Anti-CD2 (sheep red blood cell receptor) monoclonal antibodies and T cell activation. I. Pairs of anti-T11.1 and T11.2 (CD2 subgroups) are strongly mitogenic for T cells in presence of 12-O-tetradecanoylphorbol 13-acetate

A large collection of monoclonal antibodies (mAb) directed against sheep red blood cell (SRBC) receptor (cluster of differentiation 2: CD2) were classified according to three criteria: their inhibitory effect on T cell-SRBC rosette formation; the epitopic cluster recognized on the CD2 molecule; their reactivity with resting or activated T cells. All mAb were then tested in a two by two checkerboard fashion for possible T cell mitogenicity, in presence or absence of a submitogenic dose of 12-O-tetra-decanoylphorbol 13-acetate (TPA), an agent known to be comitogenic for T cells, presumably in delivering a second signal, usually accessory cell dependent. The combined data demonstrate that in the absence of TPA only few pairs of mAb directed at distinct epitopes of the CD2 molecule were mitogenic for T cells (in approximately 30% of the population tested), and in the presence of a submitogenic dose of TPA the majority of T11.1 anti-CD2-mAb (9 out of 11) were strongly mitogenic for T cells of all individuals tested when paired with T11.2 anti-CD2 mAb. The two anti-T11.1 mAb, noncomplementary to anti-T11.2 mAb, were, however, strongly mitogenic when added to mAb of the T11.3 subgroup, represented by 1-Mono-2A6. Taken together, these data strongly suggest that the main characteristic of T cell mitogenesis triggered by anti-CD2 mAb is the requirement for a signal delivered simultaneously to two different epitopes of the CD2 molecule, whether these epitopes are T11.1, T11.2 or T11.3.     

T cell co-stimulatory molecules other than CD28.

CD28 is the primary co-stimulatory receptor for inducing high-level IL-2 production and survival of na?ve CD4(+) T cells. While no other cell surface receptor can be considered fully redundant with CD28, recent developments suggest that additional co-stimulatory pathways have preferential effects at different stages of T cell activation, on different subsets of T cells or contribute to the development of different effector functions.     

IL-10 suppresses CD2-mediated T cell activation via SHP-1

Interleukin (IL)-10 is an essential suppressive cytokine and plays a key role in peripheral T cell tolerance to allergens, autoantigens, transplantation antigens and tumor antigens. However, the molecular mechanisms of direct T cell suppression by IL-10 are not fully understood. Here, we demonstrate that IL-10 directly inhibits CD2 signaling in T cells. T cell stimulation via CD2 alone induces activation and proliferation, when endogenous IL-10 sources are eliminated from cultures. IL-10 utilizes the src-homology-2 domain containing tyrosine phosphatase (SHP-1) to directly suppress T cell activation. The role of SHP-1 in IL-10-mediated suppression of CD2 co-stimulation on T cells is demonstrated by using dominant-negative SHP-1 over-expressing T cells and silencing endogenous SHP-1 by small inhibitory RNA. Findings are confirmed using both SHP-1-deficient mice and IL-10-deficient mice. CD2-induced proliferation is suppressed by exogenous IL-10 in IL-10-deficient, but not SHP-1-deficient murine T cells. In conclusion, SHP-1-mediated inhibition of CD2 signaling represents a novel mechanism for direct T cell suppression by IL-10.     

Pharmacological inhibition of CD28-stimulated T cell activation

Inflammation Research -     

CD28 and T cell co-stimulation

Over the last decade the concept of T cell co-stimulation has emerged to take a central role in the process of T cell activation. However, the exact definition of co-stimulation is still unclear. In this review, we re-examine the concept of co-stimulation. We suggest that while co-stimulation is important, there is little evidence to link co-stimulation with T cell anergy. We then suggest a framework for studying co-stimulation. Focusing on recent advances in our understanding of CD28, we discuss four areas of T cell activation where co-stimulation may play a role.     

Impaired CD28-mediated co-stimulation in anergic T cells

We have investigated a CD28 co-stimulation in anergic T cellsin staphylococcal enterotoxin Binoculated mice by stimulatingthe cells with a plate-coated anti-TCR antibody in the presenceor absence of an anti-CD28 antibody. CD28 co-stimulation increasedthe levels of IL-2 and IL-4 mRNAs in nalve CD4⁺V_ß8⁺T cells. However, it did not increase the levels of IL-4 mRNAat all and only partially increased those of IL-2 mRNA in anergicT cells. It was demonstrated that CD28 co-stimulation was impairedso that it no longer stabilized cytoklne mRNAs in anergic cells.The levels of IL-4 mRNA in response to TCR stimulation werehigher in anergic T cells than those in nalve T cells in spiteof the defective CD28 co-stimulation in the former cells. Anergyinduction and generation of a T_h2-type immune response in vivoare discussed     

