Search

We have previously established that upon CD2 activation of T cells, p56(lck) showed a transient increase in its kinase activity and was partially internalized. Here we studied the possibility that p56(lck) could retain its kinase activity in the endosomes of CD2-triggered cells. T cells were fractionated on a sucrose gradient, and the endosomal fraction was isolated. In CD2-triggered cells, part of Lck was internalized and presented a maximal kinase activity in the endosome-enriched fraction after 5 min, decreasing thereafter. In the endosomal fraction of activated cells, four tyrosine-phosphorylated proteins of apparent molecular masses of 30, 40, 56, and 70 kDa were detected. We demonstrated that the protein tyrosine kinase ZAP-70 was recruited to the endosomal fraction upon CD2 stimulation with kinetics similar to that of p56(lck), suggesting that recruitment of protein tyrosine kinases to endosomal vesicles could promote specific transduction signals at the intracellular level.

The alternative CD2-mediated pathway of T cell activation, which is independent of MHC/peptide recognition by the TCR/CD3 complex, is dependent upon two signals being received by the CD2 molecule. The natural ligand for CD2 is CD58, but controversy exists over alternative or additional ligands that could deliver the second signal in vivo. We have used rat retinal pigment epithelial cells (RPE), which lack temperature-insensitive ligands for CD2 adhesion, to study Ag-independent T cell activation. Rat RPE cells expressed high levels of CD59 and low levels of another potential CD2 ligand, CD48, both in vitro and in the in vivo model of experimental autoimmune uveoretinitis. When increasing numbers of syngeneic T cells were added to microwell cultures of rat RPE cells, the T cells, even in the absence of any exogenous stimulant in the cultures, underwent spontaneous proliferation. This effect required metabolically active RPE cells, and was IL-2 driven and enhanced in the presence of indomethacin. Proliferation was modulated by phosphatidylinositol-phospholipase C treatment of the RPE, and blocked by mAbs to CD59. Ab cross-linking of CD48 but not CD59 on the RPE was found to induce messenger RNA expression for IL-1 beta, which together with constitutively expressed IL-6 are required costimulatory factors for T cell activation through CD2. This is the first demonstration in a fully syngeneic system that bi-directional signaling involving CD59 and CD48 molecules expressed by physiologically normal, nonhematopoietic, cells can trigger T lymphocyte activation and proliferation through autocrine IL-2 production in the absence of Ag.

Short-term activated peripheral T lymphocytes are susceptible to apoptotic cell death triggered by CD2 mAbs. The aim of this study was to examine whether the CD2-mediated pathway of apoptosis is linked to the Fas death pathway, as this is the case for CD3/TCR-triggered apoptosis in several models of T cells. Using T lymphocytes from patients harboring Fas gene mutations and displaying a profound defect in Fas signaling of cell death, we show that CD2- (but not CD3-) mediated apoptosis still proceeds normally. In normal activated T cells, CD3-mediated apoptosis is prevented by reagents that block the Fas/Fas-ligand interaction, namely soluble M3 (an antagonistic anti-Fas mAb) and soluble human Fas.Fc, a fusion protein able to bind released Fas-ligand. In contrast, CD2 signaling of apoptosis resists these blocking agents. Neither new protein synthesis nor the activation of calcineurin was required for CD2- and Fas-mediated apoptosis, suggesting that latent cytoplasmic "death" molecules were activated upon stimulation of the cells. In both cases, protein tyrosine kinases were transiently activated, as is exemplified by the autophosphorylation and exokinase activity of p56lck, yielding overlapping yet nonidentical profiles of protein tyrosine phosphorylation. Pretreating the cells with herbimycin A, before the addition of the apoptotic stimuli, almost completely inhibited CD2 transmembrane signaling of apoptosis, but left intact Fas-induced apoptosis. Our data suggest that CD2 is a Fas-independent cell death pathway that might contribute directly to the elimination of T cells expanding during an immune reaction.

