also be present in a soluble form and the biological function of the soluble form of CEACAM1 with regard to NK cells has not been investigated. Here we show that the homophilic CEACAM1 interactions are abrogated in the presence of soluble CEACAM1 protein in a dose-dependent manner. Importantly, the amounts of soluble CEACAM1 protein detected in sera derived from the TAP2-deficient patients were dramatically reduced as compared to healthy controls. This dramatic reduction does not depend on the membrane-bound metalloproteinase activity. Thus, the expression of CEACAM1 and the absence of soluble CEACAM1 observed in the TAP2-deficient patients practically maximize the inhibitory effect and probably help to minimize autoimmunity in these patients.

Local tissue damage, characterized by edema, hemorrhage and necrosis, is a common consequence of envenoming by many vipers. We have investigated the contribution of inflammatory responses induced by the venom metalloproteinase jararhagin (isolated from Bothrops jararaca venom) in the development of these lesions. Local venom effects (edema, hemorrhage and necrosis) were induced experimentally in knockout mice deficient in the TNF receptors TNFR1 or TNFR2, IL-1betaR, IL-6 and iNOS. Jararhagin-induced dermal necrosis was abolished in mice deficient in the TNF receptors TNFR1 and TNFR2, and the same activity was significantly reduced in IL-6(-/-) mice. There was no significant difference in edema and hemorrhage activities following jararhagin insult between knockout and WT strains, indicating that these local venom metalloproteinase-induced effects are independent of these pro-inflammatory mediators. The contribution of both TNF receptors and IL-6 in local tissue necrosis raises important therapeutic issues regarding the treatment of local envenoming.

CD22 is an inhibitory coreceptor for B cell receptor (BCR) signaling. The inhibition is most likely mediated by activation of SHP-1. We found that SLP65/BLNK reaches maximal tyrosine-phosphorylation at earlier time points in CD22(-/-) than in wild type B cells upon BCR cross-linking, suggesting that SLP65/BLNK is a substrate of SHP-1. However, in contrast to the defective Ca(2+) mobilization of SLP65/BLNK(-/-) B cells, there was a clear Ca(2+) response in SLP65/BLNKxCD22 double-deficient B cells. This implies that SLP65/BLNK is not the sole target of SHP-1 in the regulation of the Ca(2+) signaling strength. While SLP65(-/-) mice show several blocks of B cell differentiation, in SLP65/BLNK x CD22 double-deficient mice the maturation block of B cells in the spleen was partially rescued. However, the proliferative responses of B cells from both SLP65/BLNK(-/-) and double-deficient mice were defective after IgM- or CD40-stimulation. These results show that SLP65/BLNK is not absolutely essential for Ca(2+) induction in B cells, because the deficiency of this adapter can be by-passed by the additional deletion of an inhibitory receptor. Furthermore, these experiments suggest that B cell maturation in the spleen is directly dependent on the strength of BCR-derived Ca(2+) signals.

Siglec-7 is a sialic acid binding receptor with inhibitory potential, expressed on human NK cells and monocytes. It has an unusual binding preference for alpha2,8-linked disialic acids, such as those displayed by ganglioside GD3. Here we have investigated whether siglec-7-GD3 interactions are able to modulate NK cell cytotoxicity. Using synthetic polyacrylamide glycoprobes, siglec-7 was found to be masked at the NK cell surface but it could be unmasked by sialidase treatment of NK cells. GD3 synthase-transfected P815 target cells expressed high levels of GD3 and bound strongly to recombinant siglec-7-Fc protein. Surprisingly, GD3 synthase-transfected P815 cells were killed more effectively by untreated cells in a siglec-7-independent manner. However, following sialidase treatment of NK cells, a siglec-7-dependent inhibition of killing was observed. These findings have important implications for NK cell cytotoxicity against tumor cells like melanoma that express high levels of GD3 ganglioside.

