T cell surface molecules regulating noncognate B lymphocyte activation. Role of CD2 and LFA-1.

A central event in humoral responses is the Ag-mediated interaction of Th cells and B cells. This interaction leads to the activation of both cell types and results in cytokine secretion by the T cells and proliferation and secretion of Ig by the B cells. The proliferative and differentiative responses of B cells are dependent on contact-mediated signals and cytokines provided by the activated Th cells. Although the role of cytokines in B cell activation and differentiation is understood, the nature of the signals delivered by the activated Th cells and the molecules involved in this process are not known. In this study we have examined Ag-mediated "cognate" T-B cell interactions as well as B cell activation induced by contact with preactivated and fixed Th lymphocytes. Our results indicate that both the T cell surface molecules lymphocyte function associated Ag-1 and CD2 are important in the activation of T cells by Ag presented by B lymphocytes. This indicates that B cells have similar characteristics as other APC. However, once the T cells are activated, contact-mediated stimulation of resting B lymphocytes (the noncognate phase) is dependent on CD2 but not lymphocyte function associated Ag-1. Two lines of evidence indicate this; first, it is inhibited by blocking of CD2 on the T cells and, second, such stimulation is not efficiently mediated by a CD2- Th cell line. Thus, CD2 plays an obligatory role at several discrete stages of T cell-mediated activation of resting B lymphocytes.     

Characterization of T cell proliferative responses induced by anti-Qa-2 monoclonal antibodies.

The MHC class I Qa-2 Ag are attached to the cell surface by a glycanphosphatidylinositol (GPI) anchor. Crosslinking of Qa-2 and several other cell surface Ag attached by the GPI linkage has been shown to lead to cell activation. We have developed 10 new anti-Qa-2 mAb and characterized their capacity to induce proliferation of spleen cells. In the absence of anti-Ig-mediated crosslinking, none of the mAbs alone could induce activation. However, mAb 23.1 which reacts with the alpha 3 domain of Qa-2, when combined with most of the other mAbs (alpha 1, alpha 2 domain reactive), activated cells in the absence of anti-Ig crosslinking. The mAb pair 23.1 plus 24.16 was the most proficient and induced proliferation in the absence of any exogenous second signals. Responses were greatly enhanced and equivalent to those seen with anti-CD3 by the addition of phorbol myristate acetate (PMA). Ionomycin, rIL-2, or rIL-4 also potentiated anti-Qa-2 responses but less efficiently than PMA. Significant strain variation in the magnitude Qa-2-mediated proliferative responses was observed correlating with the levels of Qa-2 expressed on the cell surface. Crosslinking of Qa-2 molecules by the mAb combinations was required because monovalent Fab fragments failed to activate cells. F(ab')2 fragments of mAb 23.1 plus 24.16 induced vigorous proliferation indicating that accessory cell presentation of the mAb via Fc receptors was not required. Immobilized (plate bound) anti-Qa-2 mAb induced proliferation suggesting that the Qa-2 pathway may be distinct from that of other GPI molecules such as Thy-1 and Ly-6. Populations enriched for T cells (approximately 95%) responded as well as whole spleen cells, whereas B lymphocytes failed to proliferate to anti-Qa-2. Both CD4+ and CD8+ cells were activated following crosslinking of Qa-2. Finally, T cell activation mediated by Qa-2 induced elevation of [Ca2+]i, IL-2R expression, and the release of IL-2. These data demonstrate that crosslinking of Qa-2 on T lymphocytes represents a potent pathway for inducing cell activation.     

Class I-specific antibodies inhibit proliferation in primary but not secondary mouse T cell responses.

Murine T and B splenocytes were incubated with antibodies that recognize CD3 or surface IgM. These antibodies induced proliferation of their respective target cells. Once stimulated via their receptors, the proliferation of both CD4+ and CD8+ T but not B lymphocytes was inhibited by class I-specific antibodies or their monovalent Fab' fragments. The inhibition of proliferation was dependent on the site on class I molecules recognized by the antibodies used, with the alpha 1/alpha 2 domains of H-2K molecules representing the major site for inhibition. Only soluble antibody-mediated proliferation could be inhibited by class I-directed antibodies; proliferation induced by CD3-specific antibody immobilized on plastic was not inhibited. Primary allogeneic MLR was also inhibited by class I-specific antibodies. In contrast, neither secondary allogeneic MLR, secondary Ag-specific responses, nor proliferation of CTL clones or tumor cell lines were inhibited by class I-specific antibodies. These results suggest a role for class I molecules in regulation of TCR/CD3- but not surface IgM-mediated cell signaling, which depends on the form of stimulation and the stage of differentiation of T cells.     

