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The existence and expansion of adaptive NK-cell subsets have been linked to HCMV infection. Phenotypically, a majority of adaptive NK cells expresses the activating receptor NKG2C and CD57. Some of the molecular factors driving the expansion of NKG2C+ CD57+ NK cells in HCMV infection have been identified. The direct interaction of adaptive NK cells with HCMV-infected cells, preceding the expansion, however, remains less studied. Recently, adaptive NK cells were reported to express higher levels of the co-activating receptor CD2. We explored whether CD2 was directly involved in the response of adaptive NK cells to HCMV. In a co-culture system of human PBMCs and productively infected fibroblasts, we observed an upregulation of CD69, CD25, and HLA-DR on all NK cells. However, only in adaptive NK cells was this increase largely blocked by antibodies against CD2 and CD58. Functionally, this blockade also resulted in diminished production of IFN-γ and TNF-α by adaptive human NK cells in response to HCMV-infected cells. Our results demonstrate that binding of CD2 to upregulated CD58 on infected cells is a critical event for antibody-mediated activation and subsequent effector functions of adaptive NKG2C+ CD57+ NK cells during the antiviral response.

Regulatory T cells (Tregs) are important for maintaining immune homeostasis, but many studies suggest that Tregs are functionally impaired in autoimmune and chronic inflammatory disorders. In addition, effector T cells may vary in sensitivity toward Treg suppression. Herein, we have studied the interplay between T effectors and Tregs in the rheumatic joint. Synovial Tregs demonstrated a high degree of FOXP3 demethylation and displayed only marginal IL-17 and virtually no IFN-γ production following in vitro stimulation, altogether indicating suppressive capacity. Still, the frequency of FOXP3 expression could not predict the degree of suppression. Instead, the inflammatory milieu in the joint, i.e. proliferative capacity of effector T cells and in situ levels of pro-inflammatory cytokines influenced Treg function. Indeed, blocking IL-6 or TNF increased the suppression by Tregs in co-cultures. Additionally, approximately 30% of the synovial FOXP3(+) T cells were Ki67(+) and hence actively dividing, but proliferation did not overlap with cytokine production, suggesting that these cells represent functional Tregs having met their cognate antigen and expanded in an attempt to alleviate joint inflammation. Overall, our data argue against a general functional deficit in joint-derived Tregs and instead emphasize the importance of the inflammatory milieu to set the threshold for immune regulation.

Bacteriophage P1 Cre/loxP based systems can be used to manipulate the genomes ofmice in vivo and in vitro, allowing the generation of tissue-specific conditional mutants. We have generated mouse lines expressing Cre recombinase in hematopoietic tissues using the vav regulatory elements, or in lymphoid cells using the hCD2 promoter and locus control region (LCR). The R26R-EYFP Cre reporter mouse line was used to determine the pattern of Cre expression in each line and enabled the assessment of Cre activity at a single-cell level. Analysis showed that the vav promoter elements were able to direct Cre-mediated recombination in all cells of the hematopoietic system. The hCD2 promoter and LCR on the other hand were able to drive Cre-mediated recombination only in T cells and B cells, but not in other hematopoietic cell types. Furthermore, in the appropriate tissues, deletion of the floxed target was complete in all cells, thereby excluding the possibility of variegated expression of the Cre transgene. Both of these Cre-transgenic lines will be useful in generating tissue-specific gene deletions within all the cells of hematopoietic or lymphoid tissues.

Since denditric cells (DC) represent the main players linking innate and adaptive immunity, their prompt generation from blood cells would be instrumental for an efficient immune response to infections. Consistent with this, CD2+ monocytes were found to express the DC maturation marker CD83, along with acquisition of high antigen-presenting activity, after a surprisingly short time in culture. This rapid process is associated with expression of IFN-alpha/beta genes and secretion of low levels of pro-inflammatory cytokines. Exposure of monocytes to IFN-alpha, but not to IL-4, induced persistence of CD2+/CD83+ cells, which were fully competent in stimulating primary responses by naive T cells. These results unravel the natural pathway by which infection-induced signals rapidly transform pre-armed monocytes into active DC.

The physiological functions of murine CD2 and its ligand CD48 are uncertain. We have examined the role of the CD2-CD48 interaction in murine T cell activation using a series of Chinese hamster ovary (CHO) cell transfectants. CHO cells expressing I-Ad together with CD48 induced more potent activation of OVA-specific, I-Ad -restricted DO11.10-transgenic T cells than CHO cells expressing I-Ad alone. CD48 augmented proliferation and IL-2 production in response to antigen. The enhancing effect of CD48 was of the same magnitude as that seen for CD80 (B7-1). Conjugate assays revealed the ability of CD48 to increase adhesion between T cells and CHO transfectants. The enhancing effects of CD48 on T cell-antigen-presenting cell adhesion and T cell activation were inhibited by anti-CD2 monoclonal antibody. This report provides the first evidence that the CD2 ligand CD48 contributes to the interactions of murine CD4+ T cells with antigen-presenting cells.

CD2 is a co-stimulatory receptor involved in T cell activation. Here we report on immunosuppressive effects of three mouse CD2 monoclonal antibodies (OX34, OX54, OX55) directed against non-overlapping epitopes of the rat CD2 receptor on various modes of T cell activation in vitro and in vivo. Although non-ligand-blocking OX54 and OX55, in concert, activated T cells through CD2 in vitro, they individually suppressed the mixed lymphocyte reaction (MLR) and significantly prolonged allograft survival after rat heart transplantation in vivo. Phenotype analysis revealed that OX55 significantly down-modulated CD2 in vivo, whereas OX54 depleted T cells. Graft rejection coincided with re-expression of CD2 and clearance of OX55 from serum, whereas T cell depletion by OX54 outlasted the period of graft survival. The most suppressive antibody, OX34, down-modulated CD2 and inhibited T cell activation through the TCR or CD2 and the MLR and prolonged median allograft survival time from 7 days in controls to 45 days in the absence of any additional treatment. Graft survival was clearly dose dependent and correlated with the duration of CD2 down-modulation and the presence of circulating CD2 antibody in serum. Importantly, the specific antibody production to a T cell-dependent antigen as demonstrated by immunization with keyhole limpet hemocyanin in vivo remained unaffected after treatment with OX34. These results demonstrate the pivotal role of CD2 signaling in mediating allogeneic immune reactions after vascularized organ transplantation while allowing specific humoral immune responses in vivo.

T cell activation requires two signals: a signal from the TCR and a co-stimulatory signal provided by antigen-presenting cells (APC). In addition to CD28, multiple molecules on the T cell have been described to deliver co-stimulatory signals. Here, we investigated whether there exist quantitative or qualitative differences in the co-stimulatory capacity between CD28 and other molecules. Anti-CD28 monoclonal antibody (mAb) and mAb against CD5, CD9, CD2, CD44 or CD11a all induced activation of naive T cells in the absence of APC when co-immobilized with a submitogenic dose of anti-CD3 mAb. [3H]Thymidine incorporation determined 2 days after co-stimulation was all comparable. In contrast to progressive T cell proliferation induced by CD28 co-stimulation, co-stimulation by other T cell molecules led to a decrease in viable cell recovery along with the induction of apoptosis of once activated T cells. This was associated with a striking difference in IL-2 production; CD28 co-stimulation induced progressively increasing IL-2 production, whereas co-stimulation by other molecules produced limited amounts of IL-2. Addition of recombinant IL-2 to the latter cultures corrected the induction of apoptosis, resulting in levels of cellular proliferation comparable to those observed for CD28 co-stimulation. These results indicate that a fundamental difference exists in the nature of co-stimulation between CD28 and other molecules, which can be evaluated by the levels of IL-2 production, but not simply by [3H]thymidine incorporation.