The cytolytic activity of human natural killer cells is induced by several triggering cell surface receptors upon interaction with specific cellular ligands. These receptors include NKp46, NKp30 and NKp44, collectively termed natural cytotoxicity receptors (NCR). Co-operation among NCR has been shown to occur for optimal recognition and killing of most tumor target cells. In this study, we show that the mAb-mediated engagement and clustering of one or another NCR results in the activation of an identical set of tyrosine kinases. These kinases are included in the signaling cascade leading to tyrosine phosphorylation of different receptor-associated signal transducing molecules i.e. CD3 zeta (associated with NKp46 and NKp30) and KARAP/DAP12 (associated with NKp44). In line with the notion that the engagement of inhibitory receptors prevents NCR-mediated responses, we show that the engagement of CD94/NKG2A virtually abrogates the tyrosine phosphorylation of the NCR-associated signaling molecules, i.e. it acts at the very early steps of the signaling cascade. Importantly, the engagement of a single NCR resulted in the activation of the signaling cascades associated with the other NCR. This "cross-talk" is confined to NKp46, NKp30 and NKp44 since neither CD16-nor KIR2DS4-associated signaling polypeptides were phosphorylated following the NCR engagement. These results suggest that a functional cross-talk specifically occurs among different NCR, possibly resulting in the amplification of the activating signals.

MRL.Fas(lpr/lpr) mice, a model for systemic lupus erythematosus (SLE) and arthritis in humans, have a Fas mutation that results in spontaneous development of systemic autoimmune diseases and a short life span. Half of them die by 5-6 months of age due to massive progression of systemic autoimmune diseases, such as lupus nephritis. However, C57BL/6 (B6).Fas(lpr/lpr) strain does not develop such disorders within the normal life span, indicating that suppressor gene(s) in B6 mice may control the onset and exacerbation of disease. Here, we show that the gene for a unique inhibitory Fc receptor for IgG (Fc gamma RIIB) is a critical SLE suppressor. Fc gamma RIIB-deficient B6.Fas(lpr/lpr) (B6.IIB(-/-)Fas(lpr/lpr)) mice developed systemic autoimmune diseases, including anti-DNA and anti-type II collagen autoantibodies and cryoglobulin production, immune complex glomerulonephritis and arthritis. They were short-lived, due to enhanced autoantibody production by B cells culminating in fatal lupus nephritis. Thus, Fc gamma RIIB deletion with Fas mutation is sufficient for the development of systemic autoimmunity in B6 mice. The inhibitory signaling cascade via Fc gamma RIIB may be critical for suppressing SLE in humans.

CD4+CD25+ regulatory T cells (Tregs) are critical for peripheral tolerance and prevention of autoimmunity. In vitro coculture studies have revealed that increased costimulation breaks Treg-mediated suppression in response to anti-CD3 or antigen. However, it was unclear whether loss of suppression arose from inactivation of Tregs or whether increased stimulation caused Th cells to escape suppression. We have investigated conditions that allow or override Treg-mediated suppression using DO11.10 TCR-transgenic T cells and chicken ovalbumin peptide 323-339-pulsed antigen-presenting cells. Treg suppression of Th proliferation is broken with potent stimulation, using activated spleen cells and high antigen dose, but is intact at low antigen dose. Costimulation with CD80 and CD86 expressed on activated dendritic cells was essential for Th cell escape from suppression at a high antigen dose. Potently stimulated Tregs were functional since they reduced levels of IL-2, IFN-gamma, IL-4 and Th CD25 expression in cocultures. Furthermore, Tregs responding to high antigen dose and activated splenocytes retained the ability to suppress proliferation, but only of Th cells responding to a sub-optimal dose of independent antigen. Together, our results demonstrate that under conditions of strong antigen-specific stimulation, Tregs remain functional, but Th cells escape Treg-mediated suppression.