Generation and characterization of continuous lines of CD8+ suppressor T lymphocytes

The ability to grow normal T lymphocytes in long term culture has advanced our understanding of T cell biology. The growth of CD4+ cell lines allowed a further evaluation and appreciation of functional subtypes within this group. Cytotoxic CD8+ T cells have been characterized as well. The routine and continuous culture of Ag-nonspecific CD8+ Ts cells has been difficult to achieve. We have found that CD8+ T cells that suppress T cell proliferation and lack cytotoxic activity against T cells can be routinely obtained from PWM or PHA-stimulated PBMC. Continuous culture of T cell blasts from PWM or PHA-stimulated PBMC resulted in the growth of CD4+ and CD8+ T cells. These lines developed suppressor cell activity within 7 days after stimulation with PWM and 3 to 4 wk after stimulation with PHA. Concomitant with the development of suppressor activity was the loss of CD4+ T cells resulting in homogeneous lines of CD8+ suppressor cells. These cell lines have been maintained in continuous culture for greater than 6 mo by addition of rIL-2 twice weekly and restimulation with feeder cells and PHA every 2 wk. Activity of these cell lines was relatively resistant to irradiation or treatment with mitomycin C. Both cell lines suppressed proliferation of autologous or heterologous CD4+ T cells stimulated with PWM, OKT3, or tetanus toxoid but failed to suppress proliferation of CD4+ T cells in a mixed lymphocyte reaction. CD4+ T cells stimulated with PWM produced equivalent amounts of IL-2 in the presence or absence of Ts cells but failed to express the IL-2R (TAC) on their surface in the presence of Ts cells. By contrast, CD4+ T cell lines or cytotoxic CD8+ T cell lines failed to suppress proliferation of CD4+ T cells. With these results we describe methods for the generation and continuous culture of Ag-nonspecific CD8+ Ts cells and define some of their properties. These cells lines should be helpful in further elucidating the functional and phenotypic repertoire of CD8+ Ts cells.     

Characterization and functionality of cell surface molecules on human mesenchymal stem cells

We have characterized adhesion molecules on the surface of multipotential human mesenchymal stem cells (hMSCs) and identified molecules whose ligands are present on mature hematopoietic cells. Flow cytometric analysis of hMSCs identified the expression of integrins: alpha1, alpha2, alpha3, alpha5, alpha6, alphav, beta1, beta3, and beta4, in addition to ICAM-1, ICAM-2, VCAM-1, CD72, and LFA-3. Exposure of hMSCs to IL-1alpha, TNFalpha or IFNgamma up-modulated ICAM-1 surface expression, whereas only IFNgamma increased both HLA-class I and -class II molecules on the cell surface. Whole cell-binding assays between the hMSCs and hematopoietic cell lines showed that T lymphocytic lines bound hMSCs with higher affinity than lines of either B lymphocytes or those of myeloid lineage. Experiments using autologous T lymphocytes isolated from peripheral blood mononuclear cells showed that hMSCs exhibited increased affinity for activated T-lymphocytes compared to resting T cells by quantitative whole cell binding and rosetting assays. Flow cytometric analysis of rosetted cells demonstrated that both CD4+ and CD8+ cells bound to hMSCs. To determine the functional significance of these findings, we tested the ability of hMSCs to present antigen to T lymphocytes. hMSCs pulsed with tetanus toxoid stimulated proliferation and cytokine production (IL-4, IL-10, and IFNgamma) in a tetanus-toxoid-specific T cell line. Maximal cytokine production correlated with maximal antigen-dependent proliferation. These data demonstrate physiological outcome as a consequence of interactions between hMSCs and human hematopoietic lineage cells, suggesting a role for hMSCs in vivo to influence both hematopoietic and immune function(s).     