The T cell and NK cell adhesion molecule CD2 interacts with different ligands, viz, CD58, CD48, and CD59. Using a fluorescent multimeric construct of rCD2, we previously identified an additional CD2 ligand (CD2L) on the erythroleukemic cell line K562. CD2L bound to a different region of CD2 than known ligands and was N-glycosylation dependent. In this study we show that mAbs specific for the carbohydrate Ag Lewis x (CD15, Gal-beta 1-4 GlcNAc alpha 1-3Fuc) inhibit multimeric rCD2 binding to CD2L. CD2L is restricted in expression to myeloid cells, where it is co-expressed with CD58 on monocytes and is the dominant, if not sole, CD2 ligand on neutrophils. Sugar specificity studies show that CD2L is not CD15. Thus, whereas soluble Lewis x inhibits binding of CD15 mAb to K562 and neutrophils, binding of multimeric rCD2 is unaffected. Furthermore, multimeric rCD2 binding to K562 is inhibited by L-fucose and following treatment of K562 with an alpha 1-6 fucosidase, whereas these treatments do not inhibit the binding of CD15 mAb. Thus, it is likely that CD2L is a carbohydrate structure closely associated with, yet distinct from, CD15, which can be sterically blocked by CD15 mAb. Functional studies revealed that CD2L is probably an important CD2 ligand in the non-MHC-restricted NK cell killing of K562 target cells, since this activity was strongly inhibited by CD15 mAb. Collectively, this study indicates that a CD15 (Lewis x)-associated carbohydrate structure(s), which has previously been shown to be a selectin ligand, also may function as an important CD2 ligand on myeloid cells.

We examined the killing activity of transmigrated lymphokine-activated killer (LAK) cells and their surface molecules associated with both transendothelial migration and cytotoxicity, using human umbilical vein-derived endothelial cell (HUVEC) monolayers on fibronectin with gelatin separating the upper chamber from the lower chamber. Migratory LAK cells were significantly more cytotoxic to Daudi target cells, expressed more LFA-1, and were more likely to be positive for CD2, compared to those LAK cells not adherent to the HUVEC monolayer. In contrast, in the absence of the HUVEC monolayer, there was no difference in LAK activity between migratory and non-adherent LAK cells. These results indicate that the interaction between LAK cells and the HUVEC monolayer allows selective migration of LAK cells with cytotoxic activity that is enhanced with respect to some surface molecules.

We demonstrate that CD2 receptor engagement, but not CD3 crosslinking, induces apoptosis in lymphocytes transformed by human T-cell lymphotrophic virus type I (HTLV-I). Mitogenic pairs of anti-CD2 monoclonal antibodies inhibited [3H]thymidine incorporation from 25 to 62% in CD2+ HTLV-I-infected lymphocytes. This inhibition was associated with a 20-40% reduction in cell number and viability over a 3-day period, morphologic evidence of apoptosis, and irreversible DNA fragmentation. While cyclosporin A abrogated CD2-mediated proliferation in peripheral blood mononuclear cells, it had no effect on CD2-induced apoptosis in the HTLV-I-infected cell lines. Since HTLV-I is mitogenic to resting lymphocytes through CD2 activation pathways, these results suggest that HTLV-I-infected lymphocytes are primed for apoptosis following additional CD2 stimulation. This CD2-mediated apoptosis might be a factor in immune regulation of HTLV-I-associated diseases or might offer a novel adjunctive approach to treatment.

Interleukin-3 (IL-3) is expressed in T lymphocytes and stimulates the growth of multipotent hematopoietic progenitors. Little is known, however, about the stimuli that lead to IL-3 protein release. We examined IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA expression and protein secretion in human T lymphocytes following activation via the TCR/CD3 complex, the CD2 receptor, and the IL-2 receptor. GM-CSF mRNA expression and protein release were found in CD3 and CD2 activated T cells with maximum GM-CSF release following stimulation with IL-2. IL-3 protein release is regulated via the CD2 receptor with virtually no IL-3 release after T cell stimulation via CD3. In contrast, IL-3 mRNA accumulation is more pronounced after CD3 activation than after CD2 activation. This suggests that upregulation of IL-3 protein release following T cell stimulation via CD-2 occurs largely at the translational or posttranslational level. These data also indicate that differential control of cytokine production can occur in response to activation of the alternative T cell receptor. Interaction of the T cell CD2-receptor with its natural ligand LFA-3 expressed on stromal cells might represent a regulatory mechanism for rapid release of IL-3, facilitating proliferation of multipotent hematopoietic cells.

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 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 is 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 up-regulation 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 effect the phenotype of activated CD4+ T cells.

