Role of SLAM in NKT cell development revealed by transgenic complementation in NOD mice.

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.

T cell-signaling network analysis reveals distinct differences between CD28 and CD2 costimulation responses in various subsets and in the MAPK pathway between resting and activated regulatory T cells.

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.

CD2 costimulation reveals defective activity by human CD4+CD25(hi) regulatory cells in patients with multiple sclerosis.

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.

Multiple microclusters: diverse compartments within the immune synapse.

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.

CD2 is involved in maintenance and reversal of human alloantigen-specific clonal anergy.

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.

CD2 sets quantitative thresholds in T cell activation.

It has been proposed that CD2, which is highly expressed on T cells, serves to enhance T cell-antigen presenting cell (APC) adhesion and costimulate T cell activation. Here we analyzed the role of CD2 using CD2-deficient mice crossed with transgenic mice expressing a T cell receptor specific for lymphocytic choriomeningitis virus (LCMV)-derived peptide p33. We found that absence of CD2 on T cells shifted the p33-specific dose-response curve in vitro by a factor of 3-10. In comparison, stimulation of T cells in the absence of lymphocyte function-associated antigen (LFA)-1-intercellular adhesion molecule (ICAM)-1 interaction shifted the dose-response curve by a factor of 10, whereas absence of both CD2-CD48 and LFA-1-ICAM-1 interactions shifted the response by a factor of approximately 100. This indicates that CD2 and LFA-1 facilitate T cell activation additively. T cell activation at low antigen density was blocked at its very first steps, as T cell APC conjugate formation, TCR triggering, and Ca(2+) fluxes were affected by the absence of CD2. In vivo, LCMV-specific, CD2-deficient T cells proliferated normally upon infection with live virus but responded in a reduced fashion upon cross-priming. Thus, CD2 sets quantitative thresholds and fine-tunes T cell activation both in vitro and in vivo.

CD2 regulates responsiveness of activated T cells to interleukin 12.

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.

Visualization of CD2 interaction with LFA-3 and determination of the two-dimensional dissociation constant for adhesion receptors in a contact area.

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.

Alternative roles for CD59.

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.

Regulation of beta 1-integrin-mediated cell adhesion by the Cbl adaptor protein.

BACKGROUND: Leukocyte activation results in a rapid increase in adhesion to the extracellular matrix due to the activation of beta 1 integrin receptors. A role for phosphatidylinositol (PI) 3-kinase in integrin activation has been proposed, as activation of integrins by many receptors can be blocked by PI 3-kinase inhibitors. One receptor that regulates integrins is the CD28 surface antigen; here, we investigated the mechanisms responsible for CD28-mediated integrin activation. RESULTS: CD28-mediated integrin activation was blocked by mutation of the binding site for the p85 catalytic subunit of PI 3-kinase in the CD28 cytoplasmic domain, and by expression of a dominant-negative form of the p85 subunit. Substitution of the Src homology 2 (SH2)-binding motif in the CD28 cytoplasmic domain for the corresponding motif in the CD28-related CTLA-4 surface antigen also blocked integrin activation but did not affect the recruitment and activation of PI 3-kinase. Mutations of the CD28 cytoplasmic domain that blocked integrin activation also impaired the tyrosine phosphorylation of the Cbl adaptor protein and the activation of the PI 3-kinase that was associated with Cbl. This Cbl-associated PI 3-kinase was distinct from the PI 3-kinase that coprecipitated with the CD28 cytoplasmic domain. CD28-mediated activation of beta 1 integrins was inhibited by expression of a mutant Cbl protein that shows reduced association with PI 3-kinase. CONCLUSIONS: Cbl is required for PI-3-kinase-dependent regulation of integrin receptors by CD28. Furthermore, CD28 is coupled to two distinct pools of PI 3-kinase, one directly associated with the CD28 cytoplasmic tail and the other associated with Cbl.

Stimulated human lamina propria T cells manifest enhanced Fas-mediated apoptosis.

Lamina propria (LP) T cells respond poorly to a proliferative stimulus delivered via TCR/CD3 pathway, but retain considerable ability to respond to a stimulus delivered via CD2 costimulatory or accessory pathway. In the present study, we showed first that unstimulated LP T cells, as compared to unstimulated peripheral blood (PB) T cells, exhibit an increased level of apoptosis which is further increased following CD2 pathway stimulation, but not following via TCR/CD3 pathway stimulation. We next showed that IL-2 had a sparing effect on apoptosis of unstimulated LP T cells in that IL-2 decreased and anti-IL-2 increased apoptosis of these cells; in contrast, IL-2 had no effect on apoptosis of CD2-pathway stimulated cells. Finally, we showed that increased apoptosis of LP T cells induced by CD2-pathway stimulation is inhibited when Fas antigen is blocked by a nonstimulatory anti-Fas antibody. These studies suggest that LP T cells are characterized by increased susceptibility to Fas-mediated apoptosis most due to a downstream change in the Fas signaling pathway. Given that IFN-gamma secretion is significantly increased in LP T cells in which apoptosis is inhibited, this feature of LP T cells may represent a mechanism of regulating detrimental immune responses in the mucosal environment.