Enhanced responses of glycosylphosphatidylinositol anchor-deficient T lymphocytes

The functions of GPI-anchored proteins in T lymphocyte activation have been controversial. This issue was addressed by studying the responses of T lymphocytes from T lymphocyte-specific GPI anchor-deficient mice to different stimuli that normally allow coligation of TCR and GPI-anchored proteins. Stimulation of GPI anchor-deficient T lymphocytes with ConA induced 2-fold higher proliferative responses than did normal cells. In response to allogeneic stimulation, proliferation of GPI anchor-deficient T lymphocytes was enhanced 2- to 3-fold. The response to ConA of a GPI anchor-deficient anti-OVA T lymphocyte clone generated from these mice was approximately 3-fold higher than that of cells from the same clone in which GPI anchor expression was restored by retroviral transduction. The response of the GPI anchor-deficient cloned anti-OVA T lymphocytes to antigenic stimulation was similar to that of the retrovirally restored cells. These results indicate that coligation with GPI-anchored proteins counteracts the response to TCR stimulation by ConA or alloantigen but not protein Ag.     

Defective clonogenic potential of CD8+ T lymphocytes in patients with AIDS. Expansion in vivo of a nonclonogenic CD3+CD8+DR+CD25- T cell population

This study examines the potential mechanism(s) responsible for the defective clonability of CD8+ T lymphocytes in patients with AIDS. By the combined use of one- and two-color fluorescence cytofluorometry we have shown an increase in the number of circulating DR+ cells due to the expression of DR on a relatively large proportion of T lymphocytes (one-third of CD3+ cells), the majority of them belonging to the CD8+ subset. In addition, the majority of CD8+DR+ cells in AIDS patients did not express CD25 Ag (the receptor for IL-2), a surface marker generally expressed on normal activated T lymphocytes. Sorted CD8+DR+ and CD8+DR- cell populations were analyzed comparatively for their ability to proliferate in response to different stimuli, including anti-CD3, anti-CD2, alone or in combination with anti-CD28 mAb and mitogens such as PHA, alone or in combination with PMA. We have demonstrated that CD8+DR+ cells were severely defective in their proliferative response to triggering via these major pathways of T cell activation even when an exogenous source of IL-2 or IL-4 was added to the microcultures 24 h after initiating the cultures. In contrast, CD8+DR- cells showed a significant proliferation in response to the different stimuli and the proliferative response was strongly enhanced by the addition of IL-2 or IL-4. At the end of the stimulation period CD8+DR+ and CD8+DR- proliferating populations were analyzed for CD25 Ag expression. Only 1 to 10% of CD8+DR+ cells expressed CD25 antigen compared with 40 to 50% of CD8+DR- cells. The proliferative defect of CD8+DR+ cells was further confirmed in experiments performed at the clonal level. The analysis of the frequency of proliferating T lymphocyte-precursors in both CD8+DR+ and CD8+DR- subsets showed that the defective clonogenic potential of CD8+ cells in AIDS patients could be in large part ascribed to CD8+DR+ cells. Five percent of CD8+DR+ cells showed a clonogenic potential compared to the 25% of CD8+DR- cells. Finally, we analyzed the surface expression of VLA-2 Ag, a marker of a chronic state of T cell activation, on circulating T lymphocytes. We have shown that a large proportion of CD3+DR+CD25- cells (50 to 80% in the different patients with AIDS analyzed) expressed VLA-2 Ag.(ABSTRACT TRUNCATED AT 400 WORDS)     

Survival and homeostatic proliferation of naive peripheral CD4+ T cells in the absence of self peptide:MHC complexes

