Costimulatory Activation Research Articles

A Role for cFLIP in B Cell Proliferation and Stress MAPK Regulation

Fas/Apo-1 signals through the FADD (Fas-associated death domain) adaptor protein, which recruits and activates the apical caspase 8 and leads to apoptosis. Cellular FLIP (cFLIP) is a homolog of caspase 8 and is also capable of binding to FADD. Previous studies suggest that cFLIP could either enhance or inhibit apoptosis and lead to NF-kappaB and Erk1/2 activation. Like FADD or caspase 8 deficiency, a lack of cFLIP disrupts embryogenesis and T cell proliferation. It has been demonstrated that B cells lacking either FADD or caspase 8 were defective in both Fas-induced apoptosis and TLR-induced proliferation, which indicates that these death-inducing proteins have an additional role in regulating innate immunity. To analyze the function of cFLIP in B cells, conditional deletion of cFLIP was induced by using CD19(Cre). The resulting B cell-specific cFLIP-deficient mice were found to have reduced numbers of peripheral B cells that were hypersensitive to Fas-induced apoptosis and impaired in proliferation induced by TLRs and the BCR. Furthermore, there was aberrant expression of costimulatory proteins and activation markers in cFLIP-deficient B cells. Whereas LPS-induced activation of NF-kappaB and Erk1/2 appears to be unaffected, p38 and Jnk were spontaneously activated and hyperinduced in cFLIP-deficient B cells. Therefore, these data revealed novel functions of cFLIP in B cells.

New Costimulatory Families: Signaling Lymphocytic Activation Molecule in Adaptive Allergic Responses

The generation of an allergic immune response requires at least two signals for complete activation of T cells. Costimulatory molecules are integral to the second signal. In this review, we analyze the costimulatory molecule signaling lymphocytic activation molecule (SLAM) and other recently described SLAM family members. We highlight recent findings that position SLAM as critical for allergic inflammation and its role in modulation of cytokine secretion. Furthermore, a possible role of SLAM as a link between the adaptive and innate immune response is also discussed. Understanding the role of costimulatory molecules, including SLAM and SLAM family members, may elucidate mechanisms involved in the allergic immune response, and suggest potential therapeutic opportunities.

Programmed Death (PD)-1:PD-Ligand 1/PD-Ligand 2 Pathway Inhibits T Cell Effector Functions during Human Tuberculosis

Protective immunity against Mycobacterium tuberculosis requires the generation of cell-mediated immunity. We investigated the expression and role of programmed death 1 (PD-1) and its ligands, molecules known to modulate T cell activation, in the regulation of IFN-gamma production and lytic degranulation during human tuberculosis. We demonstrated that specific Ag-stimulation increased CD3+PD-1+ lymphocytes in peripheral blood and pleural fluid from tuberculosis patients in direct correlation with IFN-gamma production from these individuals. Moreover, M. tuberculosis-induced IFN-gamma participated in the up-regulation of PD-1 expression. Blockage of PD-1 or PD-1 and its ligands (PD-Ls: PD-L1, PD-L2) enhanced the specific degranulation of CD8+ T cells and the percentage of specific IFN-gamma-producing lymphocytes against the pathogen, demonstrating that the PD-1:PD-Ls pathway inhibits T cell effector functions during active M. tuberculosis infection. Furthermore, the simultaneous blockage of the inhibitory receptor PD-1 together with the activation of the costimulatory protein signaling lymphocytic activation molecule led to the promotion of protective IFN-gamma responses to M. tuberculosis, even in patients with weak cell-mediated immunity against the bacteria. Together, we demonstrated that PD-1 interferes with T cell effector functions against M. tuberculosis, suggesting that PD-1 has a key regulatory role during the immune response of the host to the pathogen.