Binding of the hepatitis C virus (HCV) envelope protein E2 to CD81 provides a costimulatory signal for human T cells. This phenomenon may play a role in liver damage and autoimmune manifestations associated with HCV infection. Here we show that cross-linking of CD81 by HCV E2 induced a calcium flux in T cells that depends on Lck since it was blocked by PP1 and absent in Lck-deficient Jurkat T cells. In wild-type Jurkat cells, Lck was activated by CD81 cross-linking, and CD81, like Lck, was found in lipid rafts. Indeed, the integrity of the raft compartment was required for the induction of a calcium flux by E2, since methyl-beta-cyclodextrin abolished this response. A requirement for TCR/CD3 expression was indicated by the absence of a calcium flux following E2 stimulation of TCR/CD3-deficient Jurkat cells. CD81 cross-linking increased and prolonged the anti-CD3-induced tyrosine phosphorylation of TCR1 and of other proteins, indicating that the CD81-mediated signal converges with the TCR/CD3 signaling cascade at its most upstream step. In conclusion, we propose that the costimulatory effects of HCV E2 on T cells depend on CD81 cross-linking that activates Lck through raft aggregation and thus leads to enhanced TCR signaling.

Epitope density and organization have been shown to be important factors for B cell activation in many animal model systems. However, it has been difficult to separate the role of antigen organization from the role of local antigen concentrations because highly organized antigens are usually particulate whereas non-organized antigens are more soluble. Hence, highly organized and non-organized antigens may interact with different cell types and in different locations within lymphoid organs. In order to assess the role of antigen organization in regulating B cell responses, we immunized mice with highly repetitive virus-like particles, which exhibit different epitope densities covalently attached to them. Therefore, the same particulate structure was used to present identical epitopes that differed in their degree of organization. Induction of epitope-specific IgM titers, reflecting early B cell activation, were unaffected by the degree of epitope density. Furthermore, the absence of Th cells or CD21/CD35 did not reduce the IgM response. In contrast, the degree of organization was a critical factor influencing the magnitude of the epitope-specific IgG response. Moreover, the threshold for IgG responses was shifted in the absence of CD21/CD35, resulting in the requirement for higher epitope densities to allow efficient IgG responses. Thus, IgG but not IgM responses are regulated by epitope density and B cell costimulatory thresholds.

Tumor necrosis factor (TNF) plays a pivotal role in the pathogenesis of Crohn's disease (CD). However, little is known about the role of TNF receptors (TNF-R) in this disease. Here, we found that TNF-R2 (in contrast to TNF-R1) was significantly up-regulated on lamina propria and peripheral blood T cells in CD compared to control patients. To directly test the functional role of TNF-R2 in Th1-mediated experimental colitis in vivo, we took advantage of transgenic animals overexpressing TNF-R2 in T cells. Reconstitution of SCID mice with CD4+ CD62L+ T cells from TNF-R2 transgenic mice led to an earlier wasting syndrome, a more severe colitis and augmented Th1 cytokine production than reconstitution with cells from wild-type littermates. In addition, TUNEL staining revealed a significantly decreased apoptosis rate of lamina propria mononuclear cells in mice reconstituted with TNF-R2 transgenic T cells compared to mice reconstituted with wild-type T cells. In summary, our data suggest a critical regulatory role of TNF-R2 signaling for disease exacerbation in Th1-mediated chronic colitis. Taken together with the increased expression of TNF-R2 in CD, selective targeting of TNF-R2 signaling thus emerges as a potentially novel approach to the treatment of CD.

CD28 and CTLA-4 are opposing regulators of T cell activation, triggered by the two ligands CD80 and CD86. How these ligands promote either T cell activation via CD28 or inhibition via CTLA-4 is not understood. Using CD80 and CD86 molecules expressed on transfected cells, we have identified a major difference between these ligands in that CD80 transfectants have the ability to inhibit activation of resting human peripheral blood T cells via interaction with CTLA-4, whereas CD86 transfectants do not. Rather, CTLA-4-CD86 interactions appear to contribute towards T cell proliferation. We also observed that CTLA-4 function is strongly influenced by TCR stimulation, effects being observed only at relatively low levels of TCR stimulation. The kinetics of CD80-CTLA-4 interactions revealed that CTLA-4 inhibition took place within the first 8 h of T cell stimulation, despite there being little measurable CTLA-4 expression on the majority T cells. However, significant amounts of CTLA-4 were observed in the CD25(+) CD4(+) subset of T cells which, when removed from the cultures, accounted for the CTLA-4 inhibition observed. Overall, these data provide evidence that CD80 and CD86 differ in their interactions with CTLA-4 and that CD80 appears to be the preferential inhibitory ligand for CTLA-4 working via a population of CD4(+) CD25(+) CTLA-4(+) regulatory T cells.

