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Engagement of the T cell receptor complex reprograms T cells for proliferation, cytokine production and differentiation towards effector cells. This process depends on activating costimulatory signals and is counteracted by coinhibitory molecules. Three transcription factors, namely NF-κB, NFAT and AP-1, have a major role in inducing the transcriptional program that is required for T cell activation and differentiation. Here we describe the generation of a triple parameter reporter based on the human Jurkat T cell line, where response elements for NF-κB, NFAT and AP-1 drive the expression of the fluorescent proteins CFP, eGFP and mCherry, respectively. The emission spectra of these proteins allow simultaneous assessment of NF-κB, NFAT and AP-1 activity in response to stimulation. Ligation of the TCR complex induced moderate reporter activity, which was strongly enhanced upon coengagement of the costimulatory receptors CD2 or CD28. Moreover, we have generated and tested triple parameter reporter cells that harbor costimulatory and inhibitory receptors not endogenously expressed in the Jurkat cells. In these experiments we could show that engagement of the costimulatory molecule 4-1BB enhances NF-κB and AP-1 activity, whereas coinhibition via PD-1 or BTLA strongly reduced the activation of NF-κB and NFAT. Engagement of BTLA significantly inhibited AP-1, whereas PD-1 had little effect on the activation of this transcription factor. Our triple parameter reporter T cell line is an excellent tool to assess the effect of costimulatory and coinhibitory receptors on NF-κB, NFAT and AP-1 activity and has a wide range of applications beyond the evaluation of costimulatory pathways., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

To uncover signaling system differences between T cell stimuli and T cell subsets, phosphorylation status of 18 signaling proteins at six different time points following TCR triggering and CD28/CD2 costimulation was examined in human T cell subsets by phospho-epitope-specific flow cytometry of fluorescent cell barcoded samples, thereby providing a high-resolution signaling map. Compared with effector/memory T cells, naive T cells displayed stronger activation of proximal signaling molecules after TCR triggering alone. Conversely, distal phosphorylation events, like pErk and pS6-ribosomal protein, were stronger in effector/memory subsets. CD28 costimulation specifically induced signaling necessary for proper NF-κB activation, whereas CD2 signaled more strongly to S6-ribosomal protein. Analysis of resting regulatory T cells (rTregs; CD4(+)CD45RA(+)FOXP3(+)) and activated regulatory T cells (actTregs; CD4(+)CD45RA(-)FOXP3(++)) revealed that, although rTregs had low basal, but inducible, Erk activity, actTregs displayed high basal Erk phosphorylation and little or no Akt activation. Interestingly, the use of Mek inhibitors to block Erk activation inhibited activation-dependent FOXP3 upregulation in rTregs, their transition to actTregs, and the resulting increase in suppressive capacity. In summary, our systems approach unraveled distinct differences in signaling elicited by CD28 and CD2 costimulation and between rTregs and actTregs. Blocking rTreg transition to highly suppressive actTregs by Mek inhibitors might have future therapeutic applications.

Allelic variation of SLAM expression on CD4(+)CD8(+) thymocytes has been proposed to play a major role in NKT cell development. In this article, this hypothesis is tested by the production of subcongenic mouse strains and Slamf1 transgenic lines. The long isoform of the C57BL/6 allele of Slamf1 was transgenically expressed on CD4(+)CD8(+) thymocytes under control of an hCD2 minigene. NOD.Nkrp1b.Tg(Slamf1)1 mice, which had a 2-fold increase in SLAM protein expression on CD4(+)CD8(+) thymocytes, had a 2-fold increase in numbers of thymic NKT cells. The additional thymic NKT cells in NOD.Nkrp1b.Tg(Slamf1)1 mice were relatively immature, with a similar subset distribution to those of congenic NOD.Nkrp1b.Nkt1 and NOD.Nkrp1b.Slamf1 mice, which also express increased levels of SLAM on CD4(+)CD8(+) thymocytes and produce larger numbers of NKT cells. Transgenic enhancement of SLAM expression also increased IL-4 and IL-17 production in response to TCR-mediated stimulation. Paradoxically, NOD.Nkrp1b.Tg(Slamf1)2 mice, which had a 7-fold increase in SLAM expression, showed no significant increase in NKT cells numbers; on the contrary, at high transgene copy number, SLAM expression levels correlated inversely with NKT cell numbers, consistent with a contribution to negative selection. These data confirm a role for SLAM in controlling NKT cell development and are consistent with a role in both positive and negative thymic selection of NKT cells.

Studying the activity of homogeneous regulatory T cell (Treg) populations will advance our understanding of their mechanisms of action and their role in human disease. Although isolating human Tregs exhibiting low expression of CD127 markedly increases purity, the resulting Treg populations are still heterogeneous. To examine the complexity of the Tregs defined by the CD127 phenotype in comparison with the previously described CD4(+)CD25(hi) subpopulations, we subdivided the CD25(hi) population of memory Tregs into subsets based on expression of CD127 and HLA-DR. These subsets exhibited differences in suppressive capacity, ability to secrete IL-10 and IL-17, Foxp3 gene methylation, cellular senescence, and frequency in neonatal and adult blood. The mature, short telomere, effector CD127(lo)HLA-DR(+) cells most strongly suppressed effector T cells within 48 h, whereas the less mature CD127(lo)HLA-DR(-) cells required 96 h to reach full suppressive capacity. In contrast, whereas the CD127(+)HLA-DR(-) cells also suppressed proliferation of effector cells, they could alternate between suppression or secretion of IL-17 depending upon the stimulation signals. When isolated from patients with multiple sclerosis, both the nonmature and the effector subsets of memory CD127(lo) Tregs exhibited kinetically distinct defects in suppression that were evident with CD2 costimulation. These data demonstrate that natural and not induced Tregs are less suppressive in patients with multiple sclerosis.

The activation of classical alphabeta T cells is initiated when the T cell receptor (TCR) recognizes peptide antigens presented by major histocompatibility complex (pMHC) molecules. This recognition always occurs at the junction of a T cell and antigen-presenting cell (APC). Existing models of T-cell activation accurately explain the sensitivity and selectivity of antigen recognition within the immunological synapse. However, these models have not fully incorporated the diverse microcluster types revealed by current imaging technologies. It is increasingly clear that a better understanding of T-cell activation will require an appreciation of the diverse signaling assemblies arising within the immune synapse, the interrelationships between these structures, and the mechanisms by which underlying cytoskeletal systems govern their assembly and fate. Here, we will provide a brief framework for understanding these issues, review our contributions to current knowledge, and provide perspectives on the future of this rapidly advancing field.