Inhibiting Costimulatory Activation of T Cells

There is now good evidence that T cells play a central role in the inflammatory pathway that leads to the persistent synovitis that causes joint damage in rheumatoid arthritis (RA). T cells require two signals to become activated. The second step in the activation of T cells involves costimulatory pathways, the best described pathway being the binding of CD28 on T cells to CD80/86 on antigen-presenting cells. This observation has led to the development of a new category of biological response modifier. Abatacept is a fusion protein (cytotoxic T-lymphocyte-associated antigen-4 immunoglobulin [CTLA4Ig]); which blocks the binding of CD28 by avidly binding CD80/86. Without this costimulatory activation, the T cell becomes anergic. Abatacept has consistently been shown to improve the signs and symptoms of RA in phase II and phase III trials in patients with an inadequate response to methotrexate and anti-tumour necrosis factor (TNF) therapy. Onset of action is rapid and efficacy is maintained during the period of treatment. Recent trials have also provided evidence of improvement in quality-of-life measures and radiographic progression. The safety profile to date has also been favourable and supports the theory that targeting naive T cells early in the inflammatory pathway will lead to immunomodulation rather than immunosuppression. The evidence produced so far suggests that abatacept will be a useful addition to the available therapies for patients with RA.

Phosphorylation of the LFA-1 Integrin β2-Chain on Thr-758 Leads to Adhesion, Rac-1/Cdc42 Activation, and Stimulation of CD69 Expression in Human T Cells

Phosphorylation of the leukocyte function-associated antigen-1 (LFA-1) integrin beta2-chain on Thr-758 occurs after T cell receptor stimulation and leads to 14-3-3 recruitment to the integrin, actin cytoskeleton reorganization, and increased adhesion. Here, we have investigated the signaling effects of beta2 integrin Thr-758 phosphorylation. A penetratin-coupled phospho-Thr-758-beta2 peptide (mimicking the part of the integrin beta-chain surrounding Thr-758) stimulated adhesion of human T cells to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1). Additionally, the peptide activated the small GTPases Rac-1 and Cdc42 in T cells. Constitutively active forms of Rac-1 and Cdc42, but not Rho, could compensate for the reduction of cell adhesion to ICAM-1 caused by the T758A mutation in the beta2 integrin. Additionally, the active GTPases salvaged the cell-spreading defect of T758A integrin-transfected cells on coated ICAM-1. A dominant negative form of Cdc42, on the other hand, significantly reduced wild-type beta2 integrin-mediated cell adhesion and spreading. In a T cell stimulation system, the pThr-758 penetratin peptide acted in a similar manner to coated ICAM-1 to increase T cell receptor-induced CD69 expression. These results show that Thr-758-phosphorylated LFA-1 is upstream of Rac-1/Cdc42, cell adhesion, and costimulatory activation of human T cells, thus identifying phosphorylation of Thr-758 in beta2 as a proximal element in LFA-1 signaling.

Role for dendritic cells in immunoregulation during experimental vaginal candidiasis.

Vulvovaginal candidiasis (VVC) caused by the commensal organism Candida albicans remains a significant problem among women of childbearing age, with protection against and susceptibility to infection still poorly understood. While cell-mediated immunity by CD4+ Th1-type cells is protective against most forms of mucosal candidiasis, no protective role for adaptive immunity has been identified against VVC. This is postulated to be due to immunoregulation that prohibits a more profound Candida-specific CD4+ T-cell response against infection. The purpose of this study was to examine the role of dendritic cells (DCs) in the induction phase of the immune response as a means to understand the initiation of the immunoregulatory events. Immunostaining of DCs in sectioned murine lymph nodes draining the vagina revealed a profound cellular reorganization with DCs becoming concentrated in the T-cell zone throughout the course of experimental vaginal Candida infection consistent with cell-mediated immune responsiveness. However, analysis of draining lymph node DC subsets revealed a predominance of immunoregulation-associated CD11c+ B220+ plasmacytoid DCs (pDCs) under both uninfected and infected conditions. Staining of vaginal DCs showed the presence of both DEC-205+ and pDCs, with extension of dendrites into the vaginal lumen of infected mice in close contact with Candida. Flow cytometric analysis of draining lymph node DC costimulatory molecules and activation markers from infected mice indicated a lack of upregulation of major histocompatibility complex class II, CD80, CD86, and CD40 during infection, consistent with a tolerizing condition. Together, the results suggest that DCs are involved in the immunoregulatory events manifested during a vaginal Candida infection and potentially through the action of pDCs.