In mice, tumor necrosis factor (TNF) displays a selective species specificity. In contrast to murine TNF (mTNF), human TNF (hTNF) only induces lethality at extremely high doses of about 500 microg/mouse, whereas it still has a powerful antitumor activity in combination with interferon-gamma. The observation that hTNF does not interact with the p75 mTNF receptor seemed to provide a plausible explanation for these species-specific biological effects. Experiments in TNF receptor knockout mice and tests with hTNF muteins in baboons did not, however, support this hypothesis. We here show that an mTNF mutein selective for the p55 mTNF receptor induces lethality in a manner comparable to wild-type mTNF, and conclude that other differences between hTNF and mTNF must account for the reduced lethality of hTNF. Pharmacokinetics showed that hTNF is cleared much faster than mTNF or the mTNF mutein used. In contrast to the hardly lethal effect(s) of a bolus administration of hTNF, fractionated repetitive administration of the same total hTNF dose induced lethality. This suggests that prolonged exposure rather than peak levels determine the lethal effects of hTNF in mice. Experiments with receptor and ligand knockouts demonstrated that the difference in pharmacokinetics is independent of an interaction with (soluble) TNF receptor, TNF-induced effects or induction of endogenous TNF. These results show that manipulation of the clearance rate of TNF may broaden the therapeutic range of systemic treatments with TNF.

T cell activation and cytokine secretion are important mediators of inflammation in allergic asthma. The costimulatory pathway CD28/CD80/CD86 has been shown to play an important role in T cell activation in allergic asthma, but less is known about the effect of other costimulatory molecules in allergy. The costimulatory molecule OX40 ligand (OX40L), a member of the tumor necrosis factor superfamily, has been shown to be important in T cell priming and cytokine production. We investigated the role of OX40L in a murine model of allergic inflammation using OX40L(-/-) mice. In this model, following OVA sensitization and challenge, mice develop features of allergic inflammation including elevated levels of total serum IgE, pulmonary eosinophils, cytokines, and pulmonary inflammation. In the absence of OX40L, total serum IgE, pulmonary eosinophils, cytokines, and pulmonary inflammation were all significantly reduced compared to wild-type controls. Levels of eotaxin mRNA, an eosinophil-specific chemoattractant, were also markedly reduced, paralleling the significant reduction in pulmonary eosinophils. Levels of allergen-induced Th1 as well as Th2 cytokines were also significantly reduced. Together, the data support a critical role for OX40L signals in allergic responses.

CD95 is a major apoptosis receptor that induces caspase activation and programmed cell death in susceptible cells. CD95-induced apoptosis can be blocked by peptidic caspase inhibitors such as benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or Ile-Glu-Thr-Asp-fluoromethyl ketone. Here we show that stimulation of CD95 in the presence of these inhibitors induces necrosis and expression of various proinflammatory cytokines in primary T lymphocytes, such as TNF-alpha, IFN-gamma and granulocyte/macrophage colony-stimulating factor. In the absence of caspase inhibition CD95 stimulation did not result in cytokine expression, indicating that this proinflammatory signaling pathway is suppressed by active caspases. Further analysis with A3.01 T cells revealed that the proinflammatory signaling activity of CD95 was mediated by MEK/ERK, p38 and NF-kappaB signaling pathways. These findings point to a pivotal role of caspases not only as mediators of apoptosis but also as enzymes that prevent proinflammatory signaling during CD95-induced apoptosis. Moreover, our findings may be useful for the development of novel pharmacological strategies.