TCR-self peptide:MHC interactions play a critical role in thymic positive selection, yet relatively little is known of their function in the periphery. It has been suggested that continued contact with selecting MHC molecules is necessary for long-term peripheral maintenance of naive T cells. More recent studies have also demonstrated a role for specific self peptide:MHC complexes in the homeostatic expansion of naive T cells in lymphopenic mice. Our examination of these processes revealed that, whereas self class II MHC molecules do have a modest effect on long-term survival of individual CD4+ T cells, interactions with specific TCR ligands are not required for peripheral naive CD4+ T cell maintenance. In contrast, selective engagement of TCRs by self-peptide:MHC complexes does promote proliferation of CD4+ T cells under severe lymphopenic conditions, and this division is associated with an activation marker phenotype that is different from that induced by antigenic stimulation. Importantly, however, the ability of naive T cells to divide in response to homeostatic stimuli does not appear to be stringently dependent on TCR-self peptide:MHC interactions. Therefore, these results show that the factors regulating survival and homeostatic expansion of naive T cells in the periphery are not identical. In addition, we provide evidence for a novel form of T cell proliferation that can occur independently of TCR signaling and suggest that this reflects another mechanism regulating homeostatic T cell expansion.     

Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation.

Although resting B cells are poor accessory cells for signals transmitted through the TCR/CD3 complex, we report that these B cells can support T cell proliferation when T cell activating signals are delivered through CD2. This was first suggested when leucine methyl ester treatment of PBMC abolished proliferation induced by anti-CD3, but not by the accessory cell-dependent anti-CD2 mAb combination, GT2 and OKT11. Then we demonstrated that unstimulated, resting B cells could support the proliferation of both CD4+ and CD8+ T cells. Aggregated IgG inhibited proliferation, suggesting that anti-CD2 mAb bound to T cells were cross-linked by attachment to B cell FcR. Two lines of evidence suggested that lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction was crucial for anti-CD2-induced proliferation. First, proliferation was blocked by mAb against these adhesion molecules. Second, intercellular adhesion molecule-1 expression rapidly increased on resting B cells after the addition of anti-CD2, but not anti-CD3. This was of interest because fixed monocytes, but not fixed B cells, were able to support the proliferative response. In contrast to lymphocyte function-associated Ag-1/intercellular adhesion molecule-1, CD28/B7 interaction was not required for anti-CD2-induced proliferation, although ligation of these molecules provided important costimulatory signals for stimulation by anti-CD3. Finally, neutralizing antibodies against IL-1 alpha, IL-1 beta, and IL-6 showed only modest inhibitory effects on T cell proliferation. The addition of IL-1 and/or IL-6 to T cells failed to substitute for accessory cells and were only partially effective with fixed B cells. Further evidence of a linkage between CD2 and CD45 isoforms was obtained. Anti-CD45RA, but not anti-CD45RO, potentiated anti-CD2-induced T cell proliferation. These studies have revealed a novel role for resting B cells as accessory cells and have documented costimulatory signals that are important for this effect. Because Ag-presentation by resting B cells to T cells generally leads to T cell nonresponsiveness, it is possible that this tolerogenic signal may be converted to an activation signal if there is concurrent perturbation of CD2 on T cells.     

Human T cell activation. Synergy between CD73 (ecto-5'-nucleotidase) and signals delivered through CD3 and CD2 molecules

Interaction of the glycosyl phosphatidylinositol-linked differentiation Ag CD73 (ecto-5'-nucleotidase) with the CD73-specific mAb 1E9 generates agonistic signals that strongly synergize with T cell activation induced by CD3 and CD2 mAb. This synergy is observed only when 1E9 is immobilized on plastic and occurs in the absence of accessory cells or exogenous lymphokines. 1E9 induces a rapid (though transient) increase in [Ca2+]i in a minor proportion (20 to 30%) of unfractionated T lymphocytes (presumably CD73+ cells). However, this [Ca2+]i mobilization is not sufficient to fully activate CD73+ T cells, as shown by the requirement of additional signals such as CD3 or CD2 stimulation to initiate T cell proliferation. These signals cannot be substituted by the exogenous lymphokines, rIL-1, rIL-2, or rIL-4, or PMA (when T cells are rigorously depleted of monocytes). These data indicate that CD73 may behave as an accessory molecule regulating interactions between T cells and antigens or APC. A comparison was carried out with mAb 9.3 to the differentiation Ag CD28, another agonistic molecule with activating properties similar to CD73. Despite their lower percentage, the ability of CD73+ T cells to amplify the proliferation induced by CD3 or CD2 mAb was equivalent or even greater than that of CD28+ T cells. Once activated, CD73+ cells may recruit the remaining (CD73-) cells primed by CD3 or CD2 stimulation. Based on these data, we suggest that CD73+ T lymphocytes may be a specialized subset to amplify immune responses originated by the CD3 and CD2 activation pathways. Finally, the functional association between CD73 and integral membrane molecules like CD3 and CD2 suggests that GPI-anchored molecules may play a role in transmembrane signaling mediated by conventional second messenger systems.     