Differential responses of cord and adult blood‐derived dendritic cells to dying cells

Normal turnover of body tissues yields apoptotic cells while infections cause tissue injuries and cell necrosis. The interaction of these dying cells with dendritic cells (DCs) may provide immunological instructions leading to either immune tolerance or activation. We hypothesize that neonatal and adult DCs differ in their responses to dying cells, thereby contributing to the observed differences in immune responses between neonates and adults. We compare the outcome of interaction of cord and adult blood-derived DCs with dying Epstein-Barr-virus-transformed lymphoblastoid cells (LCLs) and the responsiveness to lipopolysaccharide. While cord DCs were able to phagocytose both apoptotic and necrotic LCLs, the subsequent responses differed significantly from those of adult DCs. Interaction of adult DCs with necrotic but not early apoptotic LCLs resulted in high expression of DC costimulatory molecules (CD80/CD86) and activation markers (CD83), production of both proinflammatory and anti-inflammatory cytokines (tumour necrosis factor-alpha, interleukin-10), and strong T-cell-stimulating activities. In contrast, in response to either necrotic or apoptotic LCLs, cord DCs had minimal up-regulation of those DC functional markers, little cytokine production and poor stimulation on T-cell proliferation. In response to lipopolysaccharide, however, both adult and cord DCs produced comparable levels of tumour necrosis factor-alpha and interleukin-10, but only adult DCs produced interleukin-12(p70). Taken together, these results suggest that neonatal DCs generally favour immune tolerance with minimal activation and cytokine production, except in extremely dangerous situations, such as bacterial sepsis, when neonatal DCs may produce certain types of cytokines and stimulate T-cell proliferation.

Hepatitis C Virus Core Protein Induces an Anergic State Characterized by Decreased Interleukin-2 Production and Perturbation of Mitogen-Activated Protein Kinase Responses

Alterations of cytokine responses are thought to favor the establishment of persistent hepatitis C virus (HCV) infections, enhancing the risk of liver cirrhosis and hepatocellular carcinoma. Here we demonstrate that the expression of the HCV core (C) protein in stably transfected T cells correlates with a selective reduction of interleukin-2 (IL-2) promoter activity and IL-2 production in response to T-cell receptor triggering, whereas the activation of IL-4, IL-10, gamma interferon, and tumor necrosis factor alpha was moderately increased. This altered cytokine expression profile was associated with a perturbation of mitogen-activated protein (MAP) kinase responses. Extracellular regulated kinase and p38 were constitutively phosphorylated in C-expressing cells, while triggering of the costimulatory c-Jun N-terminal kinase (JNK) signaling cascade and activation of the CD28 response element within the IL-2 promoter appeared to be impaired. The perturbations of MAP kinase phosphorylation could be eliminated by cyclosporine A-mediated inhibition of nuclear factor of activated T cells, suggesting that the inactivation of JNK signaling and hyporesponsiveness to IL-2 induction were downstream consequences of C-induced Ca(2+) flux in a manner that mimics the induction of clonal anergy.

Kidney Transplant Patients with Long-Term Graft Survival Have Altered Expression of Molecules Associated with T-Cell Activation

Patients with long-term functioning organ allografts may have developed different mechanisms that explain the lack of graft rejection. However, it is not known how these mechanisms interplay or whether one of them predominates in such situations. The authors analyzed the expression of T-cell surface molecules involved in alloantigen recognition, signal transduction, co-stimulation, and activation markers on circulating T cells from patients with normal kidney function 10 or more years after transplantation, short-term survival (1-4 years), and chronic rejection and from healthy adults. The percentage and the median fluorescent intensity of each marker were determined by flow cytometry. Proliferative response against specific and third-party donors and mitogenic stimulation were also determined. Peripheral blood lymphocytes from patients with long-term surviving kidneys had decreased expression of T-cell receptor (TCR)-alphabeta (P < 0.01), CD3epsilon (P < 0.05), and CD3 zeta-chains (P < 0.001); diminished percentages of CD4(+)CD28(+) (P < 0.001) cells; and increased expression of CTLA-4(+) (P < 0.01) on CD3(+) cells. CD4(+)CD25(+)CD69(+) cells were also increased in long-term surviving patients. Long-term surviving patients had decreased donor-specific proliferative responses. The decreased expression of TCR-alphabeta and epsilon- and zeta-chains on circulating T cells of long-term surviving patients suggests that these cells may have defects in alloantigen recognition or signal transduction that may result in decreased numbers of T cells expressing co-stimulatory molecules and activation markers as well as a decreased specific proliferative response. The decrease in the percentage of CD28(+) cells and the increase in CD4(+)CD25(+)CD69(+) cells suggest that regulatory mechanisms of the immune response are still active in such patients.