In this study, we investigated whether alteration in the CD2 mediated coordination of an immune response was associated with down regulation of CD4 associated Th1 cell response during Visceral Leishmaniasis (VL). Leishmania donovani (Ld) infection in VL patients markedly reduced expression of CD2 cell surface antigen on CD4+ cells. T-cells of VL patients were mostly in G0/G1 stage of the cell cycle (98.20%) with little or no activity of protein kinase C-alpha (PKC-alpha) isoform. However, pre-incubation with activating anti-CD2 monoclonal antibody (MAb) resulted in a corresponding increase up to 2.52-fold in T-cells of G2/M population supported by both activity and expression of PKC-alpha isoform. Furthermore, we observed that co-incubation of T-cell with anti-CD2 increased the lymphocyte-blast population in patients in whom the CD4 cells became more antigen responsive (CD4+ CD69+ cells). Consistent with these observations, it was shown that 59.3% of CD4 cells from patients responded to Ld by producing IFN-gamma. Even in the culture condition, when the T-cells from patients were depleted of APC, IFN-gamma production was noticed after CD2 activation. On the other hand, IL-4 production became low in the anti-CD2 antibody supplemented peripheral blood mononuclear cells (PBMNCs) culture. These findings imply that infection with L. donovani induces less CD2 on the surface of CD4+ T-cells, which once activated orchestrate the protective IFN-gamma dominant host defense mechanism via PKC-mediated signal transduction and cell cycle.

Although combination therapy allows the suppression of human immunodeficiency virus type 1 (HIV-1) viremia to undetectable levels, eradication has not been achieved because the virus persists in cellular reservoirs, particularly the latent reservoir in resting CD4(+) T lymphocytes. We previously established a simian immunodeficiency virus (SIV)/macaque model to study latency. We describe here a novel mechanism for the induction of SIV from latently infected resting CD4(+) T cells. Several human cell lines including CEMx174 and Epstein-Barr virus-transformed human B-lymphoblastoid cell lines mediated contact-dependent activation of resting macaque T cells and induction of latent SIV. Antibody-blocking assays showed that interactions between the costimulatory molecule CD2 and its ligand CD58 were involved, whereas soluble factors and interactions between T-cell receptors and major histocompatibility complex class II were not. Combinations of specific antibodies to CD2 also induced T-cell activation and virus induction in human resting CD4(+) T cells carrying latent HIV-1. This is the first demonstration that costimulatory signals can induce latent virus without the coengagement of the T-cell receptor, and this study might provide insights into potential pathways to target latent HIV-1.

CD59 was first identified as a regulator of the terminal pathway of complement, which acts by binding to the C8/C9 components of the assembling membrane attack complex (MAC), to inhibit formation of the lytic pore. Structurally, CD59 is a small, highly glycosylated, GPI-linked protein, with a wide expression profile. Functionally, the role of CD59 in complement regulation is well-defined but studies have also shown clear evidence for signalling properties, which are linked to its glycophosphatidyl inositol (GPI) anchor and its location within lipid rafts. Cross-linking of CD59 using specific monoclonal antibodies drives both calcium release and activation of lipid-raft associated signalling molecules such as tyrosine kinases. These observations clearly show that CD59 exhibits roles independent of its function as a complement inhibitor. In this review, we examine the progression of research in this area and explore the alternative functions of CD59 that have recently been defined.

Human CD2 regulates T cell activation and adhesion via mechanisms yet to be fully understood. This study focuses on CD2BP1, a CD2 cytoplasmic tail-binding protein preferentially expressed in hematopoetic cells. Structural and functional analyses suggest that CD2BP1 acts as a scaffold protein, participating in regulation of the actin cytoskeleton. In this study, using a murine Ag-specific primary T cell transduction system to assess CD69, IL-2, and IFN-gamma expression, we provide evidence that CD2BP1 directly and negatively impacts T cell activation via isolated CD2 triggering or TCR stimulation dependent on coordinate CD2 engagement. Disruption of protein tyrosine phosphatase-PEST and/or CD2BP1 association with the CD2 signalsome rescues T cells from the inhibitory effect of CD2 crosslinking. The overexpression of CD2BP1 selectively attenuates phospholipase Cgamma1, ERK1/2, and p38 phosphorylation without abrogating CD2-independent TCR stimulation. This study provides new insight on the regulation of T cell activation and may have implications for autoimmune processes known to be associated with CD2BP1 mutations.

To date, five members of the downstream of tyrosine kinase (Dok) family have been characterized. In T cells, two members, Dok-1 and Dok-2, are expressed. CD2 or CD28 stimulation, but not CD3/TCR stimulation, induces Dok phosphorylation. Recent evidence suggests that they act as negative regulators of the CD2 and CD28 signaling pathways. To identify the molecular mechanisms involved in Dok-mediated inhibition, we have identified proteins that bind to the phosphotyrosine-binding (PTB) domain of Dok-1 and Dok-2. We showed that the Dok PTB domain mediates phosphotyrosine-dependent homotypic and heterotypic interactions of Dok-1 and Dok-2. Moreover, in CD2-stimulated Jurkat cells, Dok-1 coimmunoprecipitates with tyrosine-phosphorylated Dok-2. To study the involvement of PTB-mediated oligomerization in Dok function, we have generated Jurkat clones overexpressing Dok-1 or Dok-2 with a mutation that prevents oligomerization (in either the PTB domain or Tyr146 of Dok-1 and Tyr139 of Dok-2). These mutations abrogate CD2-induced phosphorylation and the ability of Dok-1 or Dok-2 to inhibit CD2-induced ERK1/2 and NFAT activation. Moreover, overexpression of Dok-1Y146F or Dok-2Y139F interferes with CD2-induced phosphorylation of endogenous Dok, whereas overexpression of PTB mutant or wild-type Dok does not. Taken together, these data indicate that PTB-mediated oligomerization of Dok-1 and Dok-2 represents an essential step for Dok phosphorylation and function.

Membrane subdomains have been implicated in T cell signaling, although their properties and mechanisms of formation remain controversial. Here, we have used single-molecule and scanning confocal imaging to characterize the behavior of GFP-tagged signaling proteins in Jurkat T cells. We show that the coreceptor CD2, the adaptor protein LAT, and tyrosine kinase Lck cocluster in discrete microdomains in the plasma membrane of signaling T cells. These microdomains require protein-protein interactions mediated through phosphorylation of LAT and are not maintained by interactions with actin or lipid rafts. Using a two color imaging approach that allows tracking of single molecules relative to the CD2/LAT/Lck clusters, we demonstrate that these microdomains exclude and limit the free diffusion of molecules in the membrane but also can trap and immobilize specific proteins. Our data suggest that diffusional trapping through protein-protein interactions creates microdomains that concentrate or exclude cell surface proteins to facilitate T cell signaling.