Many adhesion receptors have high three-dimensional dissociation constants (Kd) for counter-receptors compared to the KdS of receptors for soluble extracellular ligands such as cytokines and hormones. Interaction of the T lymphocyte adhesion receptor CD2 with its counter-receptor, LFA-3, has a high solution-phase Kd (16 microM at 37 degrees C), yet the CD2/LFA-3 interaction serves as an effective adhesion mechanism. We have studied the interaction of CD2 with LFA-3 in the contact area between Jurkat T lymphoblasts and planar phospholipid bilayers containing purified, fluorescently labeled LFA-3. Redistribution and lateral mobility of LFA-3 were measured in contact areas as functions of the initial LFA-3 surface density and of time after contact of the cells with the bilayers. LFA-3 accumulated at sites of contact with a half-time of approximately 15 min, consistent with the previously determined kinetics of adhesion strengthening. The two-dimensional Kd for the CD2/LFA-3 interaction was 21 molecules/microns 2, which is lower than the surface densities of CD2 on T cells and LFA-3 on most target or stimulator cells. Thus, formation of CD2/LFA-3 complexes should be highly favored in physiological interactions. Comparison of the two-dimensional (membrane-bound) and three-dimensional (solution-phase) KdS suggest that cell-cell contact favors CD2/LFA-3 interaction to a greater extent than that predicted by the three-dimensional Kd and the intermembrane distance at the site of contact. LFA-3 molecules in the contact site were capable of lateral diffusion in the plane of the phospholipid bilayer and did not appear to be irreversibly trapped in the contact area, consistent with a rapid off-rate. These data provide insights into the function of low affinity interactions in adhesion.

Anti-CD3 monoclonal antibodies (mAbs) and anti-CD2 mAbs each prolong allograft survival and cause transient downmodulation of homologous receptor expression. Anti-CD2 mAbs also act synergistically with anti-CD3 mAbs to prolong allograft survival and induce tolerance. The effect of combined anti-CD2 and anti-CD3 mAb treatment on receptor expression was further analyzed with an in vitro model. The anti-CD2 mAb 12-15 caused CD2 expression on purified splenic T cells to decrease from 72.6% [mean channel fluorescence (MCF) 0.68] to 41.5% (0.45) total positive cells while CD3 expression remained unchanged [69.1% (3.47) to 76.4% (4.04)]. The anti-CD3 mAb 2C11 caused CD2 expression to increase from 72.6% (0.68) to 93.0% (1.74) while CD3 expression decreased from 69.1% (3.47) to 62.6% (2.15). The combination of anti-CD2 plus anti-CD3 preserved CD2 expression (72.6 to 71.1%) while still decreasing CD3 expression [69.1% (3.47) to 69.9% (2.37)]. Modulation of CD2 and CD3 expression was similar on mixed splenic T lymphocytes and isolated CD4 and CD8 subsets. Modulation did not change with the addition of the cytokines IL-1, IL-2, IL-4, IL-6, IL-10, TNF alpha, or TGF beta. Kinetic studies showed that modulation of CD2 was rapid, persistent, and of the same magnitude from Day 1 to Day 7 of culture while CD3 downmodulation was transient. The results of transcriptional analysis and receptor distribution suggested that downmodulation was due to receptor internalization while upmodulation was due to increased transcription. Analysis of expression of other adhesion molecules demonstrated that CD11a, CD18, CD44, CD45, CD48, CD54, and CD62L were significantly increased by either anti-CD2 or anti-CD3 mAbs while the combination was not synergistic. However, anti-CD3 significantly decreased VLA-4 alpha (CD49d) expression and anti-CD2 enhanced this decrease. Conversely anti-CD3 significantly increased IL-2R (CD25) expression and anti-CD2 profoundly inhibited the increase. These results show that anti-CD2 and anti-CD3 mAbs significantly modulate CD2 and CD3 expression on T cells and modulation is accompanied by changes in the array of other T cell surface receptors. Changes in cell surface receptor display may provide an additional explanation for the synergistic effect of anti-CD2 plus anti-CD3 in prolonging allograft survival.