Impaired interleukin-2 synthesis and T cell proliferation following antibody-mediated CD3 and CD2 or CD28 cross-linking in trans: evidence that T cell activation requires the engagement of costimulatory molecules within the immunological synapse

Although the T cell costimulatory molecules CD2 and CD28 are enriched within the immunological synapse (IS), it has been suggested that costimulatory molecules need not be localized to the contact site between a T cell and an antigen-presenting cell (APC) in order to costimulate T cell activation. To determine whether CD2 or CD28 engagement outside of the IS is sufficient to costimulate T cell activation, we compared mouse T cell responses to anti-CD3 and anti-CD2 monoclonal antibodies (mAbs) or anti-CD3 and anti-CD28 mAbs immobilized on the same, i.e., in cis, or on different, i.e., in trans, 10 micron polystyrene microspheres. In comparison to T cells that were stimulated with co-immobilized anti-CD3 and anti-CD2 or anti-CD28 mAbs, DNA synthesis, interleukin (IL)-2 production, and cellular proliferation were all severely impaired following T cell stimulation with anti-CD3 and anti-CD2 mAbs or anti-CD3 and anti-CD28 mAbs on different microspheres. Deficient cellular proliferation and IL-2 synthesis by T cells that experienced CD3 and CD2 or CD28 cross-linking in trans provides evidence that costimulatory molecules must function in the context of the IS for optimal T cell activation.     

Antigen-specific targeting of CD28-mediated T cell co-stimulation using chimeric single-chain antibody variable fragment-CD28 receptors

T cells require two distinct signals for optimal activation. One is an antigen-specific signal and is provided by engagement of the T cell receptor (TCR). The second is an antigen-independent signal mediated by engagement of the T cell surface molecule CD28 with members of the B7 family. To endow CD28 molecules with antibody-type recognition, we have constructed chimeric single-chain antibody variable fragment (scFv)-CD28 molecules; following transfection of the genes encoding such constructs into the Jurkat human T cell line we show that they are stably expressed as functional cell surface receptors. These chimeric molecules have no apparent negative effects on the expression and signaling ability of the wild-type CD28 and TCR/CD3 molecules. When combined with signaling via the TCR/CD3 complex, these antigen-specific scFv-CD28 chimeric molecules provide signals similar to those elicited by the cross-linking of the unmodified CD28 molecules. Furthermore, the generation of double transfectants simultaneously expressing scFv-CD28 and scFv-CD3ζ chimeras demonstrates that antigen-specific co-stimulatory signals can also synergize with signals mediated through chimeric CD3ζ chains to secrete maximal levels of interleukin-2. Overall, our results suggest that optimal, predefined antigen-specific activation of T cells directed by the specificity of the scFv should be possible.     

CD28-mediated costimulation impacts on the differentiation of DC vaccination-induced T cell responses

Costimulatory signals such as the ones elicited by CD28/B7 receptor ligation are essential for efficient T cell activation but their role in anti-tumour immune responses remains controversial. In the present study we compared the efficacy of DC vaccination-induced melanoma specific T cell responses to control the development of subcutaneous tumours and pulmonary metastases in CD28-deficient mice. Lack of CD28-mediated costimulatory signals accelerated tumour development in both model systems and also the load of pulmonary metastases was strongly increased by the end of the observation period. To scrutinize whether lack of CD28 signalling influences priming, homing or effector function of Trp-2(180-188)/K(b)-reactive T cells we investigated the characteristics of circulating and tumour infiltrating T cells. No difference in the frequency of Trp-2(180-188)/K(b)-reactive CD8+ T cells could be demonstrated among the cellular infiltrate of subcutaneous tumours after DC vaccination between both genotypes. However, the number of IFN-gamma-producing Trp-2-reactive cells was substantially lower in CD28-deficient mice and also their cytotoxicity was reduced. This suggests that CD28-mediated costimulatory signals are essential for differentiation of functional tumour-specific CD8+ T-effector cells despite having no impact on the homing of primed CD8+ T cells.     

Regulation of 2B4 (CD244)-mediated NK cell activation by ligand-induced receptor modulation

Natural killer (NK) cell activity can be stimulated by different surface receptors. 2B4 is a member of the signaling lymphocyte activation molecule (SLAM)-related receptor family and is important for stimulating human NK cell cytotoxicity and cytokine production. Here we show that stimulation of human NK cells by antibody-mediated 2B4 cross-linking or incubation with target cells expressing the 2B4 ligand CD48 results in a strong down-modulation of 2B4 surface expression. This down-modulation is observed in NK cell lines, purified human NK cells and NK cell clones, and is accompanied by an internalization of 2B4. The modulation of 2B4 is dependent on the activity of Src-family kinases, but independent of PI3 K activity or actin polymerization. Inhibitory receptors can interfere with 2B4-mediated signals and NK cell activation. However, co-engagement of inhibitory killer cell Ig-like receptors has no influence on the down-modulation of 2B4. This suggests that the modulation of 2B4 expression is independent of inhibitory receptors. The lower surface expression of 2B4 after ligand-induced down-modulation results in reduced 2B4-mediated NK cell activation and cytotoxicity. The modulation of activating surface receptors may therefore be another mechanism for the fine-tuning of NK cell activity and may lead to the adaptation of NK cell cytotoxicity in tissues with high ligand expression.     