In addition to the well-documented effect of NK cells on B cell differentiation via their ability to secrete IFN-gamma, NK cells can also induce, via direct cell-cell interactions, germline transcripts (Igamma2a) necessary for switch recombination to IgG2a. Analysis of the ligand-receptor pairs that could be involved in this induction revealed that the expression of CD48 on B cells is crucial for the induction. NK cells from mice with targeted deletions of either the CD2 or the CD244 gene, both of which encode ligands for CD48, are compromised in their ability to induce B cell Igamma2a expression. Interestingly, although CD244 can bind to CD48 with a higher affinity, the ability of NK cells from CD244(-/-) mice to stimulate Igamma2a is not as compromised as NK cells from CD2(-/-) mice. Despite the difference between cell surface receptors that are stimulated by NK cells vs those stimulated by the combination of LPS and IFN-gamma, we show in this study that the initiation of gamma2a germline transcription is regulated by similar cis-acting elements located at the 3' end of the IgH locus. However, NK cells cannot induce the final steps of switch recombination resulting in the production of mature mRNA from recombined DNA. Our findings suggest that these different signaling pathways converge on regulatory elements that are common to germline transcription; however, because NK induction does not result in the final steps of switch recombination, some signals initiated by LPS plus IFN-gamma are not induced by NK cells.

Recent investigations support an important role for TGF-beta in the development of colorectal cancer. However, the molecular consequences of TGF-beta signaling in the colon remains incompletely understood. In a recent study in Immunity, we analyzed the role of TGF-beta in a murine model of colon cancer. Using transgenic mice overexpressing TGF-beta or a dominant negative TGF-beta receptor II under control of the CD2 minigene, we show that TGF-beta signaling in tumor infiltrating T lymphocytes regulates the growth of dysplastic colon epithelial cells, as determined by histology and a novel system for high resolution chromoendoscopy in vivo. At the molecular level, TGF-beta signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-beta-dependent IL-6 trans-signaling prevented tumor progression in vivo. Similar to these observations in mice, here we show that human colon cancer tissue expressed only low amounts of membrane bound IL-6R. In contrast, expression and activity of the matrix metalloproteinase TACE were increased. In summary, our data provide novel insights into the role of TGF-beta signaling in colorectal cancer and suggest novel therapeutic approaches for colorectal cancer based on an inhibition of TGF-beta-dependent IL-6 trans-signaling.

The TNF superfamily cytokine, lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells (LIGHT; TNFSF14), can augment T cell responses inducing IFN-gamma production and can drive pathological gut inflammation when expressed as a transgene in mouse T cells. LIGHT expression by human intestinal T cells suggests the possibility that LIGHT may play a key role in regulation of the mucosal immune system. A nonenzymatic method was developed for the isolation of T cells from the human lamina propria, permitting analysis of native cell surface protein expression. Cell surface LIGHT was constitutively expressed on mucosal T and NK cells and a subpopulation of gut-homing CD4(+) T cells in the periphery. In addition, CD2-mediated stimulation induced efficient LIGHT expression on intestinal CD4(+) T cells, but not on peripheral blood T cells, suggesting a gut-specific, Ag-independent mechanism for LIGHT induction. By contrast, herpesvirus entry mediator expression on gut T cells was unperturbed, implicating the transcriptional regulation of LIGHT as a mechanism modulating signaling activity in the gut. Quantitative analysis of LIGHT mRNA in a cohort of inflammatory bowel disease patients indicated elevated expression in biopsies from small bowel and from inflamed sites, implicating LIGHT as a mediator of mucosal inflammation.

IFN-gamma is an important immunoregulatory protein with tightly controlled expression in activated T and NK cells. Three potential STAT binding regions have been recognized within the IFN-gamma promoter: 1) an IL-12-mediated STAT4 binding site at -236 bp; 2) a newly identified IL-2-induced STAT5 binding element at -3.6 kb; and 3) CD2-mediated STAT1 and STAT4 binding to an intronic element in mucosal T cells. However, functional activation of these sites remains unclear. In this study we demonstrate CD2-mediated activation of the newly characterized -3.6-kb IFN-gamma STAT5 binding region. CD2 signaling of human PBMC results in activation of the -3.6-kb IFN-gamma promoter, whereas mutation of the -3.6-kb STAT5 site attenuates promoter activity. Functional activation is accompanied by STAT5A but little STAT5B nucleoprotein binding to the IFN-gamma STAT5 site, as determined by competition and supershift assays. STAT5 activation via CD2 occurs independent of IL-2. Western and FACS analysis shows increased phospho-STAT5 following CD2 signaling. AG490, a tyrosine kinase inhibitor affecting Jak proteins, inhibits CD2-mediated IFN-gamma mRNA expression, secretion, and nucleoprotein binding to the IFN-gamma STAT5 site in a dose-dependent fashion. This report is the first to describe CD2-mediated activation of STAT5 and supports STAT5 involvement in regulation of IFN-gamma expression.

Factors influencing the development of CD4+CD25+ T-cells in vivo are poorly understood. In order to investigate the contribution of TGFbeta1 to the development and function of CD4+CD25+ T-cells, we generated a gain of function mutation resulting in the overexpression of an active form of TGFbeta1 in T-cells under control of the human CD2 promoter. In peripheral lymphoid organs and in the thymus, the frequency of CD4+CD25+ T-cells was increased in transgenic mice. This appeared to be due to an autocrine effect of TGFbeta on T-cells, since concomitant impairment of TGFbeta-signaling in double transgenic mice resulted in a phenotype similar to wild type. In contrast, in single transgenic mice with impaired TGFbeta-signaling in T-cells, CD4+CD25+ T-cell numbers were reduced in peripheral lymphoid organs but not in the thymus. In addition, TGFbeta was found to regulate the expression of Foxp3 in vivo, a transcription factor essential for the generation and function of regulatory T-cells. In CD4+CD25+ T-cells, TGFbeta1 increased the expression of Foxp3, whereas a decreased expression was seen in CD4+CD25+ T-cells with impaired TGFbeta-signaling. TGFbeta1 induced the expression of IL-10 in transgenic T-cells, but the increased in vitro suppressive capacity observed in transgenic CD4+CD25+ T-cells was due to the secretion of TGFbeta and not IL-10. Therefore, our study provides in vivo evidence for a role of TGFbeta in the homeostasis of CD4+CD25+ T-cells.

Few studies have addressed the consequences of physical interactions between NK and T cells, as well as physical interactions among NK cells themselves. We show in this study that NK cells can enhance T cell activation and proliferation in response to CD3 cross-linking and specific Ag through interactions between 2B4 (CD244) on NK cells and CD48 on T cells. Furthermore, 2B4/CD48 interactions between NK cells also enhanced proliferation of NK cells in response to IL-2. Overall, these results suggest that NK cells augment the proliferation of neighboring T and NK cells through direct cell-cell contact. These results provide new insights into NK cell-mediated control of innate and adaptive immunity and demonstrate that receptor/ligand-specific cross talk between lymphocytes may occur in settings other than T-B cell or T-T cell interactions.