Infection by the human T-cell lymphotropic virus type I (HTLV-I) causes T-cell activation by at least two separate mechanisms. One mechanism involves activation of the T cells harboring the virus and is exemplified by in vivo infected nonimmortalized T-cell clones that display a prolonged state of activation. This HTLV-I-induced T-cell activation is inhibited by rapamycin, a drug that inhibits p70 S6-kinase and blocks cell cycle in G1, but is not inhibited by FK506 or cyclosporin A, both of which inhibit interleukin-2 (IL-2) production. The phenotype of this pathway is consistent with an hyperactive IL-2R pathway or CD28 pathway, indicating that HTLV-I may contribute a costimulatory signal to the infected T cell. As a separate mechanism, HTLV-I-infected T cells can induce activation of uninfected T cells via T-T-cell interaction mediated by the LFA-3-CD2 pathway. This may induce IL-2 production from the uninfected T cells, leading to a more generalized activation of the immune system that potentially could provide a basis for some of the diseases associated with HTLV-I. Moreover, this THTLV-I-T-cell interaction could explain the spontaneous proliferation observed in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis.

The CD2 receptor is an important costimulatory molecule in T cell activation. Its ligand CD48 in rodents is supposed to be a homologue of human CD58, because of its similarities in structure and distribution. We evaluated the immunosuppressive activity of CD2/CD48-directed therapy in vitro and in vivo for the efficacy in prolonging rat heart allograft survival in a high responder transplant model. CD2-directed monoclonal antibody (mAb) therapy significantly prolonged median survival time to 45 days (P < 0.001). Suppression was mediated by down-modulation of CD2 below 20% on lymph node cells without considerable cell depletion. In contrast, CD48 mAb could not prolong graft survival. Rejection occurred in the presence of complete CD48 modulation and, therefore, despite disruption of the CD2-CD48 interaction. CD48 mAb failed to inhibit lymphocyte activation via a mitogenic pair of CD2 mAbs and inhibited mixed lymphocyte reaction (MLR) only by an unspecific mechanism. In conclusion, our results suggest a negative regulatory signal transduction by inhibitory CD2 mAbs and argue against a pivotal role of mere disruption of the CD2-CD48 interaction in CD2-mediated immunosuppression.

Accessory molecules play a crucial role in the development of the T cell response to antigenic challenge. We have examined the role of CD28 in modulating the 'naive' neonatal T cell response to anti-CD2-mediated activation. To compare the role of CD28, neonatal and adult T cells were stimulated with a pair of mitogenic anti-CD2 antibodies in the presence or absence of anti-CD28 MoAb. With anti-CD2 alone, neonatal T cells proliferated slightly but produced no detectable IL-2, whereas adult T cells proliferated vigorously, with significant IL-2 production. Costimulation with anti-CD28 MoAb greatly enhanced the proliferative response of neonatal T cells to levels equivalent to those of adult T cells, whereas adult T cells showed only slight increases. Although IL-2 secretion was increased in the presence of anti-CD28 MoAb, neonatal T cell IL-2 production remained lower than in adults. In contrast, enhancement of IL-2 mRNA expression in neonates was similar to adult levels. Anti-CD28 MoAb costimulation increased NF kappa B levels in neonates, albeit to levels lower than that of adults. The cellular mechanism governing the diminished proliferative response of neonatal T lymphocytes to anti-CD2 may therefore be due to decreased NF kappa B induction, reduced IL-2 mRNA expression and deficient IL-2 production. Although anti-CD28 MoAb costimulation enhances all of the above signals, NF kappa B and IL-2 levels remain lower than in adults, suggesting the need for further activation requirements in the neonate.

RBTN2 is activated by the chromosomal translocation t(11;14) (P13;p11) in some T cell leukaemias. Histologically similar T cell tumours develop with long latency in transgenic mice when either CD2 or thy1.1 promoters control rbtn2 expression. During the asymptomatic period, perturbation of T cell differentiation occurs in the thymus. The major anomalies present during this phase are an increase in the percentage of large thymocytes lacking CD4 and CD8 markers and also of small thymocytes express both the T cell marker CD3 and the B cell-specific form of CD45. These abnormal T cell populations can be clonal and thus a primary result of aberrant expression of the LIM-protein Rbtn2 is alteration of T cell differentiation preceding overt malignancy. These data provide a biological explanation for the role of Rbtn2 in tumorigenesis and presumably RBTN2 expression in T cells after the translocation t(11;14) in children has the same effect.