Costimulatory Effect of Fas in Mouse T Lymphocytes

To induce proper immune responses, T lymphocytes require two types of stimuli, antigen-specific and costimulatory signals. Among costimulatory molecules, CD28-engagement promotes the survival and proliferation of both naive and memory T cells. In addition, it is now believed that Fas may play a role in T cell activation in the human system. It is, however, controversial whether Fas can act as a costimulatory signal in the murine system. Thus, we investigated fundamental differences in the capacity to induce proliferation of T cells between Fas and CD28 in mice. Fas-mediated T cell proliferation was observed only with a full mitogenic dose of anti-CD3 antibodies, whereas CD28 engagement was able to enhance T cell proliferation in the presence of a suboptimal level of anti-CD3 antibody. Furthermore, Fas-engaged T cells showed faster response in the upregulation of CD25 and CD69 expression than CD28-engaged ones. Here, we report that Fas might play a role in mature T cell activation in the mouse system through a different mechanism from that in CD28 costimulation.     

Costimulatory Effect of Fas in Mouse T Lymphocytes

To induce proper immune responses, T lymphocytes require two types of stimuli, antigen-specific and costimulatory signals. Among costimulatory molecules, CD28-engagement promotes the survival and proliferation of both naive and memory T cells. In addition, it is now believed that Fas may play a role in T cell activation in the human system. It is, however, controversial whether Fas can act as a costimulatory signal in the murine system. Thus, we investigated fundamental differences in the capacity to induce proliferation of T cells between Fas and CD28 in mice. Fas-mediated T cell proliferation was observed only with a full mitogenic dose of anti-CD3 antibodies, whereas CD28 engagement was able to enhance T cell proliferation in the presence of a suboptimal level of anti-CD3 antibody. Furthermore, Fas-engaged T cells showed faster response in the upregulation of CD25 and CD69 expression than CD28-engaged ones. Here, we report that Fas might play a role in mature T cell activation in the mouse system through a different mechanism from that in CD28 costimulation.     

OX40 induces CCL20 expression in the context of antigen stimulation: An expanding role of co-stimulatory molecules in chemotaxis

OX40 is an inducible co-stimulatory molecule expressed by activated T cells. It plays an important role in the activation and proliferation of T lymphocytes. Recently, some co-stimulatory molecules have been shown to direct leukocyte trafficking. Chemotaxis is essential for achieving an effective immune response. CCL20 is an important chemoattractant produced by activated T cells. In this study, using DO11.10 mice whose transgenic T cell receptor specifically recognizes ovalbumin, we demonstrate that ovalbumin induces OX40 expression in CD4+ lymphocytes. Further stimulation of OX40 by OX40 activating antibody up-regulates CCL20 production. Both NF-κB dependent and independent signaling pathways are implicated in the induction of CCL20 by OX40. Finally, we primed the DO11.10 splenocytes with or without OX40 activating antibody in the presence of ovalbumin. Intranasal administration of the cell lysates derived from the cells with OX40 stimulation results in more severe leukocyte infiltration in the lung of DO11.10 mice, which is substantially attenuated by CCL20 blocking antibody. Taken together, this study has shown that activation of OX40 induces CCL20 expression in the presence of antigen stimulation. Thus, our results broaden the role of OX40 in chemotaxis, and reveal a novel effect of co-stimulatory molecules in orchestrating both T cell up-regulation and migration.     