The TNF superfamily of cytokines play an important role in T cell activation and inflammation. Sustained expression of lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells (LIGHT) (TNFSF14) causes a pathological intestinal inflammation when constitutively expressed by mouse T cells. In this study, we characterized LIGHT expression on activated human T cell subsets in vitro and demonstrated a direct proinflammatory effect on regulation of IFN-gamma. LIGHT was induced in memory CD45RO CD4+ T cells and by IFN-gamma-producing CD4+ T cells. Kinetic analysis indicated rapid induction of LIGHT by human lamina propria T cells, reaching maximal levels by 2-6 h, whereas peripheral blood or lymph node-derived T cells required 24 h. Further analysis of intestinal specimens from a 41 patient cohort by flow cytometry indicated membrane LIGHT induction to higher peak levels in lamina propria T cells from the small bowel or rectum but not colon, when compared with lymph node or peripheral blood. Independent stimulation of the LIGHT receptor, herpesvirus entry mediator, induced IFN-gamma production in lamina propria T cells, while blocking LIGHT inhibited CD2-dependent induction of IFN-gamma synthesis, indicating a role for LIGHT in the regulation of IFN-gamma and as a putative mediator of proinflammatory T-T interactions in the intestinal mucosa. Taken together, these findings suggest LIGHT-herpesvirus entry mediator mediated signaling as an important immune regulatory mechanism in mucosal inflammatory responses.

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.

CD8(+) T cells depend on the alphabeta TCR for Ag recognition and function. However, Ag-activated CD8(+) T cells can also express receptors of the innate immune system. In this study, we examined the expression of NK receptors on a population of CD8(+) T cells expressing high levels of CD44 (CD8(+)CD44(high) cells) from normal mice. These cells are distinct from conventional memory CD8(+) T cells and they proliferate and become activated in response to IL 2 via a CD48/CD2-dependent mechanism. Before activation, they express low or undetectable levels of NK receptors but upon activation with IL-2 they expressed significant levels of activating NK receptors including 2B4 and NKG2D. Interestingly, the IL-2-activated cells demonstrate a preference in the killing of syngeneic tumor cells. This killing of syngeneic tumor cells was greatly enhanced by the expression of the NKG2D ligand Rae-1 on the target cell. In contrast to conventional CD8(+) T cells, IL-2-activated CD8(+)CD44(high) cells express DAP12, an adaptor molecule that is normally expressed in activated NK cells. These observations indicate that activated CD8(+)CD44(high) cells express receptors of both the adaptive and innate immune system and may play a unique role in the surveillance of host cells that have been altered by infection or transformation.

Loss of T cell number and function during HIV infection or secondary to pharmacologic immunosuppression renders individuals susceptible to opportunistic infections, including Pneumocystis carinii pneumonia. Because costimulatory receptors are critical for optimal T cell function, we hypothesized that these proteins would regulate susceptibility to opportunistic infections. We found that despite normal T cell numbers, mice deficient in the costimulatory molecules CD2 and CD28 spontaneously developed P. carinii pneumonia. In experiments using intratracheal injection of P. carinii organisms to induce infection, the loss of CD28 alone was sufficient to render mice susceptible to acute infection; however, the organism was eventually cleared. Examination of inflammatory responses to P. carinii revealed that mice deficient in both CD2 and CD28 accumulated CD8(+) T cells in their lungs in response to infection and demonstrated markedly reduced specific Ab titers. Analysis of cytokine profiles suggested that regulation of IL-10 and IL-15 may be important elements of the response to this pathogen. Thus, costimulatory molecule function is critical in determining the initial susceptibility to infection with P. carinii. Analysis of immunologic responses in these mice may provide important insights into the defects that render individuals susceptible to opportunistic infection, and provide opportunities for novel immunologically based therapies.

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.

This study investigates the distribution of leucocytes, CD2+, CD4+, CD8+ lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium following post-ovulatory insemination in relation to clinical findings and pregnancy outcome. Crossbred multiparous sows were inseminated once either at 15-20 h after ovulation [experiment 1, slaughtered at 20-25 h (5-6 h after artificial insemination (AI), group 1-A, n = 4), at 70 h after ovulation (group 1-B, n = 4), on day 11 (group 1-C, n = 4, first day of standing oestrus = day 1) or on day 19 (group 1-D, n = 4)] or 30 h after ovulation [experiment 2, slaughtered at 5-6 h after AI (group 2-A, n = 4) or on day 19 (group 2-D, n = 3)]. The uterine horns were flushed to control for the presence of spermatozoa and neutrophils and/or for recovery of oocytes and/or embryos. Mesometrial uterine samples were plastic embedded and stained. Cryofixed uterine samples were analysed by immunohistochemistry using mAbs to lymphocyte subpopulations and MHC class II molecules. Light microscopy was used to examine surface (SE) and glandular epithelia (GE), and connective tissue layers, both subepithelially (SL) and glandular (GL). In experiment 1, group 1-A, only one sow had spermatozoa in the utero-tubal junction (UTJ). Marked/moderated numbers of neutrophils and spermatozoa were observed in the flushings of two sows. In group 1-B, altogether 23 of 48 oocytes were cleaved. Day 11 (1-C), embryos with small diameter were observed. Day 19 (1-D), no embryos were found but small pieces of foetal membrane were observed in one of the sows. In group 1-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. For T lymphocyte subpopulations, in the SE, most CD2+ cells were found in group 1-A. For both SE and GE in all groups, the number of CD8+ cells was significantly larger than that of CD4+ cells. In experiment 2, group 2-A, no sow had spermatozoa in the UTJ or in the uterine flushings. At day 19, no sow was pregnant. In group 2-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. At day 19, high E2 levels showed a hormonal prooestrous stage but the endometrial neutrophil infiltration normally expected at pro-oestrus was absent. In conclusion, post-ovulatory insemination (about 18 h after ovulation) resulted in impaired spermatozoa transport within the uterus and embryonic degeneration. In sows post-ovulatory inseminated at a later stage (30 h after ovulation), no sow was pregnant. In both experiments, disturbed immune cell patterns were observed in some individuals.