Interleukin (IL) 12 is a 70-kD heterodimeric cytokine produced by antigen-presenting cells (APCs) such as macrophages in response to infectious pathogens and interferon (IFN) gamma. The varied immunomodulatory effects of IL-12 include the stimulation of proliferation and IFN-gamma production by T cells, and it also has a central role in the development of the T helper cell type 1 immune phenotype. We undertook the production of antibodies capable of modulating the response of T cells to IL-12, and in the process we discovered two antibodies that inhibited the ability of IL-12 to stimulate T cell proliferation. In this report, we demonstrate that these anti-bodies recognize CD2, and we show how antibodies directed toward either the adhesion domain of CD2 or its ligand, CD58, specifically inhibit IL-12 induced proliferation and IFN-gamma production by phytohemagglutinin-activated T cells, leaving the response to IL-12 unaffected. A three-to fourfold reduction in proliferation and IFN-gamma production was observed at IL-12 concentrations as high as 1 nM, with complete inhibition occurring at < or = 1 pM. This novel effect is not directly mediated at the level of the IL-12 receptor, as shown by the inability of these antibodies to block IL-12 binding to activated T cells. Furthermore, by using activating pairs of CD2 antibodies, we show that CD2 stimulation strongly synergizes with IL-12, even at 0.1 pM, in inducing both T cell proliferation and IFN-gamma production. Cytolytic T lymphocyte-associated antigen 4-immunoglobulin-mediated inhibition of the B7/CD28 interaction did not affect the T cell response to either IL-12 or IL-2, but the removal of APCs selectively diminished the proliferative response to IL-12. Based on this data, we hypothesize that CD2 has a central role in an IL-12/IFN-gamma positive feedback loop between T cell and APC, providing the key functional link via a CD2/CD58 interaction that controls T cell responsiveness to IL-12. This model provides a basis for future investigations aimed at defining the signaling mechanisms that mediate this cytokine-specific regulatory effect of CD2, and it offers insight into how a cytokine receptor and distinct adhesion molecule can interact to modulate responsiveness to that cytokine. In addition, it underscores the possibility that the clinical potential of an immunomodulatory drug like IL-12 may be governed by the presence or absence of specific costimulation.

We investigated the production of cytokines by highly purified T helper cells from B-cell chronic lymphocytic leukemia (B-CLL) patients stimulated by different activation pathways, and we studied the influence of various accessory cell populations on the pattern of the secretion of cytokines, including interleukin (IL)-2, IL-4, interferon-gamma (IFN-gamma), and IL-10. Neither a qualitative nor a quantitative difference in cytokine production and proliferative capacity was observed in CLL-derived purified T cells compared with normal individuals, when T cells were stimulated by different pathways, including CD3, CD2, and costimulation with CD28. Addition of autologous accessory cells (aAC), however, dramatically influenced the cytokine pattern of normal versus B-CLL-derived T cells. CLL cells as aAC caused a marked increase of IL-2, whereas IFN-gamma was only slightly induced and IL-4 was not influenced. In contrast, in normal individuals addition of aAC, which predominantly consisted of monocytes, resulted in a significant increase of IFN-gamma and a reduction of IL-4 secretion. IL-2 production was inhibited by higher concentrations of aAC. The increased stimulation of IL-2 production by CLL cells was not specific to the leukemic cell population, as purified B cells from normal individuals had the same effect. On the other hand, purified monocytes from CLL patients and controls both induced IFN-gamma production and inhibited IL-4 secretion. After antigen-specific stimulation with tetanus toxoid, cytokine secretion was influenced by the type of aAC in a similar pattern. We conclude that T helper cells derived from patients with B-CLL are intrinsically normal and that the predominance of B cells as accessory cells in CLL significantly alters the immune function of T helper cells in vitro.

Recent studies from several laboratories suggest that the rate of postnatal maturation of T-cell function(s) associated with in vitro activation may be slower in children at high genetic risk for atopy (HR), compared to their normal (low risk; LR) counterparts. The present study compared the in vitro activity of the function-associated surface molecules CD2, CD3 and CD28 in panels of 27 HR and 13 LR infants, with a reference panel of 10 adults, employing assay systems involving T-cell stimulation with MoAbs against these molecules. The response maxima induced by saturating levels of the MoAbs were equivalent in all 3 groups, but T-cells from the HR infants required 10-50 fold higher levels of anti-CD3 stimulation to attain their maximum response, relative to adults (p = 0.02); T-cells from LR infants were also less responsive to anti-CD3 than adults, but these differences were smaller and did not attain statistical significance. It is suggested that these differences are attributable to varying proportions of competent T-memory cells (which respond to low levels of anti-CD3) in PBL from these populations, the postnatal accumulation of which proceeds slowest in the HR group.