The CD2 and CD28 adhesion molecules induce long-term autocrine proliferation of CD4+ T cells

In vitro human T lymphocyte activation requires two-signal triggering delivered by lectins, phorbol esters or antibodies directed against surface molecules. Stimulation of adhesion molecules by CD2 and/or CD28 antibodies defines alternative activation pathways. Activation by CD2 + CD28 monoclonal antibodies induces high-level, long-lasting and monocyte-independent proliferation of highly purified T cells. Limiting dilution cultures showed that CD28 in association with CD2 or CD3, without addition of exogenous cytokines, induced single-cell proliferation. CD2 + CD28 stimulation induced long-term interleukin (IL)-2-dependent autocrine proliferation of CD4⁺ T cell clones. We tried to elucidate this long-term proliferation by evaluating cytokine secretion and cytokine dependency. CD28 associated to CD3 or CD2 induced high levels of IL-2, tumor necrosis factor (TNF) and IL-4 secretion for 10 days, in contrast to CD3 alone which induced only TNF secretion. Cytokines of the monocytic lineage were also secreted, such as colony-stimulating factor-1, granulocyte macrophage colony-stimulating factor or IL-1, the latter being more specific of CD2 + CD28 activation. Blocking antibodies confirmed the crucial role of IL-2 in CD2 + CD28 activation. Anti-IL-4, anti-IL-7 receptor or anti-TNF antibodies had no effect on proliferation. Stimulation with CD2 + CD28 induced long-term autocrine (at least for IL-2) proliferation for CD4⁺ T cells, with no evidence for the implication of another cytokine among those tested other than IL-2. This represents a model for long-term autocrine growth for non-leukemic cells.     

Differential interaction of Cbl with Grb2 and CrkL in CD2-mediated NK cell activation

Natural killer (NK) cells participate in both innate and adoptive immunity by their prompt secretion of cytokines and by their ability to lyse virally infected cells or tumor cells. CD2 is surface glycoprotein receptors and crucial for NK cell activation. However, molecular events involved in CD2-mediated NK cell activation have not been fully elucidated. Cbl-Grb2 and Cbl-CrkL interactions have been implicated in T cell and B cell receptor, and cytokine receptor signaling. Here we analyzed tyrosine phosphorylation and interactions of Cbl with adapter proteins, Grb2 and CrkL, in NK3.3 cells. CD2 crosslinking results in the marked tyrosine phosphorylation of Cbl in an antibody concentration- and time-dependent manner. Immunodepletion studies reveal that Grb2-associated tyrosine phosphorylated p120 kDa protein is Cbl. In vitro binding studies using GST-fusion proteins demonstrate that Cbl constitutively associates with the SH3 domains of Grb2, with a preference for the amino-terminal domain. In addition, we demonstrate that CrkL associates with a large portion of tyrosine phosphorylated Cbl after CD2 stimulation of NK3.3 cells. In contrast to constitutive Cbl association with Grb2, tyrosine phosphorylated Cbl interacts with CrkL via its SH2 domain only after CD2 stimulation. Although the precise roles of interactions of Cbl with Grb2 and CrkL in NK cell activation remains to be elucidated, their tyrosine phosphorylation, in addition to the multiple protein interactions described here, strongly suggest that interactions of Cbl with Grb2 and CrkL may play pivotal roles in CD2-mediated NK cell activation.     

Proliferative arrest and cell cycle regulation in CD8(+)CD28(-) versus CD8(+)CD28(+) T cells

CD8(+)CD28(-) T cells have been characterized by oligoclonal expansions, impaired proliferative responses, but preserved cytotoxicity and reduced telomeres. To examine this subset further and define the underlying mechanisms of proliferation arrest, we investigated several features of this cell type compared with CD8(+)CD28(+) controls. We analyzed expression of various activation markers, thymidine incorporation upon activation, T-cell receptor (TCR) zeta-chain phosphorylation, cell cycle characteristics, and cell cycle related gene expression. Flow cytometry revealed higher expression of CD11b, CD29, CD57, and CD94, and lower expression of CD25 in CD8(+)CD28(-) compared with CD8(+)CD28(+) T cells. Sorted CD8(+)CD16(-)CD28(-) cells exhibited decreased phosphorylation of the TCR zeta-chain in three of four probands. Proliferation of these T cells was impaired, even when activated with mitogens that bypass TCR signaling. Cell cycle profiles demonstrated a lower percentage of cycling cells and significantly higher levels of cyclin dependent kinase inhibitor p16(INK4a) in the CD28(-) subset compared with the CD28(+) control. These observations suggest that expanded CD8(+)CD28(-) T cells in normal elderly individuals have reduced proliferation concomitant with increased p16(INK4a) expression. Defects in TCR signaling were associated with altered TCR zeta-chain phosphorylation.     