The low incidence of graft-versus-host disease following clinical use of umbilical cord blood compared to adult bone marrow as a source of stem cells for bone marrow reconstitution, leads to questions concerning the level of immunocompetence of newborn T cells. The maturation and functional status of newborn CD4+ T cells, which are almost exclusively CD45RA+ naive T cells, compared with their adult phenotypic counterparts, is poorly understood. We examined the proliferative response to mitogens and cytokines of CD4/CD45RA+ T cells from adults and newborns, with and without accessory cells. Newborn CD4/CD45RA+ T cells demonstrated a distinct proliferative response profile which was determined by the number of accessory cells present in co-cultures with various stimuli. Newborn CD4/CD45RA+ T cells were particularly responsive to interleukin (IL)-4, IL-4 plus anti-CD2 monoclonal antibodies (mAb) and IL-4 plus phytohemagglutinin (PHA), whereas adult CD4/CD45RA+ T cells were unresponsive under similar conditions. The mitogenic responses of newborn and adult CD4/CD45RA+ T cells to PHA and anti-CD2 mAb, which were equivalent, were directly proportional to the number of accessory cells present, whereas the responsiveness to cytokines was inversely proportional to the number of co-cultured accessory cells. Anti-CD2 responses were much more sensitive to low numbers of accessory cells than PHA. The particular sensitivity of newborn CD4/CD45RA+ T cells to IL-4 represents an antigen-independent T cell activation response which could help promote a Th2 immune response resulting in the newborn.

T cell activation via CD2 requires interaction of CD2 with several signaling molecules. To investigate the structural requirements for an association of CD2 with the tyrosine phosphatase CD45 and the zeta chain of the T cell receptor (TCR)/CD3/zeta complex, we have expressed in mouse EL4 T cells a series of human CD2 chimeric and mutant proteins. Chimeric proteins in which the CD2 transmembrane and/or cytoplasmic domains were deleted or exchanged with analogous regions of CD4, CD28 or CD58 retained association with high levels of murine CD45 phosphatase activity, suggesting that the CD2 extracellular domain largely controls interaction with CD45. To a lesser extent, the cytoplasmic domain of CD2 was also shown to interact with CD45, as demonstrated by an increase in co-immunoprecipitated phosphatase activity observed following replacement of the CD58 cytoplasmic domain with that of CD2. In contrast, the cytoplasmic domain of CD2 was found to be responsible for the majority of CD2 interaction with the zeta chain of the TCR/CD3/zeta complex. Deletion of the CD2 cytoplasmic domain, excluding the first three amino acids, removed virtually all CD2 associated zeta chain and approximately sevenfold higher levels of zeta chain were found in association with a CD58/58/2 chimera than with control human CD58 wild type. This study suggests that the CD2 extracellular and intracellular domains are differentially involved in regulating T cell activation through interaction with the tyrosine phosphatase CD45 and the zeta chain of the TCR/CD3/zeta complex.

Cultured human endothelial cells (EC) increase CD40 ligand expression on polyclonally activated human peripheral blood CD4+ helper T cells compared to T cells activated in the absence of accessory cells or in the presence of peripheral blood adherent cells or B cells. Induction of CD40 ligand expression appears to be biphasic with early induction observable at 6 h and later induction observable at 24 h. EC cause T cells to increase CD40 ligand expression during the early phase at 6 h after activation. CD40 ligand expression is restricted to the CD4+ helper T cell subset of the peripheral blood T cells, even when EC are present. Blocking monoclonal antibodies to co-stimulatory molecules on EC and T cells indicate that the CD2/LFA-3 pathway, which also contributes to induction of augmented interleukin-2 (IL-2) secretion, is involved in EC-induced upregulation of CD40 ligand. Exogenous IL-2 can also increase CD40 ligand expression. However, increased IL-2 secretion in the presence of EC can not fully account for endothelial-induced CD40 ligand up-regulation as (1) the effect of exogenous IL-2 is greater at 24 h than at 6 h, whereas the opposite is true for EC; (2) the effect of saturating levels of IL-2 is considerably smaller than that of EC; and (3) blocking of IL-2 receptors does not fully inhibit endothelial effects on CD40 ligand expression. We conclude that EC provide unique co-stimulatory signals that affect the phenotype of activated CD4+ T cells.

The ligand for the T cell antigen CD2 is CD48 in rodents, but CD58 in humans. The extracellular parts of these three antigens are structurally related in that all contain two immunoglobulin superfamily (IgSF) domains. There have been reports of alternative ligands for CD2 in the human, but not so far in rodents. We describe the analysis of ligands for rat CD2 and CD48 using fluorescent beads capable of displaying a high ligand density and detecting low-affinity interactions like that of CD2 with CD48 (Kd = 60-90 microM). Monovalent chimeric proteins containing the two IgSF domains of rat CD48 or CD2 and domains 3 and 4 of rat CD4 (CD4d3+4) were anchored to fluorescent covaspheres via a CD4 monoclonal antibody (mAb) with the CD48 or CD2 domains available for ligand binding. Multivalent CD48-CD4d3+4 covaspheres gave strong specific binding to rat CD2 expressed on the surface of transfected Jurkat cells. CD48-CD4d3+4 was compared with CD48-IgG and CD48-IgM as tools for detecting binding at the cell surface. Soluble divalent CD48-IgG and decavalent CD48-IgM bound to soluble CD2 with a Koff of around 10(-3) s-1 as determined using a BIAcore biosensor. However, binding to cells by CD48-IgG and CD48-IgM was only detectable when they were immobilized on covaspheres and represented no increase in sensitivity over CD48-CD4 covaspheres when tested for binding to cells expressing high and low levels of CD2. CD48-CD4d3+4 covaspheres only bound to rat cells expressing CD2. In the reverse orientation, bindign of CD2-CD4d3+4 covaspheres was dependent on expression of CD48. Pre-incubation of cells with CD2 or CD48 mAb abolished all binding of CD48-CD4d3+4 or CD2-CD4d3+4, respectively. The data provide no evidence for an alternative ligand for rat CD2 or CD48.

Temporal or quantitative imbalance in signals delivered to T cells via T cell antigen receptor (TCR), the CD4 co-receptor, and accessory molecules can lead to anergy, apoptosis, or both. This has been observed following ligation of CD4 by HIV gp120 prior to TCR occupancy. The ability of molecules such as CD2 and CD28, interacting with their ligands LFA-3 and B7, to provide signals that protect T cells from the induction of anergy, has been reported. Here, we demonstrate that ligation of CD2 and CD28 in conjunction with TCR occupancy rescue T cells that have been programmed for apoptotic death by prior CD4 ligation to gp120. This appears to be the result of augmented interleukin-2 and interleukin-4 release by the T cells following these molecular interactions. In conclusion, our results suggest that an impairment of antigen-presenting accessory cell functions could favor gp120-mediated apoptosis in HIV-uninfected cells.

During intrathymic T cell development, elimination of autoreactive T cell clones by programmed cell death (PCD or apoptosis) is an essential mechanism for self tolerance. The precise intracellular second messengers that lead to this process remain to be determined. In the present work, we show that treatment of freshly isolated thymocytes with an antagonist of the cAMP pathway, the Rp-cAMP, significantly decreases spontaneous death by apoptosis of human thymocytes in vitro. Addition of Rp-cAMP also rescues thymocytes from activation-induced apoptosis following the ligation of surface CD3/T cell receptor complex or CD2 antigens. A cAMP analog, the dibutyryl(Dibut)-cAMP increases PCD of human thymocytes in a dose-dependent manner. Growth and rescue from PCD of thymocytes in the presence of interleukin (IL)-2 or IL-4 are also enhanced by Rp-cAMP and inhibited by Dibut-cAMP. Finally, we detect substantial levels of intracellular cAMP in freshly isolated thymocytes. This study reveals the involvement of cAMP as a second messenger during the apoptosis of normal human thymocytes.