Adhesion receptors in lymphocyte activation.

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.

Costimulation by B7-1 and LFA-3 targets distinct nuclear factors that bind to the interleukin-2 promoter: B7-1 negatively regulates LFA-3-induced NF-AT DNA binding.

We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-kappaB nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1- and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-D-Fos complexes. In contrast, AP-1 and NF-kappaB complexes induced in B7-1- and LFA-3-costimulated T cells showed similar contents. Transient transfection of Jurkat T cells with a construct encoding the IL-2 enhancer-promoter region (position -500 to +60) linked to a luciferase reporter gene revealed that B7-1 costimulation was required to induce strong transcriptional activity. Combined B7-1-LFA-3 costimulation resulted in a synergistic increase in IL-2 transcriptional activity. Multimers of the AP-1, NF-AT, NF-kappaB, and CD28 response elements showed distinct kinetics and activity after LFA-3 and B7-1 costimulation and revealed that B7-1 and LFA-3 converge to superinduce transcriptional activity of the AP-1, NF-AT, and CD28 response elements. Transcriptional studies with an IL-2 enhancer-promoter carrying a mutation in the CD28 response element site revealed that the activity was reduced by 80% after B7-1 and B7-1-LFA-3 costimulation whereas the transcriptional activity induced by LFA-3 was unaffected. Our data strongly suggest a selectivity in induction of nuclear factors by the CD2-LFA-3 and CD28-B7-1 pathways. This selectivity may contribute to regulation of the levels of IL-2 induced by LFA-3 and B7-1 costimulation and favor autocrine and paracrine T-cell responses, respectively.

Identification of a proline-rich sequence in the CD2 cytoplasmic domain critical for regulation of integrin-mediated adhesion and activation of phosphoinositide 3-kinase.

The CD2 molecule is one of several lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in beta1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cytoplasmic domain involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of beta1 integrin activity. CD2-induced increases in beta1 integrin activity could be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the K-G-P-P-L-P motif to alanines prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation of MAP kinase but had no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2 critical for PI 3-K-dependent regulation of beta1 integrin adhesion by CD2. In addition, these studies suggest that CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion.

Assessment of costimulation and coinhibition in a triple parameter T cell reporter line: Simultaneous measurement of NF-κB, NFAT and AP-1.

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.

The oncogenic LIM protein Rbtn2 causes thymic developmental aberrations that precede malignancy in transgenic mice.

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.

Relief of cyclin A gene transcriptional inhibition during activation of human primary T lymphocytes via CD2 and CD28 adhesion molecules.

Cyclin A transcription is cell cycle regulated and induced by cell proliferative signals. To understand the mechanisms underlined in this regulation in normal human cells, we have analysed in vivo protein-DNA interactions at the Cyclin A locus in primary T lymphocytes. Stimulation of purified T lymphocytes by a combination of monoclonal antibodies directed at CD2 and CD28 adhesion molecules gives rise to a long lasting proliferation in the absence of accessory cells. Cyclin A was observed after 4 days of costimulation with anti CD2 + CD28 whereas stimulation by anti CD2 or anti CD28 alone was not effective. In vivo genomic DMS footprinting revealed upstream of the major transcription initiation sites, the presence of at least three protein binding sites, two of which were constitutively occupied. They bind in vitro respectively ATF-1 and NF-Y proteins. The third site was occupied in quiescent cells or in cells stimulated by anti CD2 or anti CD28 alone. The mitogenic combination of anti CD2 + anti CD28 released the footprint as cells were committed to proliferation. Consistent with theses results, nuclear extracts prepared from quiescent cells formed a specific complex with this element, whereas extracts prepared from cells treated with anti CD2 + anti CD28 failed to do so after cells entered a proliferative state.

Intracellular calcium levels are differentially regulated in T lymphocytes triggered by anti-CD2 and anti-CD3 monoclonal antibodies.

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.

Coengagement of CD2 with LFA-1 or VLA-4 by bispecific ligand fusion proteins primes T cells to respond more effectively to T cell receptor-dependent signals.

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.