Differential expression pattern and functioning of CD80 and CD86 in Th1 and Th2 immune responses induced by immunogenic drugs

Rationale Immunogenic drugs, of which some are known to induce hypersensitivity reactions in man, may induce Th1- or Th2-associated immune responses. Here, we study the kinetics of the expression patterns and the functional importance of costimulatory activation markers in drug-induced Th1 or Th2 immune responses. Methods Data was acquired with the popliteal lymph node assay (PLNA) using bystander antigens. BALB/c mice were s.c. injected with Streptozotocin (STZ; inducing Th1 responses) or D-Penicillamine (D-Pen; inducing Th2 responses) in the hind footpad and some groups received additional i.p. treatment with anti-CD80, anti-CD86 or a combination of both MoAb. Additionally, we exposed CD80/CD86-deficient mice to either D-Pen or STZ. We measured the numbers of Ab secreting cells, production of IL-4, IFN-γ and TNF-α, and shifts in cell subtypes. Results In Th2 responses the expression of CD86, but not CD80, was increased and abrogation of the CD80/CD86 pathway inhibited the activation of lymphocytes, IL-4, IFN-γ, IgM and IgG1 secretion. In the Th1 response, both CD80 and CD86 were increased and abrogation of CD80/CD86 signaling caused no change in macrophage numbers and levels of IgG2a and IFN-γ, and in fact increased TNF-α and IgM secretion. Conclusions Th1- and Th2-inducing immunogenic drugs give rise to distinct profiles of cytokines, shifts in cell subtypes and expression patterns of CD80 and CD86. Additionally, ligation of CD80 and CD86 is indisputably required in the induction of D-Pen-induced Th2 responses, but seems to inhibit STZ-induced Th1 phenomena.

Purification of recombinant and endogenous HSP70s

Heat shock poteins (HSPs) are powerful immunogens against the antigenic peptides they chaperone [1]. The antigenic peptides are MHC I-binding peptides and their elongated precursors derived from tumor antigens, viral antigens, minor histocompatibility antigens, or model antigens [2–6]. HSP–peptide complexes can immunize against tumors and pathogen-infected cells [7–9]. Remarkably, HSPs do not immunize after elution of the peptides they chaperone, demonstrating that HSPs are not immunogenic per se, whereas HSP–peptide complexes are [6,10]. Additionally, HSPs activate professional antigen presenting cells (APC) through specific receptor(s) to stimulate secretion of pro-inflammatory cytokines, up-regulation of co-stimulatory molecules and activation of dendritic cells [11–15]. The mechanistic exploration of the role of the HSPs on the innate and adaptive component of the immune system requires their isolation in large quantity. On one hand, isolation of naturally formed HSP–peptide complexes is key to study their specific immunogenicity. On the other hand, purification of HSPs free of endotoxin contamination is an absolute requirement for the analysis of their ability to activate APC in vitro [11]. This chapter describes a convenient and fast method of purification of endogenous and recombinant HSP of 70 kDa (HSP70) that addresses these two considerations.

Gene transfer of cytoprotective and immunomodulatory molecules for prevention of cardiac allograft rejection.