The immunotherapeutic potential of three anti-rat CD2 monoclonal antibodies (mAb) (OX34, OX54, OX55) and the combination of OX54 with OX55 was tested in Lewis rat experimental autoimmune encephalomyelitis (EAE). In actively induced EAE, a single injection of OX34 2 days before immunization with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) completely prevented or greatly attenuated EAE in all animals. Injection of OX54 acted moderately suppressive while OX55 or OX54/55 did not affect disease severity. Abrogation of EAE by OX34 was not restricted to its application before immunization. Therapeutic administration of all three mAb and the Ab combination from onset of first clinical signs efficiently blocked progression of disease and prevented all animals from developing hind limb paresis. In adoptive transfer EAE induced with in vitro activated cells of an encephalitogenic T helper line, clinical and histological signs were completely prevented by injection of OX34 on the day of cell transfer and 4 days later, underlining the strong impact of anti-CD2 mAb on the effector phase of disease. Immunocytofluorometric analysis of peripheral blood lymphocytes after a single Ab injection demonstrated that all mAb induced a variable degree of transient reduction in T cell numbers and modulation of CD2 antigens. In contrast to the other mAb, OX34 persisted on lymphocytes for at least 11 days, which may explain its unique suppressive effect on EAE after a single injection before immunization. The assumption that prophylactic administration of OX34 also inhibits MBP-induced EAE, due to persistence into the effector phase, was substantiated by the finding that none of the mAb prevented generation of an antigen-specific cellular response in MBP/CFA-immunized animals. Since none of the Ab induced T cell unresponsiveness or inhibited T cell activation by antigen- or Ab-mediated stimulation of the T cell receptor, we suggest that their marked action on the effector phase of EAE may rely on inhibition of T cell infiltration into the central nervous system. The demonstrated efficacy of these anti-CD2 mAb in EAE suggests a potential therapeutic role that may be equal to that of anti-CD4 or anti-T cell receptor Ab.

Receptors for the invariant chain of immunoglobulins (FcR) define the cellular response to specific antigens. Fc gamma R recognize IgG and so elicit a variety of effector functions including phagocytosis. We are interested in the structural determinants for Fc gamma R-mediated phagocytosis, specifically Fc gamma RI(p135) and Fc gamma RIIa isoforms. The low-affinity receptor, Fc gamma RIIa, is found on macrophages and its cytoplasmic domain contains a tyrosine activation motif which has previously been shown to regulate endocytosis. In contrast, Fc gamma RI has no known signaling motifs, though a functional interaction has recently been demonstrated with the gamma chain of the high-affinity receptor for IgE, Fc epsilon RI. This accessory molecule has a cytoplasmic tyrosine activation motif implicated in signal transduction. Here we demonstrate that although Fc gamma RI transiently expressed on COS-7 cells is able to rosette opsonized SRBC, it cannot phagocytose them. If the cytoplasmic domain of either gamma chain or Fc gamma RIIa replaces that of Fc gamma RI in a chimeric receptor, efficient phagocytosis occurs. This particle ingestion is sensitive to the tyrosine kinase inhibitor genistein. Chimeric receptors where the extracellular domain of either Fc gamma RI or Fc gamma RIIa is replaced with that of CD2, a T cell antigen, indicate that Fc gamma R-mediated phagocytosis is ligand independent. We conclude that phagocytosis is dependent upon close particle apposition, tyrosine kinase activity, and that the process is ligand independent.

Through complex interplay with APCs, subsets of NK cells play an important role in shaping adaptive immune responses. Bovine tuberculosis, caused by Mycobacterium bovis, is increasing in incidence and detailed knowledge of host-pathogen interactions in the natural host is essential to facilitate disease control. We investigated the interactions of NK-cell sub-populations and M. bovis-infected DCs to determine early innate mechanisms in the response to infection. A sub-population of NK cells (NKp46(+) CD2(-) ) selectively expressing lymphoid homing and inflammatory chemokine receptors were induced to migrate towards M. bovis-infected DCs. This migration was associated with increased expression of chemokines CCL3, 4, 5, 20 and CXCL8 by M. bovis-infected DCs. Activation of NKp46(+) CD2(-) NK cells and secretion of IFN-γ was observed, a response reliant on localised IL-12 release and direct cellular interaction. In a reciprocal manner, NKp46(+) CD2(-) cells induced an increase in the intensity of cell surface MHC class II expression on DCs. In contrast, NKp46(+) CD2(+) NK cells were unable to secrete IFN-γ and did not reciprocally affect DCs. This study provides novel evidence to demonstrate distinct effector responses between bovine NK-cell subsets during mycobacterial infection.

So far little is known about natural killer (NK) cells in the pig due to the lack of NK cell-specific markers. In this study, we identified the activating receptor NKp46 (CD335) in swine with newly developed monoclonal antibodies (mAbs) for more detailed studies on NK cells in this species. The NKp46 mAbs showed a specific reactivity with a distinct population of perforin(+) CD2(+) CD3(-) CD8α(+) CD16(+) lymphocytes. In spleen and liver, an additional subset of CD8α(dim/-) lymphocytes with increased NKp46 expression was observed. Surprisingly, we could identify NKp46(-) cells with an NK cell phenotype in all animals analyzed. These lymphocytes showed comparable cytolytic activity against xenogeneic and allogeneic target cells as NKp46(+) NK cells. In contrast, NKp46(+) NK cells produced several fold higher levels of interferon-γ (IFN-γ) than the NKp46(-) cells after cytokine stimulation. Furthermore, an activation-dependent induction of NKp46 expression in formerly NKp46(-) cells after stimulation with interleukin-2 (IL-2), IL-12, and IL-18 could be shown. In summary, our data indicate that NKp46 is not expressed by all porcine NK cells and that NKp46 discriminates porcine NK cells differing in regard to cytokine production, which challenges the paradigm of NKp46 as a comprehensive marker for NK cells across different mammalian species.

Many lymphocytes enter tissues such as peripheral lymph nodes, and Peyer's patches through high endothelial venules (HEV). It is known that HEV differ in the expression of adhesion molecules as lymphocyte subsets do. Through the interaction of these molecules B and T lymphocyte subsets are thought to be preferentially directed into lymphoid organs. However, it is unclear which role these mechanisms play in vivo, since there are no studies demonstrating that blood lymphocyte subsets preferentially interact with different types of HEV in vivo. Therefore, in the present study the frequency of B, T, CD4+ and CD8+ lymphocytes in the wall of the HEV of rat peripheral lymph nodes and Peyer's patches was analyzed by immunohistology. In addition, the expression of CD44, VLA-4, LFA-1, ICAM-1, CD2 and L-selectin on B and T lymphocyte subsets of the blood was determined by flow cytometry. Although B and T lymphocytes showed significantly different levels of expression for each adhesion molecule investigated, the relation of B and T lymphocytes within the HEV of peripheral lymph nodes and Peyer's patches was strikingly comparable (38.0 +/- 5.2% vs. 40.6 +/- 5.7% and 62.0 +/- 5.2% vs. 59.4 +/- 5.7%, respectively). The same was true for CD4+ and CD8+ cells. Thus, although HEV and the blood lymphocyte subsets differ markedly in their expression pattern of adhesion molecules, the existing levels are sufficient to mediate comparable entrance of B and T lymphocyte subsets into both types of HEV.