So far, clinical benefit of hematopoietic stem cell induced donor-specific tolerance across major histocompatibility complex (MHC) barriers was hampered by either graft rejection or graft-versus-host disease. An alternative approach focuses on the use of donor-derived cells that bear an inherent mechanism to circumvent allospecific rejection upon injection into non-immunosuppressed hosts. Using a myeloablative conditioning model in the rat, full donor chimeric recipients were generated and their potential to induce long-term cardiac allograft survival was compared with the fate of grafts transferred to non-immunosuppressed host rats pretreated with donor-macrophages derived from the peritoneal cavity in the LEW to DA inbred strain combination. The induction of full multilineage long-term donor-chimerism (> 150 days) after initial host conditioning using two doses of cyclophosphamide and one shot of busulphan prevented acute graft rejection, whereas non-chimeric animals experienced acute and complete rejection. Conversely, vigorous T-cell depletion is required to protect conditioned animals from lethal graft-versus-host disease. Instead, the use of donor intraperitoneal macrophages achieved a state of transient chimerism and subsequent long-term graft survival in fully immunocompetent rats without the need of lethal conditioning regimens. In conclusion, the complex immunologic interactions, observed after allogeneic bone marrow transplantation as a means to induce donor chimerism, and subsequent long-term graft acceptance can be avoided if appropriate cell populations can be identified that, by themselves, induce a transient state of donor chimerism prevailing long enough to deviate allospecific immune responses, as outlined in this study.

The development of T cells in the thymus is regulated by a series of stage-specific transcription factors. Deregulated expression of these factors can lead to alterations in thymocyte development with the production of aberrant cell subsets and predispose to tumor formation. The three genes of the Runx family are multilineage regulators of differentiation that have been reported to be expressed in the T cell lineage. However, their roles in thymocyte development and T cell function are largely unknown. While the Runx2/Cbfa1/AML3/Pebp2alphaa gene plays a primary role in osteogenesis and regulates a number of key bone regulatory genes, we show here that Runx2 is also expressed during the earliest phase of thymic development, in the double-negative subset. Furthermore, enforced expression of Runx2 in transgenic mice under the CD2 promoter was found to affect T cell development at a stage coincident with beta-selection, resulting in an expansion of double-negative CD4 and CD8 immature single-positive cells. Unlike wild-type controls this preselection population (CD4-CD8+heat-stable Ag+TCR-) is in a nonproliferative state, but appears to be primed for further transformation events. Overall the data suggest that Runx2 accelerates development to the CD8 immature single-positive stage, but retards subsequent differentiation to the double-positive stage. Thus, Runx2 joins a small group of transcription factors that can interfere with early T cell development, cause an expansion of a specific subset, and predispose to lymphoma.

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.

We investigated the functional role of large-scale molecular segregation at the T cell-APC contact site during T lymphocyte Ag recognition. Inhibition of CD2-CD58 interaction markedly affected segregation of CD2 and CD2AP from CD45. Under these conditions, Ag-induced calcium mobilization, PKC theta; clustering at the immunological synapse, and IFN-gamma production also were inhibited. However, early TCR signaling and T cell polarization toward APCs were unaffected. Our results indicate that the "raison d'être" of a large-scale segregation of surface molecules and intracellular enzymes and adapters, in Ag-stimulated T cells, is to reinforce the assembly of the signal transduction cascade rather than favor TCR engagement and triggering.

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.

In order to establish the mechanism(s) of immunomodulatory and anti-tumor properties of sheep erythrocytes (SRBC), the T11 target structure (T11TS) or CD58 molecule as a pertinent component of SRBC was isolated, purified and finally administered in rats with experimentally induced brain tumor. Results showed inhibition and/or abrogation of tumor growth. Subsequent studies on cellular immunity also revealed potentiation of lymphocytes and PMNs at peripheral level. Presence of activated lymphocytes as revealed through flowcytometric analysis of CD25 expression evidenced infiltration of activated lymphocytes in the brain tumor tissues. The analysis of data suggests that T11TS or sheep form of LFA3 is capable of inhibiting/preventing tumor growth in rat brain by way of immunopotentiation (CMI) at the peripheral immune system and thereby facilitating infiltration of the activated lymphocytes into the brain cavity through the blood brain barrier.

The role of CD2 in murine CD4 helper T cell differentiation and polarization was examined using TCR-Cyt-5CC7-I transgenic recombination activating gene-2-/- H-2(a) mice on CD2+/+ or CD2-/- backgrounds. In the absence of CD2, thymic development was abnormal as judged by reduction in the steady state number of total, double-positive, and CD4 single-positive (SP) thymocytes, as well as a defect in their restorative dynamics after peptide-induced negative selection in vivo. In addition, in CD2-/- animals, lymph node CD4 SP T cells manifest a 10- to 100-fold attenuated activation response to cytochrome c (CytC) agonist peptides as judged by induction of CD25 and CD69 cell surface expression or [(3)H]TdR incorporation; differences in the magnitude of responsiveness and requisite molar peptide concentrations were even greater for altered peptide ligands. Although the presence or absence of CD2 did not impact the final Th1 or Th2 polarization outcome, CD2 expression reduced the CytC peptide concentration threshold necessary to facilitate both Th1 and Th2 differentiation. In vivo administration of CytC peptide to CD2-/- animals yielded an impaired CD4 SP T cell effector/memory phenotype compared with similarly treated CD2+/+ mice. Analysis of TCR-Cyt-5CC7-I human CD2 double-transgenic mice similarly failed to reveal a preferential Th1 vs Th2 polarization. Collectively, these results indicate that CD2 is important for the efficient development of CD4 SP thymocytes and TCR-dependent activation of mature CD4 lymph node T cells, but does not direct a particular helper T cell subset polarity.

Recent data suggest that mast cells (MCs) in patients with systemic mastocytosis or mast cell leukemia express a CD2-reactive antigen. To explore the biochemical nature and function of this antigen, primary MCs as well as the MC line HMC-1 derived from a patient with mast cell leukemia were examined. Northern blot experiments revealed expression of CD2 messenger RNA in HMC-1, whereas primary nonneoplastic MCs did not express transcripts for CD2. In cell surface staining experiments, bone marrow (BM) MCs in systemic mastocytosis (n = 12) as well as HMC-1 cells (30%-80%) were found to express the T11-1 and T11-2 (but not T11-3) epitopes of CD2. By contrast, BM MCs in myelodysplastic syndromes and nonhematologic disorders (bronchiogenic carcinoma, foreskin phimosis, uterine myeomata ) were consistently CD2(-). All MC species analyzed including HMC-1 were found to express LFA-3 (CD58), the natural ligand of CD2. To study the functional role of CD2 on neoplastic MCs, CD2(+) and CD2(-) HMC-1 cells were separated by cell sorting. CD2(+) HMC-1 cells were found to form spontaneous aggregates and rosettes with sheep erythrocytes in excess over CD2(-) cells, and a T11-1 antibody inhibited both the aggregation and rosette formation. Moreover, exposure of CD2(+) HMC-1 cells to T11-1 or T11-2 antibody was followed by expression of T11-3. In addition, stimulation of neoplastic MCs through T11-3 and a second CD2 epitope resulted in histamine release. These data show that neoplastic MCs express functionally active CD2. It is hypothesized that expression of CD2 is associated with pathologic accumulation and function of MCs in systemic mastocytosis.