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.

In addition to the antigen receptor, resting T cells express a number of receptors that can be stimulated to generate proliferative signals. These "accessory" receptors require co-expression of the T cell receptor (TCR), suggesting that they channel their signals via secondary activation of the signal transduction function of the CD3-TCR complex. Little is known about how different receptors control each other's function when one or more stimuli are presented at the same time. In order to study the regulation of accessory receptors by the CD3-TCR and vice versa, we have investigated the activation of the CD2 cell adhesion molecule receptor and the pertussis toxin receptor, a 43 kDa plasma membrane protein. Both receptors can activate signal transduction pathways in T cells similar to that of the CD3-TCR, including increases in Ca2+ and phosphatidylinositol turnover. They are also similar in that they utilize the antigen receptor to transmit their signals to the cell since CD3-TCR(-) mutants cannot be activated via either CD2 or the toxin receptor. We have previously shown that submaximal stimulation of the CD3-TCR blocks second messenger generation and proliferation in response to pertussis toxin. This heterologous desensitization was unidirectional since activation of the toxin receptor had no effect on CD3-TCR function. Here we extend these studies to show that activation of both CD2 and the toxin receptor led to rapid tyrosine phosphorylation of three similar proteins. Submaximal stimulation of the CD3-TCR completely inhibited toxin receptor-stimulated tyrosine protein kinase activity but did not desensitize CD2 function as determined by activation of tyrosine protein phosphorylation. Furthermore, CD2 stimulation did not lead to desensitization of the pertussis toxin receptor. These data support a system of complex regulatory relationships between different signaling receptors and suggest a model for signal integration and inter-receptor cross-talk in T cell activation.

Antigen and/or mitogen-driven T-cell activation is mediated by a rise in intracellular free Ca2+, as second messenger. A regulatory key role for this process is represented by membrane-associated [Ca2+/Mg2+] ATP-ase that is mainly devoted to extrusion of intracellular ion excess. In the present study we have investigated the kinetics of CA2+ fluxes in both resting and already activated (Jurkat T-cell line) T lymphocytes after CD3 and CD2 (T11(2) and T11(3)) triggering and focused our attention on plasma membrane [Ca2+/Mg2+] ATP-ase activity. In both resting T cells and Jurkat cell line, the CD2 stimulation was able to determine a rise in intracellular free Ca2+ higher than that observed after CD3 triggering. In addition, this calcium signal was independent of negative feedback control exerted by [Ca2+/Mg2+] ATP-ase, as well as of IP3 generation. Thus the CD2 molecular system may, together with cell-adhesion properties, act as an amplifier of Ca2+ signals that, if delivered in the context of other molecular systems, such as CD3 or MHC class II antigens, are essentially devoted to the polyclonal co-stimulatory recruitment of a larger cellular repertoire.

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.

CD27neg T cells are found only among CD4pos-CD45ROpos T cells and represent a T cell subset functionally distinct from CD27pos T cells. We examined CD4posCD45ROpos T cells that were sorted into CD27neg and CD27pos populations for their cytokine production in response to different activation pathways. We found that CD27neg T cells are characterized by high IL-4 and low IL-2 production, regardless of whether the cells were activated through CD3 plus CD28, CD2 plus CD28, or PHA plus PMA. However, subpopulation-specific patterns of cytokines were the clearest demonstrable following CD2 plus CD28 stimulation. We conclude from these data that high IL-4 production is a stable phenotype of CD27neg T cells.

The present results support a role for epithelial cells in the activation of T cells in an apparent antigen-independent manner. The transient expression of CD25 indicates a short acting T cells activation. Possibly, this event primes T cells to respond swiftly upon antigen-specific stimulation or to synthesize mediators that affect the local milieu. The molecular mechanism of interaction, although not well defined possibly involves LFA3-CD2 interactions. In T cell activation, via LFA3-CD2 interaction, the density of presented LFA3 molecules is critical. With the increase in the level of expression of LFA3 by epithelial cells this critical density may have been reached. However, based on what is known about T cell activation and CD25 expression in particular it is likely that additional signals such as soluble mediators are required for T cell activation by epithelial cells. Whether this mode of activation occurs in vivo remains to be established by studying ex vivo and in situ material. Not much is known about the expression of LFA3 by epithelial cells in vivo, nor about the stimuli that induce the upregulation of LFA3. In preliminary experiments with fluorescence microscopy we found that neither TNF-alpha nor IL-1 beta induce LFA3 in the same fashion as IFN-gamma. In conclusion, T cell activation by epithelial cells could be an important feature in inflammatory and immunological processes in mucosal systems such as the bronchi and deserves further research.