Antigen receptor engagement initiates clonal expansion and antibody secretion in B lymphocytes in response to foreign antigens. However, binding of self antigen to antigen receptors targets self-reactive B cell clones for elimination or inactivation. The antigen-triggered biochemical events and the eventual response of the cells are dependent on the simultaneous occupancy of co-stimulatory receptors. CD2 is an intercellular adhesion molecule implicated in cell activation and expressed in human T and natural killer cells as well as in mouse B lymphocytes. Mouse B cells specific for allogeneic major histocompatibility complex (MHC) class I initiate a suicide program that leads to DNA fragmentation and cell death when confronted with soluble MHC class I while undergoing clonal expansion when the antigen is present on mitomycin C-treated cells. Here we show that occupancy of CD2 in mouse B cells by the presence of either monoclonal antibody (mAb) specific for CD2, or soluble recombinant mouse CD48, its natural ligand in mouse, prevents the induction of apoptosis. Furthermore, the in vitro activation by mitomycin C-treated allogeneic cells, is abrogated in the presence of anti-CD48 mAb (OX78). These results indicate that a CD2-CD48 interaction is involved in the control of B cell activation.

Previous studies have reported an association of the cell surface adhesion molecule CD2 with the T cell receptor and with CD45 on mouse and human T lymphocytes. In this study the association of CD2 with cell surface molecules was investigated using cell surface biotinylation of T lymphocytes, coupled with immunoprecipitation using two CD2-specific monoclonal antibodies (mAb) (RM2-5 and 12-15) and analysis by SDS-PAGE. Although both CD2 mAb immunoprecipitated CD2 from lysates of murine lymphocytes, it was found that mAb 12-15, but not RM2-5, co-precipitated two other molecules of 95 and 180 kDa. Subsequent studies revealed that the 95- and 180-kDa molecules were associated with a subspecies of CD2 (approximately 5%) on thymocytes, the antigen-specific T cell line D10, and splenic T cells but not B cells. Two lines of evidence were obtained consistent with the 95- and 180-kDa molecules being the beta and alpha chains of LFA-1. Firstly, an analysis of 12-15 mAb immunoprecipitates on 4-12% gels under reducing and nonreducing conditions shows that the 95- and 180-kDa molecules have a molecular weight and migration pattern identical to LFA-1. Secondly, depletion of LFA-1 from lysates with LFA-1 mAb abolished the ability of CD2 mAb 12-15 to co-precipitate the 95- and 180-kDa molecules, thereby identifying these as the beta and alpha chains of mouse LFA-1, respectively. These results provide evidence for the first time for an association of LFA-1 and CD2 on mouse T lymphocytes, and suggest that the association occurs with an immunologically distinct subspecies of CD2 molecules.

Retinoic acid related orphan receptor gamma-t (RORγt) is known to be a master regulator of Th17-cell development. In this study, we generated RORγt-overexpressing transgenic (RORγt Tg) mice in which transgene expression was driven by the CD2 promoter, and found that these mice developed polyclonal plasmacytosis and autoantibody production. RORγt Tg mice were generated on a C57BL/6 background, and also were intercrossed with BALB/c mice. BALB/c F1 (BALB/F1) RORγt Tg mice developed massive polyclonal plasma-cytosis, and had shorter life spans. Splenomegaly and infiltration of plasma cells into the lung were observed. Hyperglobulinemia, anti-double-stranded DNA antibodies, anti-erythrocyte antibodies, and anti-platelet antibodies were detected in BALB/F1 RORγt Tg mice. In the present study, polyclonal plasmacytosis in BALB/F1 RORγt Tg mice appeared to be due to the induction of excessive IL-6 production by IL-17. We detected increased numbers of CD11b(+) cells that produced IL-6. We also generatedIL-6-deficient RORγt Tg BALB/F1 background mice, which displayed high levels of serum IL-17, but did not develop severe hyperglobulinemia. Excessive IL-6 production by several cell types, including macrophages, in BALB/F1 RORγt Tg mice, might effect the development of plasma-cytosis. These results suggest that RORγt plays important roles in the development of plasmacytosis and autoantibody production.

The human adhesion receptor CD58 (LFA-3) is expressed on most human cell types. Here we report on a soluble form of CD58 (sCD58) in human serum, human urine, and culture supernatants of several cell lines. sCD58 partially purified from human serum, from supernatant of the Hodgkin cell line L428, and purified sCD58 from human urine were found to have a molecular mass of 40-70 kDa under denaturating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting). However, gel filtration of sCD58 purified from human urine gave a molecular mass of 118-166 kDa, suggesting a noncovalent homotrimer conformation or its association with other molecules. Using an enzyme-linked immunosorbent assay specific for CD58 we found that sera from patients suffering from different forms of hepatitis contained elevated sCD58 levels (n = 108). Accordingly, there was a fivefold increase of supernatant sCD58 when the hepatocellular carcinoma cell line Hep G2 was incubated with 25 ng/ml recombinant tumor necrosis factor-alpha in vitro. In contrast, sCD58 serum levels of 337 additional patients suffering from various other immunological disorders were not found to be raised. At high concentrations sCD58 binds to CD2-positive cells and inhibits rosette formation of human T cells to human erythrocytes. Thus, local release of large quantities of naturally occurring sCD58 may interfere with intercellular adhesion in vivo.

Activation of immature thymocytes or transformed (i.e. leukemic) T lymphocytes via CD3/T cell receptor (TcR) signaling can induce programmed cell death (apoptosis). Recent data indicate that anti-CD3/TcR monoclonal antibodies (mAb) also trigger apoptosis in activated (but not resting) mature peripheral LT cells. We now report that interleukin-2 (IL-2) dependent human polyclonal T cell lines as well as T cell clones undergo programmed cell death when triggered via the alternative CD2-dependent activation pathway. In the presence of exogenous IL-2, a pair of mitogenic anti-CD2 mAb suppressed the IL-2-driven proliferative response. Growth inhibition was associated with cell death and DNA fragmentation as revealed by propidium iodide staining and gel electrophoresis, respectively. Induction of apoptosis by anti-CD2 mAb was prevented by cyclosporine A and FK 506. We conclude that programmed cell death can be initiated in activated human T cells by signaling via the CD2 pathway.

The murine equivalent to CD2, previously known as a T cell marker, is expressed on mouse B cells. The monoclonal anti-CD2 antibody 12-15 was found to induce B cell homotypic adhesion. When treated with F(ab')2 fragments of 12-15, purified, resting B cells aggregate within 2 h of incubation and the response is optimal after 20 h. Anti-CD2-induced aggregation is a dose-related active process, dependent on temperature, metabolic energy and divalent cations. Aggregation is inhibited by two different Fab monomers of anti-CD2, implying that it is the CD2 molecule itself that functions as an adhesion molecule. We also report that interleukin 4-induced B cell homotypic adhesion involves CD2-mediated cell binding, since the antibodies specific for mouse, CD2, inhibited interleukin-4-induced cell aggregation.

The effect of influenza (FLU) infection on heterotypic conjugate formation between antigen-presenting cells and T lymphocytes has been studied with FLU-specific T cell clones and FLU-infected B-lymphoblastoid cells (B-LCL). Conjugate formation between FLU-infected B-LCL (FLU+ B-LCL) and T cells was found to be consistently enhanced in comparison with peptide-sensitized or uninfected B-LCL. Treatment of B-LCL with exogenous neuraminidase (NA-NAse) similarly enhanced conjugate formation indicating that increased conjugate formation may be mediated by the viral neuraminidase. Monoclonal antibody blocking experiments revealed that the contribution by CD2/LFA-3 is increased relative to that of LFA-1/ICAM-1 in conjugates between FLU+ B-LCL or NANAse-treated B-LCL and T cell clones. In contrast, both pathways of adhesion contributed equally to conjugate formation between peptide-sensitized B-LCL or control B-LCL and T cell clones. Thus, FLU infection causes increased conjugate formation between antigen-presenting cells and T cells and skews towards CD2/LFA-3-dependent adhesion, independent of T cell receptor signalling.