Human retinal pigment epithelial (RPE) cells are capable of presenting bacterial superantigens (SAg) to T cells in vitro by ligation of MHC class II molecules on RPE cells with the T cell receptor. The purpose of this study was to evaluate the involvement of adhesion molecules in presentation of SAg. Cultured human fetal and adult RPE cells were treated with interferon-gamma (IFN-gamma, 500 U ml(-1) for 72 hr) and afterwards pulsed with the SAg staphylococcal enterotoxin A (SEA, 500 ng ml(-1) for 2 hr) followed by coculture with freshly obtained T cells isolated from peripheral blood. Proliferation was measured by (3)H-thymidine incorporation assay. In selected experiments, either RPE or T cells were pre-treated with blocking antibodies specific for cell surface molecules. For comparison, dendritic cells were used as superantigen presenting cells for T cells. This study showed that presentation of SEA by RPE cells to resting T cells was dependent on the presence of the molecules CD2, CD58 and CD18, CD54. The cycling status of T cells was decisive, thus resting T cells but not activated T cells were capable to proliferate in response to SEA presentation. Proliferation of T cells induced by adult RPE cells was comparable to the proliferation induced by dendritic cells at concentrations of SAg above 100 ng ml(-1), but at concentrations of SAg below 10 ng ml(-1) the response was significantly lower for SAg presented by RPE cells compared to dendritic cells. The results demonstrate that CD2-CD58 and CD18-CD54 interactions are critical for SAg presentation by RPE cells to T cells. The findings thus suggest that also presentation of peptides to resting T cells by RPE cells may be dependent upon these interactions., ((C) 2001 Academic Press.)

The protein tyrosine kinase ZAP-70 plays a pivotal role involved in signal transduction through the T cell receptor and CD2. Defects in ZAP-70 result in severe combined immunodeficiency. We report that Herpesvirus saimiri, which does not code for a ZAP-70 homologue, can replace this tyrosine kinase. H. saimiri is an oncogenic virus that transforms human T cells to stable growth based on mutual CD2-mediated activation. Although CD2-mediated proliferation of ZAP-70-deficient uninfected T cells was absent, we could establish H. saimiri-transformed T cell lines from two unrelated patients presenting with ZAP-70 deficiencies. In these cell lines, CD2 and CD3 activation were restored in terms of [Ca(2+)](i), MAPK activation, cytokine production, and proliferation. Activation-induced tyrosine phosphorylation of zeta remained defective. The transformed cells expressed very high levels of the ZAP-70-related kinase Syk. This increased expression was not observed in the primary T cells from the patients and was not due to the transformation by the virus because transformed cell lines established from control T cells did not present this particularity. In conclusion, wild type H. saimiri can restore CD2- and CD3-mediated activation in signaling-deficient human T cells. It extends our understanding of interactions between the oncogenic H. saimiri and the infected host cells.

Induction and maintenance of peripheral tolerance is an important phenomenon for the control of homeostasis in the immune system. There is now compelling evidence for CD4(+) T cells that prevent immune pathology, both in autoimmunity and in transplantation. However, the mechanisms involved in the specific differentiation of these T cells are unknown. We had previously shown that repetitive stimulations of naive T cells in the presence of IL-10 induce the differentiation of T regulatory cells 1. We further dissected the mechanism of IL-10 function and demonstrated that IL-10 acts by the down-regulation of most costimulatory molecules without modifying the expression of CD58. Using artificial APCs expressing various costimulatory molecules, we demonstrated that, in contrast to other costimulation patterns, costimulation via CD2 alone, in the absence of costimulations through CD28- or LFA-1, induced T cell anergy in an IL-10-independent pathway along with the differentiation of Ag-specific regulatory T cells. T regulatory cell-1 differentiation via CD2 was very efficient as both high IL-10 secretion and regulatory function were observed after the first stimulation of naive T cells with CD32-CD58 L cells. The possibility to rapidly induce the differentiation of Ag-specific regulatory T cells will certainly accelerate their characterization and their potential use as regulators of T cell-mediated diseases.

We have analyzed the murine CD40 ligand promoter with regard to stimulation of transcriptional activity in Jurkat T cells after signaling via the TCR and the costimulatory molecules CD28 and CD2. TCR engagement was necessary for the induction of transcriptional activity from the CD40 ligand promoter, and costimulation through either CD28 or CD2 further increased the activity. Analysis of promoter deletants showed that the DNA elements needed for transcriptional activity induced by costimulatory molecules were located within two regions containing previously identified transcription factor NFAT sites. Further studies of the proximal NFAT site showed that it was not dependent on AP-1 binding for transcriptional activity induced by costimulation through CD28. Instead, a region between the TATA box and the proximal NFAT site was shown to bind proteins of the early growth response family and to contribute to NFAT-mediated transcriptional activation.

p62(dok) belongs to a newly identified family of adaptor proteins. In T cells, the two members that are predominantly expressed, p56(dok) and p62(dok), are tyrosine phosphorylated upon CD2 or CD28 stimulation, but not upon CD3 ligation. Little is known about the biological role of Dok proteins in T cells. In this study, to evaluate the importance of p62(dok) in T cell function, we generated Jurkat clones overexpressing p62(dok). Our results demonstrate that overexpression of p62(dok) in Jurkat cells has a dramatic negative effect on CD2-mediated signaling. The p62(dok)-mediated inhibition affects several biochemical events initiated by CD2 ligation, such as the increase of intracellular Ca(2+), phospholipase C gamma 1 activation, and extracellular signal-regulated kinase 1/2 activation. Importantly, these cellular events are not affected in the signaling cascade induced by engagement of the CD3/TCR complex. However, both CD3- and CD2-induced NF-AT activation and IL-2 secretion are impaired in p62(dok)-overexpressing cells. In addition, we show that CD2 but not CD3 stimulation induces p62(dok) and Ras GTPase-activating protein recruitment to the plasma membrane. These results suggest that p62(dok) plays a negative role at multiple steps in the CD2 signaling pathway. We propose that p62(dok) may represent an important negative regulator in the modulation of the response mediated by the TCR.

Cluster of differentiation 2 (CD2) is a cell surface glycoprotein expressed on most human T cells and natural killer (NK) cells and plays an important role in mediating cell adhesion in both T-lymphocytes and in signal transduction. The understanding of the biochemical basis of molecular recognition by the cell adhesion molecule CD2 has been advanced greatly through the determination of structures and the dynamic properties of the complexes and their individual components and through site-directed mutagenesis. A number of general principles can be derived from the structural and functional studies of the extracellular domains of CD2 and CD58 and their complex. Significant electrostatic interactions within the protein-protein interfaces contribute directly to the formation of macromolecular complexes of CD2 and CD58. Also, residues located on the protein-protein interface demonstrate a certain degree of conformational change upon the formation of a complex. Structural analysis of CD2 has revealed that this adhesion molecule exhibits strong conformational flexibility with a partial non-native helical conformation at high temperatures and in the presence of an organic solvent. In addition, it can be converted into a domain swapped dimer, or trimer and tetramer through hinge deletion. Thus, the conformational status of the adhesive proteins contributes to the regulation of cell adhesion and the folding of CD2.