B7 costimulatory molecules play an important role in inducing autoimmunity, tumor immunity, and transplant rejection, and therapeutic manipulation of B7 is being investigated in human diseases. To determine whether B7 costimulation is essential for inducing autoimmunity on different genetic backgrounds, we backcrossed B7.1/B7.2 deficient ((-/-)) mice on to the C57BL/6 (B6) and SJL backgrounds and induced experimental autoimmune encephalomyelitis (EAE) in these mice. B7.1/B7.2(-/-) mice on the B6 background were resistant to EAE induced with MOG 35-55, whereas the SJL B7.1/B7.2(-/-) mice were susceptible to PLP 139-151 or PLP 178-191-induced EAE. The SJL B7.1/B7.2(-/-) mice had a qualitatively different lesion pattern in that they showed increased white matter vacuolation compared to wild-type SJL mice when immunized with either PLP 139-151 or PLP 178-191. (B6xSJL)F1 B7.1/B7.2(+/+) mice were susceptible to EAE whereas (B6xSJL)F1 B7.1/B7.2(-/-) mice were resistant to EAE induced with either encephalitogenic peptide. Thus, genetic background determines the B7 requirement for inducing autoimmunity. These data have important implications for developing B7-based immunotherapies for human diseases.

BOB.1/OBF.1 (also called OCA-B), a B lymphocyte-specific transcriptional coactivator, is recruited to octamer-containing promoters by interacting with the Oct-1 or Oct-2 proteins. BOB.1/OBF.1-deficient mice show impaired secondary immunoglobulin isotype secretion and complete absence of germinal centers. Furthermore, numbers of splenic B cells are reduced due to a developmental block at the transitional B cell stage in the bone marrow. We found that surface expression of CD22 is selectively increased on B lineage cells in the bone marrow of BOB.1/OBF.1-deficient mice. CD22 is known as a negative regulator of B cell receptor signaling. We therefore investigated whether defects in B cell development in the BOB.1/OBF.1-deficient mice might be due to CD22 up-regulation. Mice were generated lacking both genes. In BOB.1/OBF.1xCD22 double-deficient mice, numbers of transitional B cells in the bone marrow were normal. Consequently, double-deficient mice also had normal B to T cell ratios in the spleen. We show that BOB.1/OBF.1(-/-) B cells were incapable to induce BCR-triggered Ca(2+) mobilization. This Ca(2+)-signalling defect was restored in BOB.1/OBF.1xCD22 double-deficient B cells. Nevertheless, double-deficient animals were unable to mount humoral immune responses and to form germinal centers. Finally, we demonstrate that CD22(-/-) splenic B cells proliferate independently of BOB.1/OBF.1 upon stimulation with LPS. These studies suggest that the B cell differentiation defect observed in BOB.1/OBF.1(-/-) mice is BCR-signal dependent. However, the impairment in germinal center formation is caused by a different mechanism.

The contribution of the costimulatory molecules B7-1 and B7-2 to the in vivo differentiation of Th cells remains controversial. The infection of resistant and susceptible strains of mice with the parasite Leishmania major provides a well-established model for studying in vivo differentiation of CD4+ T cells. We have infected B7-1/B7-2-deficient mice on the BALB/c and 129 background with L. major and subsequently examined different parameters of infection and cytokine responses upon restimulation of lymph node cells in vitro. BALB/c B7-2-deficient and B7-1/B7-2-double deficient mice are resistant to L. major, whereas BALB/c B7-1-deficient mice remain as susceptible as wild-type BALB/c mice. Differential expression of B7-1 and B7-2 can explain the distinct roles observed for these B7 costimulators in L. major infection.