Regulation of interleukin (IL)-4 production, but not IL-2 production, was found to be quite different in either freshly isolated T cells or T cell clones. Both fresh T cells and T helper 2-like clones produced IL-4 when stimulated with anti-CD2 in combination with anti-CD28. However, whereas T cell clones showed enhanced IL-4 production when phorbol 12-myristate 13-acetate (PMA) was used in addition to anti-CD2 and anti-CD28, IL-4 production by fresh T cells was inhibited by the presence of PMA. Prestimulation of fresh T cells led to the following observations: (a) activation in the absence of PMA led to a reversal of the PMA effect and (b) within 2 days these cells resembled T cell clones in that IL-4 production was no longer inhibited by PMA. When prestimulation was carried out in the presence of PMA, the inhibition of IL-4 production seemed irreversible. Removal of PMA after 3 days did not lead to renewed capability of IL-4 production, whereas IL-2 production was unimpaired. Our data show that the capacity of cultured T cells to produce IL-4 is determined and fixed during the first 2-3 days of stimulation.

CD2-associated molecules were identified by means of in vitro kinase assays of CD2 immunoprecipitates obtained from nontransformed human T lymphocytes lysed in the detergent brij 58. Under these conditions CD2 was found to be associated with the protein tyrosine kinases p56lck and p59fyn as well as two low molecular weight phosphoproteins. The latter were identified as the zeta and epsilon chains, the major signaling components of the CD/7 TcR complex. Importantly, induction of T cell unresponsiveness towards CD2-mediated stimuli by means of CD3/T cell receptor (TcR) modulation results in uncoupling of zeta and epsilon from the CD2 molecule, while its associations with p56lck and p59fyn remain unaffected. Moreover, despite the incapacity of T lymphocytes to undergo DNA synthesis in the CD3/TcR-modulated state, CD2 triggering still results in tyrosine phosphorylation of some unknown protein substrates. Thus, the same zeta and epsilon chains which are components of a functional TcR complex appear to also couple to the CD2 molecular complex. Moreover, dissociation of TcR and CD2 complexes in intact cells seems to block CD2-mediated T cell growth but does not result in complete abolishment of the signal transducing capacity of the CD2 receptor.

T cell activation by triggering the T cell receptor (TcR)-CD3 complex leads to a dramatic increase in tyrosine phosphorylation of multiple cellular proteins. To date, there has been no direct evidence on the identity of the tyrosine kinase activity implicated in this signaling pathway. In this study, we demonstrate that activation of human T cells with anti-CD3 monoclonal antibody increases tyrosine kinase activity of p56lck. This extends our previous findings which demonstrated the involvement of p56lck kinase activity in the CD2 signal transduction pathway. The results from peripheral blood lymphocytes and Jurkat cell line showed in both cases an early and transient change in the specific activity of p56lck, followed by a shift to a higher apparent molecular mass. Therefore, to test directly the role of TcR-CD3 in CD2-induced activation of p56lck, we utilized mutant variants of the Jurkat cell line lacking in cell surface TcR-CD3. We found that cell surface expression of TcR-CD3 is not required for the activation of p56lck via CD2 but is necessary for the appearance of the reduced-electrophoretic-mobility form of p56lck observed after CD2 triggering. By isolating CD45- mutants from Jurkat cells, we observed that surface expression of the tyrosine phosphatase CD45 is required in order to increase p56lck activity following CD2 stimulation, while CD4-induced activation of the kinase remained unchanged. These data provide evidence for a specific functional linkage between CD2 and p56lck, in which CD45 may play an essential role.

Natural killer (NK) cells can be differentiated into lymphokine-activated killer (LAK) effectors following stimulation with interleukin (IL)-2. This induction can be negatively regulated by IL-4. In this study, we demonstrate that the stimulation of NK cells through the CD2 pathway with (9-1 + 9.6) monoclonal antibodies can also induce these cells to secrete tumor necrosis factor-alpha (TNF-alpha) and to differentiate into LAK effectors. More importantly, our data indicate that, in contrast to the IL-2-induced LAK generation, the anti-CD2-triggered LAK activity was not regulated by IL-4. IL-4 was found to enhance the LAK activity as well as NK cell proliferation following activation with anti-CD2 by a mechanism involving, at least in part, an increased TNF-alpha production. Using immobilized monoclonal antibodies against the Fc receptor (Fc gamma RIII or CD16) for NK stimulation, we also observed that the anti-CD16-induced LAK activity was not inhibited by IL-4. These data further point to a pivotal role of TNF-alpha as a regulatory cytokine in anti-CD2-induced LAK generation, and suggest that IL-4 could serve as a discriminatory factor between two distinct pathways involved in the activation of non-MHC-restricted cytotoxicity.

Appropriate experimental conditions were devised to demonstrate that CD58 (LFA-3), CD54 (ICAM-1) and CD11a/CD18 (LFA-1) adhesion molecules are the source of signals that regulate nonspecific major histocompatibility complexunrestricted and CD3/T cell receptor (TcR)-triggered cytotoxicity. Using anti-LFA-3 monoclonal antibody (mAb)-treated, interleukin-2 (IL-2)-cultured peripheral blood lymphocytes (PBL) or cloned CD3+/CD8+ cells as lymphocyteactivated killer (LAK) effectors, and ligand (CD2)-negative tumor cell lines as targets, a down-regulation of CD3− and CD3+ cell-mediated LAK activity was consistently observed. Anti-LFA-3 mAb also down-regulated tumor cell lysis when T cell clones were triggered to kill P815 cells through stimulation of the CD3/TcR complex by an anti-CD3 mAb. The inhibitory effect of anti-LFA-3 mAb was not prevented by stimulatory anti-CD2 mAb. Anti-ICAM-1 mAb treatment of IL-2-cultured PBL consistently up-regulated LAK cytotoxicity against tumor target cells. However, this effect was only exerted on CD3− LAK effectors. Anti-LFA-1 mAb blocked conjugate formation between effector cells and tumor target cells, thus rendering this model unsuitable to evaluate the regulatory role of LFA-1. Therefore, a cytotoxicity model system was applied in which a hybrid anti-CD3/anti-human red blood cell (HuRBC) mAb triggers cytolytic T cells to lyse HuRBC. In these experiments, anti-LFA-1 mAb markedly up-regulated the lytic ability of IL-2-cultured PBL. We conclude that mAb against LFA-3, ICAM-1 and LFA-1 molecules deliver regulatory signals for LAK cells and cytotoxic T lymphocytes. As these stimuli may be delivered by ligands expressed on tumor targets as well as on other immune competent and inflammatory cells, the present observations are relevant in the context of both the host's immune response against tumors and the general functioning of the immune system.