To examine the function of CD2 in vivo, N15 TCR transgenic (tg) RAG-2(-/-) H-2(b) mice bearing a single TCR specific for the vesicular stomatitis virus octapeptide bound to the H-2K(b) molecule were compared on a wild-type or CD2(-/-) background. In N15tg RAG-2(-/-) CD2(-/-) mice, thymic dysfunction is evident by 6 wk with a pre-TCR block in the CD4(-)CD8(-) double-negative thymocytes at the CD25(+)CD44(-) stage. Moreover, mature N15tg RAG-2(-/-) CD2(-/-) T cells are approximately 100-fold less responsive to vesicular stomatitis virus octapeptide and unresponsive to weak peptide agonists, as judged by IFN-gamma production. Repertoire analysis shows substantial differences in Valpha usage between non-tg C57BL/6 (B6) and B6 CD2(-/-) mice. Collectively, these findings show that CD2 plays a role in pre-TCR function in double-negative thymocytes, TCR selection events during thymocyte development, and TCR-stimulated cytokine production in mature T cells.

Objective: T lymphocytes can be activated by soluble factors such as cytokines or through direct cell-cell interactions. Although cytokine receptors are known to signal through STAT family transcription factors, the mechanisms by which other cell-surface molecules, such as CD2, transduce signals is unclear. The goal of this study was to determine whether stimulation of T cells through CD2 recapitulates aspects of cytokine-induced T-cell activation by use of STAT transcription factors., Materials and Methods: T cells were treated with anti-CD2 antibodies or cells bearing the natural CD2 ligand CD58, after which signaling through STAT transcription factors was assessed., Results: Stimulation of CD2 on primary T lymphocytes leads to the tyrosine phosphorylation, nuclear translocation, and DNA binding of STAT1. In contrast to stimulation by cytokines, the activation of STAT1 in response to CD2 ligation is delayed and does not involve Jak kinases. Furthermore, while STAT phosphorylation induced by cytokines is generally transient, STAT1 phosphorylation following CD2 stimulation persists for a period of days. Transcription of key target genes such as IRF1 and c-fos proceeds with delayed kinetics following CD2 stimulation, suggesting that this unique pattern of STAT activation may lead to a distinct cellular response following CD2 ligation. This pathway appears to be restricted to T cells, as stimulation of CD2 on NK cells does not lead to STAT1 activation., Conclusion: Stimulation of T cells through cell-surface molecules such as CD2 involves activation of STAT transcription factors, thus recapitulating elements of cytokine signaling.

Stimulation of B cells by surface immunoglobulin (sIg) triggering, or through the mitogenic combination of phorbol ester and calcium ionophore, is accompanied by activation of STAT transcription factors. The mechanism responsible for the delayed nuclear accumulation of phosphorylated STAT3 was examined in detail, focusing on the role of B cell-derived lymphokines. sIg-induced activation of STAT3 was partially inhibited in B cells obtained from IL-6- or IL-10-deficient mice, and was partially blocked by neutralizing antibodies directed against either of these lymphokines. sIg-induced STAT3 activation was completely inhibited by combining IL-6- and IL-10-specific neutralizing antibodies, or by adding individual neutralizing antibodies to B cells obtained from lymphokine-deficient animals. In contrast, IL-10 alone appeared to account for STAT3 activation resulting from B cell stimulation with phorbol ester and calcium ionophore. In keeping with these results, soluble IL-6 and IL-10 were found in supernatant fluid obtained from stimulated B cells. This work indicates that a lymphokine pathway is responsible for STAT3 activation that occurs late after B cell stimulation, and points out differences in B cell activation that result from stimulation through the antigen receptor and through pharmacological mimicry of signaling mediators.

The CD2 co-receptor expressed on the surface of T lymphocytes is able to stimulate T cell activation, proliferation and cytokine production in the absence of direct engagement of the antigen-specific TCR. Engagement of human CD2 by mitogenic pairs of anti-CD2 mAb induces tyrosine phosphorylation of a number of intracellular proteins including a 120 kDa phosphoprotein that we identify as the proto-oncogene c-Cbl. Rapidly tyrosine phosphorylated following stimulation of a number of cell surface receptors, c-Cbl is an adaptor protein that has been shown to associate with a complex of intracellular signaling molecules, and to mediate both positive and negative regulatory effects. Here we show that, like TCR-CD3 stimulation, stimulation of CD2 enhanced the association of c-Cbl with both Crk(L) and the p85 subunit of phosphatidylinositol-3 kinase. Overexpression of wild-type c-Cbl protein inhibited both CD2and CD3-induced NF-AT transcriptional activity, suggesting that CD2 signaling is also negatively regulated by c-Cbl. The inhibitory effect of c-Cbl depended upon its N-terminal phosphotyrosine-binding domain, the domain that has been shown to be required for inhibition of the Syk/ZAP-70 family kinases. In Syk(-) Jurkat T cells stably expressing wild-type ZAP-70, CD2 stimulation induced only a minimal increase in ZAP-70 tyrosine phosphorylation. Nevertheless, ZAP-70 kinase was required for CD2-mediated NF-AT transcriptional activity. Thus, CD2-mediated NF-AT transcriptional activity appears to depend upon ZAP-70/Syk kinases and to be negatively regulated by c-Cbl.

TCR- but not CD2-triggered IL-2 production is p56(lck) dependent. To test the hypothesis that p59(fyn), a second src-family protein tyrosine kinase (PTK) expressed in T lymphocytes, might be an essential upstream component of the CD2 signaling pathway, we generated human (h) CD2 transgenic (tg) fyn(+/+) and fyn(-/-) mice. Clustering of hCD2 molecules on resting peripheral T lymphocytes results in Ca(2+) mobilization, activation of MAPK and cellular proliferation. In contrast, in the absence of p59(fyn), these CD2-initiated activities are markedly reduced, while TCR-triggered proliferation is unaffected. Several CD2 pathway components regulated by p59(fyn) have been identified including phospholipase C-gamma1 (PLC-gamma1), Vav, protein kinase C-theta isoform (PKC-theta), docking protein (Dok), focal adhesion kinase (FAK) and Pyk2. Decreased inducible PKC-theta catalytic activity and Vav phosphorylation likely account for diminished p38 and JNK activation in hCD2tg fyn(-/-) mice. Moreover, deficiency in fyn-dependent PLC-gamma1 catalytic activity may contribute to reduced PKC-alpha-dependent ERK activation. Of note, CD2-dependent Dok but not linker from activated T cells (LAT) tyrosine phosphorylation requires p59(fyn). Furthermore, that FAK and Pyk2 are target substrates implies that p59(fyn) may be an important regulator of T cell adhesion as well. Collectively, these data identify p59(fyn) as a key PTK in CD2-mediated activation of mature T lymphocytes.