CD84 is a member of the CD2 subset of the immunoglobulin superfamily of cell surface receptors. Several members of this family are involved in the activation of T cells and NK cells. Although CD84 was originally cloned from a human B cell line cDNA library, very little is known regarding its biology on primary human leukocytes. We investigated the expression and biochemistry of CD84 on human B cells. CD84 was expressed on B cells in peripheral blood, spleen and cord blood. Two populations of splenic B cells could be resolved, CD84(lo) and CD84(hi). CD84(hi) B cells represented a subset of memory B cells as demonstrated by increased cell size, co-expression of the memory B cell-specific marker CD27, somatically mutated Ig variable region genes, and increased proliferation compared to CD84(lo) B cells. CD84 became rapidly phosphorylated on tyrosine residues following ligation with a specific monoclonal antibody and recruited the cytoplasmic adaptor proteins SAP and EAT-2. The ability of CD84 to undergo tyrosine phosphorylation and to recruit these SH2 domain-containing proteins suggests it may function in the activation of B cells, particularly memory cells, and its signal transduction pathway may utilize SAP and/or EAT-2. Thus, investigation of expression and function of CD84 and CD27 is likely to contribute to a greater understanding of the development and biology of memory B cells in normal and immunocompromised hosts.

Activation of the complement system generates the anaphylatoxic peptide C5a, which elicits a broad range of inflammatory activities. The biological activities of C5a are mediated through its binding to the widely expressed C5a receptor (C5aR), a G-protein-coupled seven transmembrane domain receptor. In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, the C5aR is expressed on monocytes/macrophages, reactive astrocytes and T cells infiltrating the central nervous system (CNS). To investigate the role of the C5aR in this T cell-driven autoimmune model, we induced EAE in C5aR-deficient mice (C5aR(-/-)) and wild-type mice using a myelin oligodendrocyte glycoprotein (MOG) peptide as the immunogen. We found that C5aR(-/-) mice were fully susceptible to MOG-induced EAE with no difference in disease onset or severity in C5aR(-/-) mice compared to control mice. Cellular infiltrates (macrophages and T cells) were similar in the spinal cords of both animal groups and splenic T cells from C5aR(-/-) mice and control mice responded identically to MOG in T cell proliferation assays. Ribonuclease protection assays demonstrated no significant differences in pro-inflammatory gene expression between receptor-deficient and sufficient mice. These results indicate that the C5aR is not an essential mediator in the induction and progression of EAE.

Experimental autoimmune thyroiditis (EAT) is inducible in mice by immunization with thyroglobulin and adjuvant. Previous studies have shown that EAT is an autoimmune Th1-mediated disease but its characteristics differ with the adjuvant. Granulomatous lesions with marked follicular disruption develop following administration of thyroglobulin (Tg) and complete Freund's adjuvant (CFA) whereas when lipopolysaccharide (LPS) is used as the adjuvant only focal infiltrates of mononuclear cells are observed. The pro-inflammatory cytokine, TNF-alpha, is associated with Th1 autoimmune-mediated conditions. Cytokine antagonists have been used as potential therapeutic agents in several experimental autoimmune models. Soluble cytokine receptors belong to this category and may naturally be shed from cell membranes to inhibit cytokine activity. We show that the administration of the soluble TNF receptor type I (sTNFR I) in the induction of EAT has very different effects on the two models of induced autoimmune thyroiditis. sTNFR I treatment inhibits the induction of EAT only when mouse Tg is given with LPS not with CFA, suggesting an important difference in the pathogenic processes.

Engagement of CTLA-4 is critical for inhibiting T cell immune responses. Recent studies have shown that CTLA-4 plays a key role in regulating peripheral T cell tolerance. It has been suggested that one mechanism by which CTLA-4 performs this function is by regulating cell cycle progression. Here, we investigate in depth the role of CTLA-4 in regulating cell cycle progression in naive T cells by comparing the immune responses in the absence or presence of CTLA-4. In the absence of CLTA-4, T cells exhibit marked increases in T cell proliferation, IL-2 mRNA and protein secretion, and cells cycling in the S and G2-M phase. Analyses of cyclins, cyclin-dependent kinases, and cell cycle inhibitors involved in the transition from the G1 to S phase reveal that cell cycle progression is prolonged in the absence of CTLA-4. This is due to the early exit from the G1 phase, entry into the S phase, and prolonged S phase period. Re-expression of the cell cycle inhibitor p27(kip1) is delayed in the absence of CTLA-4. These studies demonstrate that the B7 : CTLA-4 pathway exerts its major effects on T cell immune responses via regulation of the cell cycle.