We have examined the regulation of interleukin (IL)-4 production by human peripheral blood T cells. Production of IL-4 was shown to be regulated differently from IL-2 and interferon(IFN)-gamma production. Stimulation of peripheral blood lymphocytes with anti-CD3, anti-CD2, anti-CD28, Phorbol 12-myristate 13-acetate (PMA) or IL-2 as a single stimulant did not induce IL-4 production. However, combinations of anti-CD2 with either anti-CD28 or IL-2 resulted in IL-4 production, peaking at days 3-4. Stimulation with anti-CD3 instead of anti-CD2 gave similar results, but was less potent. After days 3-4, IL-4 levels decreased, most likely due to consumption of IL-4. PMA profoundly affected cytokine production, it enhanced IL-2 production by at least tenfold, whereas, in the same cell population, IL-4 production was almost completely inhibited. This was observed at the protein as well as at the mRNA level. In contrast, agents that increase intracellular cAMP levels inhibited IL-2 production but left IL-4 production unaffected. IFN-gamma production behaved similar to IL-2 production but the effects were less outspoken.

We have identified a new T cell activation pathway mediated by the lymphocyte homing receptor/CD44 molecule, 8B2.5, a local monoclonal antibody (mAb), which recognizes two glycoproteins of 85 and 220 kDa with wide tissue distribution, is shown by sequential immunoprecipitations and competitive antibody-binding inhibition experiments with several CD44 reference mAb to recognize the CD44 molecule. The 8B2.5 mAb, but not reference CD44 mAb, is able to induce resting peripheral blood lymphocytes to proliferate in the presence of phorbol esters. This proliferation is monocyte dependent but Fc independent and results from 8B2.5 mAb binding to CD44 molecules both expressed by both T cells and monocytes. In the absence of monocytes, proliferation can be restored by solid-phase 8B2.5 mAb, or, to a lesser extent, by adding interleukin 2. Although CD3 and CD44 surface molecules are found physically independent, T cell activation via the CD44 pathway is inhibited by CD3 modulation. In addition to the direct role of CD44 molecules in T cell proliferation, CD44 mAb can up- or- down-regulate the CD3 and CD28 pathways, depending on the presence of monocytes. These results suggest that T cell and monocyte binding to high endothelial venule or extracellular matrix proteins could further promote clonal expansion of resting T cells migrating in certain specific anatomic sites.

An immediate consequence of T cell activation via the T cell receptor (TcR)/CD3 complex and CD2 antigen is the hydrolysis of phosphatidylinositol-(4,5)-bisphosphate and the generation of inositol-(1,4,5)-trisphosphate and diacylglycerol which then regulate intracellular calcium and protein kinase C. Changes in cellular levels of phosphoinositides phosphorylated on the D-4 and D-5 position during T cell activation have been well documented. Recently it has been proposed that phosphoinositides phosphorylated on the D-3 position of the inositol ring by a novel phosphoinositide (PI) 3 kinase may also be important in cell activation. In the present study we have examined the levels and regulation of D-3 phosphoinositides in T cells activated by the TcR/CD3 complex and CD2 antigens. The data show the existence of phosphatidylinositol-(3)-monophosphate [PtdIns(3)P], phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol-(3,4,5)-trisphosphate [PtdIns(3,4,5)P3] in T cells. Activation of the TcR/CD3 complex or CD2 antigen results in modulation of PtdIns(3,4)P2 and a putative PtdIns(3,4,5)P3 in T cells but does not change levels of PtdIns(3)P. These data provide the first evidence that lipid products of a PI 3 kinase exist in T cells.

We report the consequences of low expression of the T cell receptor (TcR)/CD3 complex by T lymphocytes from a 4-year-old boy with a mild immunodeficiency. TcR/CD3 expression was found to be deficient on both resting and activated T cells, using both anti-CD3 and anti-TcR alpha/beta monoclonal antibodies. As shown by immunofluorescence and immunoprecipitation studies, residual expression (corresponding to about 10% of normal) was detectable on resting and activated TcR alpha/beta+ T cells. Other T cell membrane receptors were normally expressed. The functional consequences of this TcR/CD3 expression deficiency included an absence of T cell proliferation, interleukin 2 receptor expression and calcium flux following anti-CD3 and anti-CD2 antibody-triggered T cell activation. Antigen (tetanus toxoid, Candida and allogeneic cell)-induced proliferation was detectable. In contrast, cytotoxic T cell activity towards allogeneic cells was deficient. These findings shed light on the function of the TcR/CD3 complex and indicate that the expression of a limited number of TcR/CD3 receptors may be sufficient to trigger antigen-specific T cell activation (and, possibly, differentiation) and that anti-CD3 antibody-induced T cell activation differs somewhat from antigen/major histocompatibility complex molecule-induced activation. These results also confirm that the CD2 pathway of T cell activation is CD3 dependent.

Cell-cell and cell-extracellular matrix (ECM) binding mediated by integrin molecules has been implicated in lymphocyte migration and adhesion. We describe here that ECM binding triggers antigen-independent activation of T cell functions. Fibronectin and vitronectin, when coated on plates, not only acted synergistically on anti-CD3-induced serine esterase release in a murine cytotoxic T lymphocyte clone and interleukin 2 production in a murine helper T cell hybridoma but also could trigger these responses alone. All these stimulatory effects of ECM were abrogated by a monoclonal antibody which reacts with a unique very late activation antigen-like integrin and this monoclonal antibody, when coated on plates, exhibited a similar synergistic effect to that of ECM proteins. Therefore, ECM receptors expressed on activated T cells appear to play an important role in triggering T cell effector functions in localized tissues abundant in ECM proteins.

Acute P. falciparum malaria is associated with loss of in vitro T cell responsiveness to antigenic stimulation, and with high plasma levels of soluble interleukin 2 receptor (IL 2R). In the present study peripheral T cells from acute P. falciparum malaria patients from a malaria-endemic area of Sudan were analyzed for expression of cell surface antigens associated with T lymphocyte adhesion, activation and maturation. The results were compared to results from T cells obtained from the same donors either before the attack, or during convalescence. Most donors showed a remarkable loss of T cells with high expression of the surface marker LFA-1 (CD11a/CD18) during the clinical episode, in addition to the functional changes described above. Two donors that did not show phenotypic changes were furthermore characterized by having an unabated proliferative response and normal plasma IL 2R levels. All peripheral CD3+ T lymphocytes expressed LFA-1, which had a clearly bimodal distribution on these cells. The T cell subpopulation having high LFA-1 expression (LFA-1++) was composed of both memory and unprimed T cells, according to their expression of CD45RA and CD45R0. Analysis of expression of membrane-bound IL 2R (CD25) and ICAM-1 (CD54) did not reveal in vivo activated T cells in the peripheral blood of the patients. Taken together, these data suggest that circulating T cells recognizing parasite antigens are temporarily withdrawn from peripheral circulation during P. falciparum malaria.

The influence of T cell receptor (TcR) triggering on T cell adhesion function has been systematically investigated in the present studies; we show that the adhesion function of LFA-1 is minimal in non-activated T cells but is augmented within minutes following TcR-mediated activation. In contrast, CD2 function is essentially optimal in non-activated T cells and undergoes no detectable modification within 12 h of TcR stimulation. Protein kinase C activation augments LFA-1 but not CD2 adhesion function and cyclic AMP reduces LFA-1 adhesion without affecting CD2-LFA-3 interactions. Up-regulation of the LFA-1 pathway occurs in the absence of any detectable surface redistribution of this molecule, suggesting an activation dependent modification leading to a high-affinity ICAM-1 binding state. The TcR independence of CD2 adhesion function implies a critical role of the CD2 pathway in initiating cell-cell interactions prior to TcR engagement and LFA-1-ICAM-1 binding and underscores the complementary nature of the CD2 and LFA-1 adhesion pathways during the immune response.