Resting T cells can be activated by selected pairs of anti-CD2 MoAb. Activation is dependent on the presence of accessory cells, which can be replaced by either anti-CD28, or by the combination of IL-1 beta and IL-6. The present study was undertaken to investigate accessory signalling by B7-1, the natural ligand of CD28, in this pathway of T cell activation. 3T6 mouse fibroblasts were transfected with human B7-1 and used as accessory cells in cultures of purified resting human T cells. In the presence of a stimulating pair of anti-CD2 MoAb, T cell proliferation, production of cytokines (IL-2, IL-4, IL-10, GM-CSF, IFN-gamma and TNF-alpha), and generation of cytotoxic T lymphocytes were all supported by B7-1(+) 3T6 cells but not by control 3T6 cells. Blocking studies with anti-IL-2 + anti-IL-2R MoAb revealed both IL-2-dependent and IL-2-independent CTL generation after B7-1-mediated costimulation. Moreover, a partial or complete resistance to inhibition with CsA was observed for IL-2 production and CTL generation respectively in the presence of the costimulatory signal derived from B7-1-CD28 interaction. Anti-CD2 MoAb with B7-1 costimulation could directly induce proliferation, IL-2 production and generation of CTL activity in highly purified CD8+ T cells without the help of CD4+ T cells. We conclude that CD28 ligation with the natural ligand B7-1 provides a strong accessory signal for CD4 and CD8 cell activation through CD2.

Both increased T cell numbers and their increased activation state have implicated an important role for T cells in chronic inflammatory reactions seen in the airways of (allergic) asthmatics. Airway epithelial cells are frequently exposed to stimuli that cause the release of mediators and the expression of cell adhesion molecules. We have examined whether human airway epithelial cells can activate lung-derived T cells. Clonal lung T cells showed an increased adherence to transformed airway epithelial cells that had been exposed previously for 2 h to human recombinant interferon-gamma (IFN-gamma; 100 U/ml). After an additional 16-24 h of culturing in the absence or presence of epithelial cells, T cells expressed increased levels of both the alpha-chain of the interleukin-2 receptor (IL-2R, CD25) and the transferrin receptor (CD71), both markers of T cell activation. T cells apparently activated by epithelial cells, however, did not produce IFN-gamma or IL-4 nor showed an increased proliferation on the addition of IL-2 (5-50 U/ml). The induced adherence to and the activation of T cells by epithelial cells is mediated largely by CD2 and its ligand lymphocyte functional antigen-3, a pathway known to up- and downregulate T cell functions.

Induction and maintenance of a state of T cell unresponsiveness to specific alloantigen would have significant implications for human organ transplantation. Using human histocompatibility leukocyte antigen DR7-specific helper T cell clones, we demonstrate that blockade of the B7 family of costimulatory molecules is sufficient to induce alloantigen-specific T cell clonal anergy. Anergized cells do not respond to alloantigen and a variety of costimulatory molecules, including B7-1, B7-2, intercellular adhesion molecule-1 (ICAM-1), and lymphocyte function-associated molecule (LFA)-3. However, after culture in exogenous interleukin (IL)-2 for at least 7 d, anergized cells can respond to alloantigen in the presence of LFA-3. LFA-3 costimulation subsequently restores responsiveness to alloantigen in the presence of previously insufficient costimulatory signals. Expression of CD2R epitope is downregulated on anergic cells and is restored after 7 d of IL-2 culture. The loss of the CD2R is temporally associated with the inability of anergized cells to respond to LFA-3. These results suggest that in addition to blockade of B7 family members, inhibition of CD2 and, potentially, other costimulatory pathways that might reverse anergy will be necessary to maintain prolonged alloantigen-specific tolerance.