The leukocyte-associated Ig-like receptor-1 (LAIR-1), a surface leukocyte receptor containing two immune receptor tyrosine-based inhibitory motif (ITIM) is expressed on acute myeloid leukemia (AML) blasts isolated from peripheral blood or bone marrow of 17 patients (2 M0, 3 M1, 5 M2, 2 M4 and 5 M5 according to French, American and British classification). Further, we provide evidence thatLAIR-1 engagement inhibits granulocyte-monocyte colony-stimulating factor (GM-CSF)-induced proliferation of AML blasts. Indeed, leukemia cells stimulated with GM-CSF were blocked in the G0/G1 phase of the cell cycle and underwent apoptosis within 4 days after the engagement of LAIR-1. Remarkably, LAIR-1 was functional also in AML blasts which do not express CD33, mainly M4 and M5. Importantly, the LAIR-1 ligation led to a strong inhibition of both GM-CSF receptor-mediated intracellular calcium increases, phosphorylation and activation of Akt1/protein kinase B alpha, a substrate of the phosphatidylinositol-3 kinase. This last inhibitory effect was prevented by a synthetic peptide spanning the ITIM portion of LAIR-1, suggesting the involvement of SHP-1 phosphatase in LAIR-1-mediated inhibitory signal. Altogether, these findings indicate that the engagement of LAIR-1 can down-regulate GM-CSF-mediated survival and proliferation of AML blasts, suggesting an additional therapeutic approach to the treatment of AML patients.

Human CMV (HCMV) has evolved several strategies to evade the immune system of the infected host. Here, we investigated the role of the HCMV-encoded protein UL40 in the modulation of NK cell lysis. UL40 carries in its leader sequence a nonameric peptide similar to that found in many HLA class I molecules leader sequences. This peptide up-regulates the expression of HLA-E, the ligand for the NK cell inhibitory receptor CD94/NKG2A. The UL40-encoded HLA-E-binding peptide was present in all HCMV clinical (4636, 13B, 109B, 3C) and laboratory (AD169) strains analyzed. However, transfection of UL40 in different cell lines (293T, 721.221, K562) did not consistently confer protection from NK lysis (as measured using NKL and the newly generated NK line Nishi), despite a moderate up-regulation of HLA-E. Interestingly, combined transfection and treatment with IFN-gamma increased the inhibitory effect, via an HLA-E- and CD94/NKG2A-dependent mechanism. Although cells transfected with UL40 derived from either AD169 or 3C showed protection from NK cell lysis, infection of fibroblasts with the viruses resulted in a strong inhibition only with the clinical strain 3C. Our results suggest that UL40 and IFN-gamma-dependent up-regulation of HLA-E is only one possible mechanism to avoid NK cell recognition of HCMV infected cells.

The ability of CD8+ cytotoxic T lymphocytes (CTL) to clear viral infections may be limited when high avidity CTL encounter supra-optimal antigen density on antigen-presenting cells (APC) and undergo antigen-dependent apoptosis of CTL (ADAC). Previously, we have shown ADAC in CD8+ populations to be Fas independent, TNF-alpha receptor 2 (TNFR2) mediated, caspase dependent, and accompanied by a decrease in Bcl-2. We now employ flow cytometry to follow ADAC within individual CD8+ cells to demonstrate that the intense TCR signal induced in high avidity CTL by supra-optimal antigen density results 8 - 16 h later in a caspase-independent TNFR2 down-modulation that is directly related to the stimulating APC antigen density and concludes in a rapid onset of apoptosis by 18 - 24 h. Individual CTL undergoing apoptosis exhibit a dramatic and concurrent: (1) positive staining with Annexin V and propidium iodide; (2) transformation to a smaller cell size characteristic of apoptosis; and (3) a nearly complete loss of Bcl-2, c-IAP1, and TRAF2. We conclude that the antigen-dependent apoptosis of CD8+ CTL occurs when a tandem TCR/TNFR2 signal initiates an abrupt and concordant onset of multiple apoptotic events.