Structurally unrelated, FK-506 and cyclosporin (CsA) bind to and inhibit the action of distinct cytoplasmic receptors, FK-506-binding protein (FKBP) and cyclophilin (CyP), respectively. These receptors, termed immunophilins, share no sequence similarity, and yet both have been demonstrated to be capable of catalyzing the cis-trans isomerization of peptidyl-prolyl bonds (rotamase activity). Because FK-506 and CsA bind to different intracellular target structures, we investigated the spectrum of action of FK-506, in comparison to CsA, on T cell activation. We have shown that FK-506, like CsA, is able to inhibit T cell activation mediated not only by the T cell receptor-CD3 complex, but also via another surface molecule, CD2. T cell proliferation, stimulation of interleukin 2 production, and induction of apoptosis were all sensitive to inhibition by both FK-506 and CsA. With each parameter of activation, FK-506 is approximately 10-100-fold more effective than CsA. In contrast, FK-506 did not affect T cell proliferation induced by anti-CD28 monoclonal antibody in the presence of phorbol 12-myristate 13-acetate. This CD28 pathway, however, was inhibitable by a structural homolog of FK-506, rapamycin, demonstrating that the mechanism of action of FK-506 has specificity. These data suggest that immunophilins or the complex of drug coupled to immunophilin (i.e. FK-506/FKBP, CsA/CyP) are involved in and regulate selective pathways of T cell stimulation.

In this study, we demonstrated that antibodies reactive with a common beta 1 subunit (CD29) of the VLA family of antigens could synergize with CD3 but not CD2 in inducing CD4 cell proliferation in a serum-free culture system. The extent of activation was similar to that obtained when CD4 cells were cultured with anti-CD3 plus fibronectin. The proliferative response induced via the CD3-CD29 pathway was associated with the interleukin (IL) 2 autocrine pathway, including IL 2 production and IL 2 receptor expression. Specifically, cross-linking of VLA-5, but not other members of the VLA family of antigens with the CD3 molecule induced strong CD4 cell activation. However, cross-linking of CD3 with other cell surface antigens such as CD4 did not induce CD4 cell activation in this serum-free culture system. The above results strongly suggest that the VLA/CD29 family of antigens may play an important role in regulating CD4 T cell activation via the CD3-T cell receptor pathway.

The relationship of dendritic cells (DC) isolated from the peripheral blood to those of lymphoid tissue is, in terms of maturation and function, incompletely understood. In our present study, we have explored the molecular basis of adhesion of T cells to blood DC. Analysis of the expression of adhesion receptors on the cell surface of blood DC revealed that these cells express lymphocyte function-associated antigen (LFA)-1 (CD11a/18), ICAM-1 (CD54), LFA-3 (CD58) and CD44, but are very late antigen (VLA)-4 (CD49d) and vascular cell-adhesion molecule (VCAM)-1 negative. The LFA-1 pathway was found to play a key role in T cells-blood DC adhesion; monoclonal antibodies (mAb) against both LFA-1 and ICAM-1 strongly inhibited adhesion between those cells. Moreover, a T cell clone from an LFA-1-deficient patient showed poor binding to blood DC. The important role of LFA-1 in T cell-blood DC adhesion was also supported by the metabolic energy and divalent cation dependence of the interaction. mAb against LFA-3 and CD2 did not inhibit T cell-blood DC binding. In contrast to the strong inhibition by antibodies to LFA-1 and ICAM-1, antibodies to CD44 enhanced conjugate formation between T cells and blood DC. Together, our results show that the LFA-1/ICAM-1 pathway plays a central role in T cell-blood DC adhesion, a situation like that in T cell adhesion to lymphoid DC. However, unlike lymphoid DC, blood DC do not express VCAM-1 nor use LFA-3 for T cell binding.

Human lamina propria lymphocytes were recovered from surgical specimens of unaffected large bowel and compared with peripheral blood lymphocytes (PBL) from the same individual. We investigated their capacities to respond to triggering stimuli delivered by mitogenic monoclonal antibodies directed at the T cell receptor/CD3 complex or, alternatively, the CD2 glycoprotein. In marked contrast to PBL, expression of the interleukin 2/interleukin 2 receptor system was markedly reduced in LPL following activation through the CD3/T cell receptor. Perhaps, more important, responses to CD2 triggering were similar or even stronger in LPL as opposed to PBL. This indicates that the CD2-dependent alternative pathway of T cell activation might serve a functional role in local immunity in the gut.

Three types of T cell receptors (TcR) gamma/delta were identified in swine, all of which are disulfide-bonded cell surface heterodimers. The first, a 38/40-kDa molecule, is related to the TcR gamma/delta of sheep and cattle, and defines in swine a subset of CD4-CD8- T lymphocytes that is enriched in the circulating T lymphocyte pool. The other two receptors are private to swine. The second, a 37/40-kDa molecule, is expressed by the major subset of CD2-CD4-CD8- T lymphocytes that is neither enriched in nor excluded from lymphoid tissue. By contrast, the third type of TcR gamma/delta, a 40/46-kDa molecule, is found on CD2+CD4-CD8- T lymphocytes accumulating in lymphoid tissue. Thus, TcR gamma/delta T lymphocyte subsets differing in homing propensity are typified by the expression of distinct molecular forms of the TcR gamma/delta.

In this study we examined the effect of interleukin 4 (IL 4) on T cell activation and proliferation via the alternative CD2 pathway. To this end highly purified human resting T cells were cultured with a stimulating pair of anti-CD2 monoclonal antibodies in the absence of accessory signals from monocytes. Addition of either recombinant (r)IL 2 or rIL 4 resulted in proliferation of the anti-CD2-stimulated T cells. The growth-promoting effect of rIL 4 on preactivated. T cells was shown to be partly a direct effect. rIL 4 also induced IL 2 production and, as a consequence, the effect of rIL 4 on T cell growth was enhanced by endogenously produced IL 2. Moreover, rIL 4 acted in synergy with exogenously added rIL 2 in promoting growth of anti-CD2-stimulated T cells. The synergistic effect of IL 2 and IL 4 could be explained by IL 2-induced up-regulation of IL 4 responsiveness. In contrast, preincubation with rIL 4 did not enhance IL 2 responsiveness and rIL 2 but not rIL 4 up-regulated IL 2R expression on anti-CD2-stimulated T cells. Finally we could demonstrate that monocyte-produced cytokines (IL 1 and IL 6) enhance the proliferative response to rIL 4 of anti-CD2-stimulated T cells. It can be concluded that IL 4 can act as a paracrine growth factor for T cells activated in the alternative CD2 pathway, and that it acts synergistically with IL 2, IL 1 and IL 6. Moreover, IL 4 is a helper signal for IL 2 production. Thus, IL 2 and IL 4 are involved in a bidirectional regulatory network, with IL 4 as an inducer of IL 2 production, and IL 2 as an enhancer of IL 4 responsiveness.

International audience; NK-T-B antigen (NTBA) is a CD2 family member that functions as a coreceptor in human NK cell activation. Several receptor/ligand interactions occur between different members of this molecular family. In this study, in order to identify the natural ligand of NTBA, we produced a chimeric protein formed by the NTBA extracellular region fused with the Fc portion of human IgG1 (termed NTBA-Fc*). NTBA-Fc* specifically binds to NTBA cell transfectants but not to cells transfected with other CD2 family members including CD2, CD48, CD84, CD150, CD229, and CD244. Moreover, NTBA-Fc* also binds to NTBA(+) but not to NTBA(-) T cell lines. Enzyme-linked immunosorbent assays, plasmon resonance analysis, as well as NTBA-Fc*-mediated down-regulation of NTBA surface expression further confirmed the occurrence of NTBA/NTBA homophilic interaction. Functionally, in NK cells, NTBA-Fc* promoted a strong production of IFN-gamma and TNF-alpha. Moreover, NTBA-transfected targets displayed increased susceptibility to NK-mediated killing as compared to untransfected cells and this effect was specifically inhibited by anti-NTBA mAb. Altogether our data indicate that NTBA is characterized by self recognition.