Current treatments of heart transplantation are limited by incomplete effectiveness, significant toxicity, and failure to prevent chronic rejection. Genetic manipulation of the donor heart at the time of removal offers the unique opportunity to produce a therapeutic molecule within the graft itself, while minimizing systemic effects. Cytoprotective approaches including gene transfer of heme oxygenase (HO)-1, endothelial nitric oxide synthase, and antisense oligodeoxynucleotides specific for nuclear factor (NF)-kappa B or intercellular adhesion molecule (ICAM)-1 reduced ischaemia-reperfusion injury and delayed cardiac allograft rejection in small animals. Exogenous overexpression of immunomodulatory cytokines such as interleukin (IL)-4, IL-10 and transforming growth factor-beta, as well as gene transfer of inhibitors of pro-inflammatory cytokines also delayed graft rejection. Gene transfer-based blockade of T-cell costimulatory activation with CTLA4-Ig or CD40-Ig resulted in long-lasting graft survival and donor-specific unresponsiveness, as manifested by acceptance of a second graft from the original donor strain but rejection of third-party grafts. Similar results were obtained with donor major histocompatibility complex class I gene transfer into bone marrow cells. Gene therapy approaches to chronic rejection included gene transfer of HO-1, soluble Fas, tissue plasminogen activator and antisense oligodeoxynucleotides specific for the anti-apoptotic mediator Bcl-x or the E2F transcription factor. Despite major experimental advances, however, gene therapy for heart transplantation has not entered the clinical arena yet. Fundamental questions regarding the most suitable vector, the best gene, and safety issues remain unanswered. Well-controlled studies that compare gene therapy with established treatments in non-human primates are needed before clinical trials can be started.

Selective stimulation of collagen synthesis in the presence of costimulatory insulin signaling by connective tissue growth factor in scleroderma fibroblasts.

To examine the mechanism of collagen induction by connective tissue growth factor (CTGF), a profibrotic cytokine overexpressed in the skin of patients with systemic sclerosis (SSc). Dermal fibroblasts from 7 SSc patients and 7 matched healthy adult donors were stimulated with CTGF in the presence or absence of the culture-medium supplement, insulin-transferrin-selenium (ITS). Expression of collagen protein was analyzed by a (3)H-proline incorporation assay. To identify the signaling pathways mediating CTGF induction of collagen, pharmacologic inhibitors were used, including rottlerin, a protein kinase C delta (PKC delta) inhibitor. Collagen levels in both SSc and normal fibroblasts were increased after treatment with transforming growth factor beta in serum-free medium, whereas no stimulation was observed following addition of CTGF. In the presence of ITS, CTGF (2.5 ng/ml) potently stimulated collagenous protein levels in SSc cell lines (n = 5); however, CTGF was not stimulatory in the majority of normal fibroblasts (n = 6). ITS alone induced collagen levels in normal fibroblasts to the levels observed in SSc skin fibroblasts, thereby diminishing the hallmark difference in basal collagen levels in these cell types. Insulin was the ITS component responsible for promoting the basal and CTGF stimulation of collagenous proteins. Rottlerin, the PKC delta inhibitor, down-regulated collagen synthesis in normal and SSc fibroblasts cultured in ITS, and inhibited the stimulatory effects of CTGF in cooperation with insulin or of insulin (500 ng/ml) alone. Increased responsiveness of SSc fibroblasts to CTGF-mediated collagen synthesis requires the costimulatory activation of insulin signaling pathways to induce matrix production. Blockade of this effect via rottlerin may suggest that PKC delta is a downstream signaling molecule necessary for CTGF stimulation of collagen synthesis.

New insights into the interactions between T-cell costimulatory blockade and conventional immunosuppressive drugs.

To determine the precise in vivo interaction between T-cell costimulatory blockade and conventional immunosuppression in transplantation. Blocking B7 or CD154 T-cell costimulatory activation pathways prevents allograft rejection in small and large animal transplant models and is considered a promising strategy for clinical organ transplantation. A fully MHC-mismatched vascularized mouse cardiac allograft model was used to test the interactions between anti-CD154 or CTLA4Ig monotherapy and conventional immunosuppressive drugs in promoting long-term graft acceptance. The frequency of alloreactive T cell was measured by ELISPOT. Chronic rejection was examined by histology. Cyclosporine, tacrolimus, and anti-IL-2R monoclonal antibody therapy abrogated the effect of a single-dose protocol of anti-CD154 therapy. In contrast, rapamycin acted synergistically with anti-CD154 therapy in promoting long-term allograft survival. The addition of calcineurin inhibitors did not abolish this synergistic effect. Intense CD154-CD40 blockade by a multiple-dose schedule of anti-CD154 resulted in long-term graft survival and profound alloreactive T-cell unresponsiveness and overcame the opposite effects of calcineurin inhibitors. CTLA4Ig induced long-term graft survival, and the effect was not affected by the concomitant use of any immunosuppressive drugs. The widespread view that calcineurin inhibitors abrogate the effects of T-cell costimulatory blockade should be revisited. Sufficient costimulatory blockade and synergy induced by CD154 blockade and rapamycin promote allograft tolerance and prevent chronic rejection.