This study addresses the role of the tyrosine kinase ZAP-70 in CD2-mediated T cell activation. Patients lacking ZAP-70 have few mature CD8+ T cells and high numbers of CD4+ T cells that are nonfunctional upon TCR triggering. Such a patient with a homozygous deletion in the zap-70 gene that resulted in the complete absence of ZAP-70 protein expression has been identified. Expression of the tyrosine kinases Lck, Fyn, and Syk was normal. The patient's T cells were activated with two different pairs of mitogenic mAbs. CD2-induced phosphorylation of the zeta-chain and influx of Ca2+ was defective in the ZAP-70-deficient T cells, whereas CD2-induced phosphorylation of several other proteins, including Syk, was not affected. CD2-induced proliferation as well as production of TNF-alpha and IFN-gamma was abrogated in ZAP-70-deficient T cells, whereas PMA plus ionomycin induced normal activation of these cells. Together, this study shows that CD2-activation triggers ZAP-70-dependent and -independent pathways. Deletion of ZAP-70 affected CD2- and CD3-mediated proliferation and cytokine production in a similar way, suggesting that one of the different CD2 pathways converges with a CD3 pathway at or upstream of the activation of ZAP-70.

Reorganization of the cortical cytoskeleton is a hallmark of T lymphocyte activation. Upon binding to antigen presenting cells, the T cells rapidly undergo cytoskeletal re-organization thus forming a cap at the cell-cell contact site leading to receptor clustering, protein segregation, and cellular polarization. Previously, we reported cloning of the human lymphocyte homologue of the Drosophila Discs Large tumor suppressor protein (hDlg). Here we show that a novel protein termed GAKIN binds to the guanylate kinase-like domain of hDlg. Affinity protein purification, peptide sequencing, and cloning of GAKIN cDNA from Jurkat J77 lymphocytes identified GAKIN as a novel member of the kinesin superfamily of motor proteins. GAKIN mRNA is ubiquitously expressed, and the predicted amino acid sequence shares significant sequence similarity with the Drosophila kinesin-73 motor protein. GAKIN sequence contains a motor domain at the NH(2) terminus, a central stalk domain, and a putative microtubule-interacting sequence called the CAP-Gly domain at the COOH terminus. Among the MAGUK superfamily of proteins examined, GAKIN binds to the guanylate kinase-like domain of PSD-95 but not of p55. The hDlg and GAKIN are localized mainly in the cytoplasm of resting T lymphocytes, however, upon CD2 receptor cross-linking the hDlg can translocate to the lymphocyte cap. We propose that the GAKIN-hDlg interaction lays the foundation for a general paradigm of coupling MAGUKs to the microtubule-based cytoskeleton, and that this interaction may be functionally important for the intracellular trafficking of MAGUKs and associated protein complexes in vivo.

Expression of the L1 family cell adhesion molecule neuroglian in Drosophila S2 cells leads to cell aggregation and polarized ankyrin accumulation at sites of cell-cell contact. Thus neuroglian adhesion generates a spatial cue for polarized assembly of ankyrin and the spectrin cytoskeleton. Here we characterized a chimera of the extracellular and transmembrane domains of rat CD2 fused to the cytoplasmic domain of neuroglian. The chimera was used to test the hypothesis that clustering of neuroglian at sites of adhesion generates the signal that activates ankyrin binding. Abundant expression of the chimera at the plasma membrane was not a sufficient cue to drive ankyrin assembly, since ankyrin remained diffusely distributed throughout the cytoplasm of CD2-neuroglian-expressing cells. However, ankyrin became highly enriched at sites of antibody-induced capping of CD2-neuroglian. Spectrin codistributed with ankyrin at capped sites. A green fluorescent protein-tagged ankyrin was used to monitor ankyrin distribution in living cells. Enhanced green fluorescent protein-ankyrin behaved identically to antibody-stained endogenous ankyrin, proving that the polarized accumulation of ankyrin was not an artifact of fixing and staining cells. We propose a model in which clustering of neuroglian induces a conformational change in the cytoplasmic domain that drives polarized assembly of the spectrin cytoskeleton.

Stimulation of endothelial cells (EC) with IFN-gamma generates selective enhancement of T(h)1 cell transmigration and induction of MHC class II expression on EC. In the present study, we tested whether antigen presentation by EC could influence transmigrating T cells in an in vitro system. Bacterial antigen presentation by EC from primary culture and after cloning induced antigen-specific anergy of transmigrating T(h)1 clone cells in a MHC class II-dependent manner as characterized by non-responsiveness to subsequent antigen presentation and inability to produce IL-2. This T cell transmigration anergy induced by EC was abrogated by anti-rat CD28 mAb, suggesting that lack of B7 co-stimulatory signals by EC might be related to the induction of anergy. While MHC class II expression on primary and cloned EC was observed after IFN-gamma stimulation, these cells never expressed B7. B7-1 gene-transfected endothelial clone cells (ECC/B7-1) were developed to elucidate the influence of B7 co-stimulation by EC. ECC/B7-1 induced proliferation of T(h)1 clone cells, whereas ECC did not induce proliferation in co-culture of T(h)1 clone cells and EC stimulated with IFN-gamma and antigen. In the transmigration assay, ECC/B7-1 did not induce transmigration anergy of T(h)1 clones or T(h)1 lines unless anti-rat B7-1 blocking mAb was added. Therefore, in rats, the T cell anergy induced during transmigration across a layer of EC seemed to be due to antigen presentation in the absence of B7 on the EC. We introduce the concept of transmigration anergy in this manuscript. Thus, EC can play a critical immune regulatory role in the context of antigen presentation by MHC class II to transmigrating T cells.

T cell activation requires co-engagement of the TCR with accessory and costimulatory molecules. However, the exact mechanism of costimulatory function is unknown. Mice lacking CD2 or CD28 show only mild deficits, demonstrating that neither protein is essential for T cell activation. In this paper we have generated mice lacking both CD2 and CD28. T cells from the double-deficient mice have a profound defect in activation by soluble anti-CD3 Ab and Ag, yet remain responsive to immobilized anti-CD3. This suggests that CD2 and CD28 may function together to facilitate interactions of the T cell and APC, allowing for efficient signal transduction through the TCR.