Activation-induced natural killer (NK) cell death is very rapid compared to activation-induced T or B cell death. Here we show that NK cell activation is accompanied by the leakage of granzymeB from intracellular granules into the cytoplasm. Evidence for granzyme B leakage includes the formation of granzyme B/serine proteinase inhibitor 9 (PI-9) complexes that are detected by immunoprecipitation as well as colocalization of granzyme B and PI-9 detected by immunocytochemistry. The pro-apoptotic molecule Bid, a specific substrate for granzyme B, was cleaved within 2 min following CD2-induced NK cell activation, suggesting that granzyme B triggers apoptosis by directing Bid to mitochondrial membranes. The granzyme B/PI-9 protein ratio was found to mirror the percentage of CD2-induced NK cell death, suggesting that an excess of leaked granzyme B over its inhibitor is a major determinant of cell death. We suggest that granzyme B leakage-induced cell death is an important determinant of activation-induced NK cell death and that this process may be important for the fate of NK cells which encounter malignant or virus-infected cells.

The pathways leading to activation of mucosal lamina propria (LP) T cells differ from those of peripheral T cells. LP T cells exhibit enhanced IFN-gamma secretion when activated through the CD2 pathway. This study demonstrates CD2 signaling is followed by activation of STAT proteins in both peripheral blood mononuclear cells (PBMC) and lamina propria mononuclear cells (LPMC), although, distinct differences exist in regulation of IFN-gamma promoter gene expression. Both PBMC and LPMC exhibit enhanced secretion and transactivation of the -2.7 kb IFN-gamma promoter region following CD2 signaling, but the IFN-gamma STAT-binding region (within the first intron) serves as an orientation-independent enhancer of promoter activity only in LPMC. Mutation of the STAT site impairs enhancer activity. In LPMC, but not PBMC, CD2 mediates binding of STAT1 and STAT4 to the IFN-gamma intronic element. Unstimulated LMPC exhibit low levels of phosphotyrosine-STAT4 and STAT1 and phosphoserine-STAT1, which increase substantially following CD2 activation. In PBMC, CD2-mediated phosphorylation is primarily restricted to enhanced levels of phosphotyrosine-STAT1. Thus, these results indicate that both common as well as unique molecular mechanisms are involved in CD2 signaling and activation of the STAT pathway in LP T cells which are critical for regulation of IFN-gamma expression in the gut.

Natural killer (NK) cells participate in both innate and adaptive immunity through the prompt secretion of cytokines and ability to lyse virally infected cells or tumor cells. Although it has been well understood that lipid rafts (rafts) and a raft-associated linker for activation of T cells (LAT) plays a central role in TCR signal transduction, there are still great gaps in our knowledge of the molecular events involved in NK cell activation. We show here that CD2 and rafts became polarized to the site of NK cell activation by CD2 cross-linking or target cell binding using confocal microscopy, and LAT and a significant amount of CD2 colocalized in raft fractions of sucrose-density gradient from an NK cell line, NK3.3. CD2 cross-linking strongly induced tyrosine phosphorylation of LAT, resulting in increased association with phosphatidylinositol 3-kinase (PI 3-K) and phospholipase C-gamma1 (PLC-gamma1). In vitro binding studies using glutathione S-transferase fusion proteins demonstrated that a large portion of the association between LAT and PI 3-K or PLC-gamma1 was mediated through their SH2 domains in tyrosine phosphorylation-dependent manner. Furthermore, disruption of lipid rafts by cholesterol depletion from cell membranes using methyl-beta-cyclodextrin markedly reduced LAT tyrosine phosphorylation and NK cell functions, including cytotoxicity and granule exocytosis. These results document that modulation of raft integrity by aggregation of NK cell activating receptors, which leads to the formation of complexes of LAT with PI 3-K and PLC-gamma1, is essential for the NK cell lytic mechanisms.

T lymphocytes can be activated and induced to proliferate through stimulation of the CD2 glycoprotein with functional combinations of CD2 antibodies. However, this mechanism of signal transduction via CD2 is still not fully understood. We have investigated which molecules on the T cell surface preferentially associate in Cis with CD2 and may regulate its signaling properties. Though a quantification method we found that CD5 represents the antigen capable of co-precipitating a larger proportion of CD2. Using co-capping assays and immunoprecipitations from cell lysates, we show that an association between CD2 and CD5 can be found in rat thymocytes, T lymphocytes and in a thymoma cell line. Possibly, this interaction is a direct one, since CD2 and CD5 transiently expressed in Cos7 cells co-precipitate each other. Furthermore, using CD2 chimeric proteins containing different domains of CD2, expressed in Cos7 cells as well as in stably transfected Jurkat cells, we show that the interaction between CD2 and CD5 is held at both the intra- and extracellular levels, but does not involve the transmembrane domain. The fact that both the extracellular and the cytoplasmic domains of CD2 interact with CD5 suggests a specific and tight association between the two molecules, possibly relevant for the fine-tuning of signal transduction in T lymphocytes.

The phosphatidylinositol phosphatase gene PTEN is a dual specific phosphatase acting on phospho amino acids but also on three phosphorylated inositol phospholipids. Present results demonstrate that PTEN is inducible by costimulatory signals in human CD4(+) T cells. PTEN expression was up-regulated on RNA and protein level in freshly isolated human CD4(+) T cells following stimulation with CD28 or CD2. In contrast, PTEN expression was high but remained CD28 and CD2 unresponsive in lymphoma cells. Intracellular staining revealed PTEN expression in CD4(+) T cell populations stimulated with anti-CD28 or anti-CD28 / anti-CD3. Stimulation with anti-CD3 alone did not induce PTEN expression. Inhibition of PTEN expression by antisense oligonucleotides in CD4(+) T cells stimulated with non-mitogenic anti-CD28 resulted in massively increased proliferation, which was sensitive to the phosphatidylinositol 3-kinase (PI3 K) inhibitor wortmannin. Although CD28 and CD2 induce PI3 K signal transduction, wortmannin did not block PTEN up-regulation by CD28 or CD2 indicating that PTEN gene expression is PI3 K independent. These results demonstrate that PTEN negatively controls costimulatory signals by antagonizing PI3 K activity in the absence of TCR engagement.

Glucocorticoids are agents endowed with powerful immunosuppressive and anti-inflammatory properties partially related to the inhibition of adhesion-related processes. We have previously demonstrated that glucocorticoids inhibit LFA-1 and CD2 expression in human peripheral blood mononuclear cells (PBMC) by down-regulating mRNA steady-state levels. In this study, we investigated whether glucocorticoids could also act indirectly by modulating the effect/function of cytokines whose expression are known to inhibit. To test this hypothesis, we replenished the following cytokines IL-2, IL-7, IL-15, TNF-alpha, IL-1beta, IL-4 and IL-10, in an in vitro PBMC culture system. Our results indicate that only the IL-2Rgamma-chain-dependent cytokines IL-2, IL-7 and IL-15, among the cytokines of this panel, could reverse the inhibition of glucocorticoids on PBMC adhesion molecule expression and the related functions of intercellular aggregation and proliferation. Furthermore, we also demonstrated that IL-2, IL-7 and IL-15 could induce de novo the synthesis of LFA-1 and CD2. Taken together, these data suggest that glucocorticoids inhibit PBMC LFA-1 and CD2 expression not only directly by modulating transcriptional events, but also indirectly through the inhibition of IL-2Rgamma-dependent cytokines.