L-selectin, LFA-1 and alpha(4) integrins play important roles in the homing of naïve T cells into peripheral lymphoid tissues. L-selectin- or LFA-1-deficient lymphocytes cannot effectively home to lymph nodes (LN), and antibody blockade of alpha(4) integrins also hinders lymphocytes homing. The present study was initiated to explore whether it is feasible to ameliorate acute graft-versus-host disease (aGVHD) by modulating the homing process of donor cells in the recipient in a mouse model. Using a fluorescence labeling method, we found that two monoclonal antibodies directed at L-selectin and alpha(4) integrins, respectively, when used in combination, could delay half of the donor C57BL/6J mouse spleen cells homing into the LN of recipient BALB/c mouse 15 h after injection. Spleen cells (1 x 10(7)) derived from C57BL/6J (H-2(b)) mice were injected into each C.B-17 SCID recipient mouse (H-2(d)) with or without prior incubation with 10 microg each of the two antibodies. T cell repopulation in the blood was observed in both groups of mice at a comparable level 14 days after injection of the donor cells. Eight control mice started to show aGVHD signs 7 - 14 days after the injection, and all died by day 31. However, among the ten mice that received the antibody-treated donor cells, two died before day 29, four survived between 36 and 78 days, and the remaining four survived more than 150 days, with two of them aGVHD free. It is apparent that the temporarily reduced lymphocyte homing into LN reduced the alloreactivity of the donor T cells, thus providing a simple way of modifying aGVHD. This novel approach may shed light on the prevention of aGVHD associated with clinical bone marrow transplantation.

Chronic hepatitis C virus (HCV) infection frequently develops into liver disease and is accompanied by extra-hepatic autoimmune manifestations. The tetraspanin CD81 is a putative HCV receptor as it binds the E2 envelope glycoprotein of HCV and bona fide HCV particles. Here we show that HCV E2 binding to CD81 on human cells in vitro lowers the threshold for IL-2 receptor alpha expression and IL-2 production, resulting in strongly increased T cell proliferation. HCV E2-induced co-stimulation also enhances the production of IFN-gamma and IL-4 and causes increased TCR down-regulation. This suggests that binding of HCV particles to CD81 on T cells in vivo may lead to activation by otherwise suboptimal stimuli. Therefore, co-stimulation of autoreactive T cells by HCV may contribute to liver damage and autoimmune phenomena observed in HCV infection.

Human CD46, or membrane cofactor protein, is a regulator of complement activation and is used as a cellular receptor by measles virus. Using a series of 13 single point mutants, the region of short consensus repeat (SCR) 2 domain involved in the regulation of complement activation was mapped to residues E84, N94, Y98, E102, E103, I104 and E108. Molecular modelling localized all residues, with the exception of E84, close to each other on the external lateral face of the molecule, away from the residues important for the binding of measles virus, which are localized on the top of the molecule. The E84 residues is localized in the SCR1-2 hinge and the deleterious effect of its substitution by an alanine residue could affect the relative orientation and / or tilt of SCR1 on SCR2. Taken together, the results suggest that the measles virus binding and cofactor activity of CD46 map to distinct areas on the SCR2 module.

CD99/E2 is an integral transmembrane protein which forms, together with Xga, a distinct family whose genes are located in the pseudoautosomal region. The number of T cells that firmly bound to vascular endothelial cells under physiological shear stress increased 2-14-fold upon CD99 stimulation, and bound cells became much more resistant to detachment forces and spread. T cell arrest occurred within 1 min and was dependent on the alpha4beta1-VCAM-1 pathway. In contrast, the alphaLbeta2-ICAM-1 pathway remained unactivated. This was observed with T cell lines and with activated peripheral blood lymphocytes, and was limited within the resting peripheral CD4+ T cells to the memory subset, while virgin cells were unaffected. This discloses a stepwise regulation of the T cell extravasation cascade.