Inhibition of CD28-mediated T cell costimulation by the phosphoinositide 3-kinase inhibitor wortmannin

T lymphocyte activation requires at least two signals, one via the antigen-specific T cell receptor and a second via the surface molecule CD28 which provides signals critical to interleukin-2 (IL-2) production and T cell proliferation. We have previously shown (Ward S. G., Westwick J., Hall N. and Sansom D. M. Eur. J. Immunol. 1993. 23: 2572) that CD28 stimulates phosphoinositide (PI) 3-kinase activity, indicating that D-3 phosphoinositides may act as mediators of CD28-induced T cell costimulation. Here, we report that immunoprecipitation of CD28 molecules from Jurkat cells stimulated with the CD28-ligand B7, results in a ligand-dependent association of CD28 with PI 3-kinase. This association correlates with the appearance of PI 3-kinase enzymatic activity in CD28 immunoprecipitates and the formation of D-3 phosphoinositides. Consistent with the hypothesis that D-3 phosphoinositides are important mediators of CD28 signaling, treatment of T cells with the PI 3-kinase inhibitor wortmannin, inhibited both T cell proliferation and production of IL-2, but not the response of T cells to exogenous IL-2. Hence, abrogation of PI 3-kinase activity by wortmannin, appears sufficient to disrupt the costimulatory pathway utilized by CD28, indicating a central role for this enzyme in the CD28 signaling pathway.     

Gamma delta T cells of the murine vagina: T cell response in vivo in the absence of the expression of CD2 and CD28 molecules

While little is known about their activation requirements and function, the intraepithelial T cells of the murine vagina express TCR complexes in which the antigen recognition components and the signaling components have unusual features. These vaginal T cells express an invariant V gamma 4/V delta 1 TCR and appear to be the only intraepithelial gamma delta T cells that exclusively use FcR gamma chains in their TCR complex. To further characterize the vaginal gamma delta T cells we isolated them from normal mice and from mice injected systemically with an activation-inducing dose of anti-TCR mAb. The isolated gamma delta T cells were examined by flow cytometry for their surface expression of a panel of adhesion, proteins, activation antigens and cellular interaction molecules (CD44, CD62L, CD45RB, LFA- 1, CD2 and CD28). The patterns of expression observed indicate that the vaginal gamma delta T cells of normal mice show the phenotype of effector T cells. The adhesion/co-stimulatory molecules CD28 and CD2 were not detected on vaginal gamma delta T cells, an interesting finding since the absence of CD2 from other T cells has been suggested to result in anergy. However, vaginal gamma delta T cells are responsive to TCR-mediated signals since injection of normal mice with pan-anti-TCR antibody or stimulating anti-gamma delta TCR antibody resulted in an increase in cell number and increased expression of transferrin and IL-2 receptors. These results indicate that vaginal gamma delta T cells might utilize other co-stimulatory molecules, if any, in connection with TCR-induced activation and differentiation. While the physiological function of vaginal gamma delta T cells remains unknown, the expression of an invariant V gamma 4/V delta 1 TCR, their exclusive use of gamma chain homodimers in their TCR, and the absence of CD2 and CD28 co-stimulatory molecules are a novel combination of properties that suggests specialized functional properties. Although vaginal gamma delta T cells share some features in common with gamma delta T cells that reside in other epithelial tissues, such as skin and intestine, the present studies provide additional evidence that vaginal gamma delta T cells are a highly specialized and distinct T cell population.      

T cell activation induced by cross-linking CD3 and CD28 leads to silencing of Epstein-Barr virus/C3d receptor (CR2/CD21) gene and protein expression

Complement receptor II (CR2) also known as CD21 is the receptor for C3d on immune complexes. In humans it serves as a receptor for the Epstein-Barr virus (EBV). CR2 is expressed on B cells and in low density in the T cell lineage. EBV can infect T cells and EBV-positive T lymphomas have been described. Although CR2 mRNA is readily detectable in T cells, the function of CR2 in human T lymphocytes remains elusive. Here we have analyzed the expression of CR2 in normal and activated T cells. PCR analyses and immunofluorescence/confocal microscopy of peripheral blood T cells and of activated T cells shows considerable reduction in CR2 mRNA and protein expression upon activation. The downregulation of CR2 expression may modulate life span or immunological reactivity of T cells and the susceptibility of cells to infection by lymphotropic viruses.