The ability of cultured human endothelial cells (EC) to activate resting T cells depends on signals provided by EC that augment T cell IL-2 synthesis. EC-mediated augmentation of IL-2 secretion by PHA-activated T cells is resistant to inhibition by cyclosporin A (CsA) or by ascomycin. Specifically, the 50% inhibitory dose of these drugs is increased over 100-fold in the presence of EC. Although rapamycin also inhibits IL-2 secretion, there is no effect of EC on the sensitivity of T cells to this drug. Resistance to CsA requires cell contact between the EC and T cells and develops 8 h after initiation of coculture. Experiments with blocking Abs implicate T cell CD2 and its endothelial cell ligands, both LFA-3 and CD59, but not T cell CD28 and its putative ligands, in EC-induced resistance to CsA. However, CD2 signals are not sufficient to induce resistance to CsA, and other EC signals remain to be identified. These observations suggest that interactions of graft EC with host T cells may contribute to CsA-resistant reactions in vivo.

We investigated the adhesion to B cells of CD4+ T cells both in the resting state and following activation by CD3 cross-linking or stimulation by PMA/ionomycine/IL2 for 6 days. Both resting and activated CD4+ T cell adhesion were inhibited by anti-LFA-1, -CD2, -VLA-5/CD29, and -CD43 antibodies, suggesting coordinated upregulation of T cell adhesion. The CD2 and LFA-1 adhesion pathways were found to act independently, as CD2 was functional in T cells not expressing LFA-1, and vice versa, and as specific antibodies had additive effects. In contrast, LFA-1- and VLA-5/CD29-specific antibodies did not have an additive blocking effect on CD4+ T cell adhesion, suggesting that efficient adhesion requires a competitive association of integrins with cytoskeleton elements. Although the involvement of fibronectin (coated to B cells via VLA-4) in VLA-5-mediated T cell adhesion to B cells is feasible, an anti-fibronectin and a VLA-4-specific antibody had no blocking effect. The involvement of an unidentified B cell ligand can also be envisaged.

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.

To examine the effects of ligand engagement and accessory molecule juxtaposition on T cell receptor (TCR) signaling, we prepared LFA-3/ICAM-1 Rg and LFA-3/VCAM-1 Rg bispecific immunoglobulin fusion proteins (Rg, recombinant globulin). These novel fusion proteins allowed us to examine the effects of ligand driven co-engagement of T cell proteins CD2 and LFA-1 or CD2 and VLA-4 on TCR-dependent mobilization of intracellular Ca2+. We observed that preincubation of resting T cells with LFA-3/ICAM-1 Rg or LFA-3/VCAM-1 Rg fusion proteins resulted in significantly enhanced mobilization of intracellular Ca2+ following TCR-accessory molecule cross-linking relative to T cells preincubated with each of the monospecific Rgs alone or with combinations of the monospecific Rg fusion proteins. In addition, such coengagement stimulated TCR-dependent activation and tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1). These results suggest that when T cells interact with antigen presenting cells the engagement of multiple cell adhesion molecules such as CD2, LFA-1, and VLA-4 primes the T cell to respond more effectively to signals delivered through the TCR.

The past several years have seen significant progress in understanding the role of T lymphocyte coreceptors in adhesion and activation. New insights have been gained in several areas: the avidity regulation of beta 1 and beta 2 integrins and their role in signal transduction; the regulation of CD8 avidity; the role of Lck in CD4 coreceptor activity; and the novel role for CD2 adhesion in the T cell antigen response.

We showed previously that the TCR and CD2 fail to couple efficiently with their signal transduction machinery in J45.01, a CD45-deficient variant of the Jurkat T-cell line. Transfection into J45.01 of a cDNA encoding a chimeric membrane protein containing the cytoplasmic sequence of CD45 and extracellular and transmembrane sequences derived from the A2 allele of MHC class I rescues proximal signaling events after TCR stimulation. In this report, we describe rescue of CD2-mediated signaling and evaluate further the characteristics of TCR signaling in J45.01 after expression of the chimeric protein. Cells expressing the chimeric molecule demonstrate TCR- and CD2-mediated increases in PTK activity and PI turnover. Stimulation of the TCR and CD2 on the transfected cells also results in the expression of CD69 on the cell surface, a more distal signaling event. Although these measures of signal transduction via the TCR and CD2 are restored in the transfected cells, the magnitude of the responses are less than those seen in the wild-type Jurkat cells. These findings demonstrate that the cytoplasmic domain of CD45, expressed as a chimeric membrane protein, is sufficient for mediating signal transduction through CD2 as well as through the TCR complex. In addition, these results suggest that the extracellular and/or transmembrane domains of CD45 may contribute to the efficiency of signal transduction.