Stimulation of human T cells via anti-T cell receptor monoclonal antibody BMA031: distinct cellular events involving interleukin-2 receptor and lymphocyte function antigen 1

We have analyzed activation of resting human T cells by anti-T cell receptor (TCR) monoclonal antibody (mAb) BMA031, a murine mAb of the G2b isotype. Human peripheral blood lymphocytes (PBL) respond to anti-TCR mAb by short-term proliferation in vitro and by acquisition of responsiveness to interleukin 2 (rIL-2) in the absence of detectable IL-2 production. Cell depletion and limiting dilution experiments indicate that anti-TCR mAb +/- rIL-2 stimulation covers a substantial portion of human T cells, including CD4+ and CD8+ cells. Enhancement by rIL-2 of anti-TCR mAb-induced proliferation is blocked by anti-IL-2 receptor (IL-2R, p55) mAb, while anti-TCR mAb-induced proliferation is not. In contrast, anti-TCR mAb-induced proliferation is blocked by anti-lymphocyte function antigen 1 (LFA-1, CD11a) mAb and is not demonstrable in PBL from two patients with severe congenital LFA-1 deficiency, not even in the presence of irradiated LFA-1+ PBL. We conclude that stimulation of resting human T cells by anti-TCR mAb BMA031 enables dissociation of distinct steps in T cell activation that specifically require participation of IL-2R (p55) and LFA-1 cell surface molecules in a mutually exclusive way.     

T-deficient transmembrane signaling in CD4+ T cells of retroviral-induced immune-deficient mice.

The defective virus found in the LP-BM5 mixture of murine leukemia viruses induces a severe immune deficiency disease in C57BL/6 mice that is characterized by the activation and expansion of T and B cells that become unresponsive to normal immune stimuli. The nature of the biochemical lesion in these defective lymphocyte populations remains unknown. Flow cytometric analysis of the T cell population in infected animals has demonstrated expansion of both CD4+ and CD8+ subsets. Despite chronic expansion in vivo, CD4+ T cells by wk 4 postinfection failed to up-regulate cell surface IL-2R expression, produced IL-2, or proliferate in vitro in response to either Con A, Staphylococcal enterotoxin super-antigens, or anti-CD3 stimulation. Exogenous IL-2 did not restore the proliferative response and also failed to up-regulate IL-R expression on CD4+ T cells from infected mice, even though basal IL-2R expression was initially elevated compared to normals. In contrast, CD4+ T cells from infected mice could be induced to proliferate by stimulation with PMA and ionomycin resulting in IL-2R up-regulation, IL-2 production, and proliferation. Moreover, proliferation could also be induced by anti-CD3 plus PMA, although anti-CD3 plus ionomycin was without effect. These studies suggest that chronic expansion of CD4+ T cells in infected mice is probably not maintained by normal TCR signaling, which appears defective in these cells. In addition, the lesion in biochemical signaling appears to result in defective activation of protein kinase C, which can be overcome by direct activation with PMA.     

Heterologous cross-linking of Lyt-2 (CD8) to the alpha beta-T cell receptor is more effective in T cell activation than homologous alpha beta-T cell receptor cross-linking

Ag recognition of Lyt-2 (CD8)-positive T lymphocytes requires the presentation by APC of a suitably processed Ag in association with MHC class I molecules. In previous studies we have obtained evidence that, for optimal activation, both the alpha beta-TCR and Lyt-2 have to participate in this recognition process. In the current study we investigate the functional consequences of limited cross-linking of these cell surface molecules by using soluble, dimeric hetero- and homoconjugates of mAb to Lyt-2 and to the TCR beta-chain (F23.1). Heterologous cross-linking of Lyt-2 to the TCR induced a vigorous, selective Lyt-2+ T cell proliferative response. Functionally active cytotoxic cells were generated, and a high frequency of responding cells was observed in limiting dilution analyses. In contrast, homologous TCR cross-linking initiated a less pronounced proliferation with a relatively low frequency of response, whereas Lyt-2 cross-linking resulted in no cellular proliferation. Significant T cell activation occurred with exposure to anti-Lyt-2: F23.1 mAb dimers at concentrations an order of magnitude lower than those required for stimulation by F23.1:F23.1 mAb dimers. The induction of proliferation by mAb dimers occurred in the absence of Fc components and in rigorously APC depleted, purified T cell preparations. Effective stimulation of resting T cells could be induced also by heterodimers of monovalent Fab fragments. Heterologous cross-linking of Lyt-2 to the TCR was superior to homologous TCR cross-linking primarily with respect to proliferation in IL-2 containing media and to IL-2R expression, whereas proliferation in response to other lymphokines and the production of IL-2 itself were similar under both cross-linking regimens. Thus, when linked to the TCR, Lyt-2 contributed a strong, positive signal toward IL-2-dependent growth of resting T cells. We assume that in the case of Ag-driven T cell activation, the class I MHC molecule acts as the physiologic cross-linking ligand for Lyt-2 and the TCR.     