Modulation of Th2 type cytokine production from human peripheral blood leukocytes by a macrolide antibiotic, roxithromycin, in vitro

The influence of a macrolide antibiotic, roxithromycin (RXM), on Th1 and Th2 cytokine productions from human peripheral blood T cells was examined under stimulation with co-stimulatory molecules. Peripheral blood T cells prepared from both healthy and allergic rhinitis donors were cultured in the presence of RXM on anti-CD3 mAb and anti-CD26 mAb-coated wells, anti-CD3 mAb and anti-CD28 mAb-coated wells, and anti-CD3 and PMA. T-cell proliferation, along with the concentration of interleukin (IL)-2, interferon (IFN)-γ, IL-4 and IL-5 were measured. RXM did not affect T-cell proliferation induced by several ways of co-stimulatory activation as assessed by 3H-thymidine incorporation into DNA. RXM also had no effect on IL-2 and IFN-γ secretion by T cells prepared from both healthy and allergic rhinitis donors. On the other hand, RXM markedly inhibited both IL-4 and IL-5 secretions under each of the co-stimulatory conditions in a dose-dependent manner. These results indicate that RXM inhibits specifically Th2 cytokine secretion from T cells induced by co-stimulatory molecule stimulations. This inhibitory action of RXM may be partially responsible for attenuating effect of the agent on the inflammatory diseases.

T cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3.

Agents that increase intracellular cAMP inhibit the activation and function of T cells and can lead to cell death. Recently, it has been postulated that cAMP inhibits T cell function in large part by acting as a brake on the T cell receptor and costimulatory receptor pathways. Therefore, for full activation of the T cell to occur, this inhibitory influence must be removed. One likely mechanism for accomplishing this is by up-regulation and/or activation of specific cyclic nucleotide phosphodiesterases (PDEs), and such a mechanism for one phosphodiesterase, PDE7A1, has been reported. In this paper, we extend this mechanism to another isozyme variant of the same PDE family, PDE7A3. We also report the full-length sequence of human PDE8A1 and show that it also is induced in response to a combination of T cell receptor and costimulatory receptor pathway activation. However, the time course for induction of PDE8A1 is slower than that of PDE7A1. The basal level measured and, therefore, the apparent fold induction of PDE7A1 mRNA and protein depend in large part on the method of isolation of the T cells. On the other hand, regardless of the isolation method, the basal levels of PDE7A3 and PDE8A1 are very low and fold activation is much higher. Constitutively expressed PDE8A1 and PDE7A3 also have been isolated from a human T cell line, Hut78.

Specific inhibition of TH2-type cytokine production from human peripheral T cells by terfenadine in vitro.

Cytokine imbalance is thought to be one of the causes for allergic diseases. The effect of anti-allergic drugs on cytokine production from T cells should be examined in a convenient way. To study the in vitro effect of terfenadine, a prototype non-sedating H1 receptor antagonist, on cytokine production from activated T cells. T cells were cultured in the presence of terfenadine on anti-CD3 mAb and anti-CD26 mAb-coated wells, anti-CD3 mAb and anti-CD28 mAb-coated wells, and anti-CD3 mAb wells with PMA. T-cell proliferation, along with the concentrations of interleukin (IL) -2, interferon (IFN) -gamma, IL-4, and IL-5 were measured. Terfenadine inhibited T-cell proliferation and IL-4 and IL-5 production under each costimulatory condition tested, whereas it had no effect on IL-2 and IFN-gamma production. These results indicate that terfenadine has a specific inhibitory effect on TH2-type cytokine production induced by several ways of costimulatory activation.

Finally, CTLA4Ig graduates to the clinic.

Finally, CTLA4Ig graduates to the clinic.

RAT CD28 SPECIFIC MONOCLONAL ANTIBODY: ITS POTENTIAL ROLE IN ACTIVATION INDUCED CELL DEATH AND PREVENTION OF ACUTE VASCULARIZED CARDIAC ALLOGRAFT REJECTION

501 Full activation of T cells requires two distinct signals. Signal one is provided by the engagement of the TCR with the MHC + antigenic peptide complex, while signal two is provided by the interaction of specific T cell costimulatory molecules with their ligands on antigen presenting cells. In particular, the CD28-B7 pathway plays a critical role in T cell costimulatory activation. Numerous studies indicate that B7 blockade by CTLA4Ig (which binds B7-1 and B7-2) or by anti-B7 monoclonal antibodies prevents allograft rejection and induces donor specific tolerance in some experimental models. The exact mechanisms of CD28-B7 blockade in vivo remain unclear, although anergy, deletion and a regulatory Th2 switch have all been proposed. More recently, studies in IL-2 gene knockout mice suggest that activation induced cell death (AICD) may be an important mechanism of action of B7 blockade in vivo. JJ319, a rat anti-CD28 specific monoclonal antibody, is known to costimulate T cell proliferation effectively in resting rat T cells, leading to subsequent IL-2 secretion (J Immunol 1995 154: 5121-5127). When it was added to a LEW×WF rat MLR culture, increased lymphocyte proliferation in vitro was noted. We then tested the effect of this antibody in the WF into LEW vascularized cardiac allograft model. LEW recipients received a single 0.5 mg iv injection of the antibody on day 0 or day 2 post-transplant. Graft survival is shown below: (Table)TableInterestingly, these findings demonstrate that administration of a single injection of a signaling anti-CD28 monoclonal antibody prevents acute allograft rejection and prolongs graft survival. Day 0 administration appears to be more effective than day 2. These observations suggest that CD28 costimulation may be responsible for enhanced IL-2 production. Since IL-2 is known to promote Fas-mediated activation induced cell death, this may explain the prolongation of graft survival. Understanding the mechanisms of action of signaling anti-CD28 monoclonal antibodies in vivo may lead to the development of novel strategies that induce tolerance through specific deletion of alloreactive T cells in the periphery.

Analysis of Raf-1 Activation in Response to TCR Activation and Costimulation in Murine T-Lymphocytes: Effect of Age

Stimulation of the ERK (MAPK) pathway in T-lymphocytes contributes to cell activation and IL-2 production. The ERK pathway is initiated by the activation of the serine/threonine kinase Raf-1 in a Ras-dependent manner. Raf-1 activates the dual-specific kinase MEK, which in turn activates ERK. To see if aging leads to an alteration of Raf-1 kinase activity we performedin vitrokinase assays on Raf-1 isolated from CD4+T-cells from young and old mice. We found an age-related impairment in the kinase activity of Raf-1 in T-cells stimulated by a combination of antibodies to the CD3ϵ chain of the T-cell receptor and CD4. Aging led to a two- to fourfold decline in Raf-1 activity (depending on the stimulation time) without a change in the kinetics of enzyme activation. We also found that Raf-1 activation by CD3/CD4 costimulation is lower in memory cells than in naı̈ve cells from mice of the same age. However, aging also leads to a decline in Raf-1 activity in the naı̈ve subset of CD4+T-cells, suggesting that two mechanisms lead to the age related decline in Raf-1 function. Finally, we found that antibodies to the costimulatory molecule CD28 trigger Raf-1 activation and enhance anti-CD3-mediated Raf-1 activation but cannot restore Raf-1 activation levels from old T-cells to those seen in young mice. Our data suggest that age-dependent declines in T-cell ERK function are caused by alterations in the signals that activate Raf-1 and that age-dependent defects in T-cell cytokine production and proliferation may be caused at least in part by defects in signals that activate Raf-1.