Roscovitine Suppresses CD4+ T Cells and T Cell-Mediated Experimental Uveitis

Background

T cells are essential for the development of uveitis and other autoimmune diseases. After initial activation, CD4+ lymphocytes express the co-stimulatory molecule OX40 that plays an important role in T cell proliferation. Cyclin dependent kinase 2 (CdK2) plays a pivotal role in the cell cycle transition from G1 to S phase. In addition, recent research has implicated CdK2 in T cell activation. Thus, we sought to test the immunosuppressive effect of roscovitine, a potent CdK2 inhibitor, on CD4+ T cell activation, proliferation, and function.

Design and Methods

Mouse CD4+ T cells were activated by anti-CD3 and anti-CD28 antibodies. The expression of OX40, CD44, and CdK2 were analyzed by flow cytometry. In addition, cell cycle progression and apoptosis of control and roscovitine-treated T lymphocytes were measured by BrdU incorporation and annexin V assay, respectively. Furthermore, the immunoregulatory effect of roscovitine was evaluated in both ovalbumin-induced uveitis and experimental autoimmune uveitis (EAU) models.

Results

In this study, we found that T cell activation induced OX40 expression. Cell cycle analysis showed that more CD4+OX40+ cells entered S phase than OX40- T cells. Concurrently, CD4+OX40+ cells had a higher level of CdK2 expression. Roscovitine treatment blocked activated CD4+ cells from entering S phase. In addition, roscovitine not only reduced the viability of CD4+ lymphocytes but also suppressed T cell activation and cytokine production. Finally, roscovitine significantly attenuated the severity of T cell-dependent, OX40-enhanced uveitis.

Conclusion

These results implicate CdK2 in OX40-augmented T cell response and expansion. Furthermore, this study suggests that roscovitine is a novel, promising, therapeutic agent for treating T cell-mediated diseases such as uveitis.     

The role of protein kinase C in transmembrane signaling by the T cell antigen receptor complex. Effects of stimulation with soluble or immobilized CD3 antibodies

Phorbol esters, such as phorbol myristate acetate (PMA), are known to be potent co-stimulants with calcium ionophores for activation of T lymphocytes. The most extensively studied intracellular effect of PMA is its ability to activate the cytoplasmic enzyme protein kinase C (pkC). Herein, we examined the role of pkC activation during T cell activation. During physiologic activation, this enzyme is activated by diacylglycerol which is generated through the hydrolysis of polyphosphoinositides. Therefore, we studied the activation of T lymphocytes induced by a synthetic diacylglycerol, dioctanoylglycerol. In contrast to PMA, this compound can be metabolized in T cells and presumably more closely mimics physiologic activation of pkC. Dioctanoylglycerol together with reagents that induce increases in intracellular free Ca2+ concentration, Ca2+ ionophores, or anti-cluster designation (CD)3 monoclonal antibodies (mAb) were able to induce interleukin 2 receptor expression and proliferation of T lymphocytes. Previous studies have demonstrated that the stimulation of T cells via the CD3/T cell antigen receptor complex by mAb against CD3 leads to an increase in cytoplasmic free Ca2+ and to an activation of pkC. Paradoxically, however, soluble CD3 antibodies do not cause proliferation of resting purified T cells. Inasmuch as immobilization of CD3 mAb has been shown to influence the agonist properties of such antibodies, we compared the ability of soluble and immobilized CD3 mAb to activate pkC. We demonstrated herein that soluble CD3 mAb cause only a very transient activation of pkC in the T cell leukemic line Jurkat. This pkC activation is markedly prolonged when Jurkat cells are stimulated with immobilized rather than soluble CD3 antibodies. These studies suggest that activation of pkC plays a major role in T cell activation and that the activation of pkC is influenced by the form in which CD3 mAb is presented to T cells.