B7/CD28 costimulation of T cells induces a distinct proteome pattern

Effective immune strategies for the eradication of human tumors require a detailed understanding of the interaction of tumor cells with the immune system, which might lead to an optimization of T cell responses. To understand the impact of B7-mediated costimulation on T cell activation comprehensive proteome analysis of B7-primed T cell populations were performed. Using this approach we identified different classes of proteins in T cells whose expression is either elevated or reduced upon B7-1- or B7-2-mediated CD28 costimulation. The altered proteins include regulators of the cell cycle and cell proliferation, signal transducers, components of the antigen processing machinery, transporters, cytoskeletal proteins, and metabolic enzymes. A number of differentially expressed proteins are further modified by phosphorylation. Our results provide novel insights into the complexity of the CD28 costimulatory pathway of T cells and will help to identify potential targets of therapeutic interventions for modulating anti-tumor T cell activation.     

Rho kinase promotes alloimmune responses by regulating the proliferation and structure of T cells

Coordinated rearrangements of the actin-myosin cytoskeleton facilitate early and late events in T cell activation and signal transduction. As many important features of cell shape rearrangement involve small GTP-binding proteins, we examined the contribution of Rho kinase to the functions of mature T cells. Inhibitors of the Rho kinase pathway all had similar actions to inhibit the proliferation of primary lymphocyte cultures. Likewise, transfection of the human Jurkat T cell line with a dominant negative, kinase-defective mutant of Rho kinase diminished Jurkat cell proliferation. Furthermore, inhibition of Rho kinase substantially attenuated the program of cytokine gene expression that characterizes T cell activation, blocked actomyosin polymerization, and prevented aggregation of the TCR/CD3 complex colocalized with lipid rafts. These actions are relevant to immune responses in vivo, as treatment with a Rho kinase inhibitor considerably prolonged the survival of fully allogeneic heart transplants in mice and diminished intragraft expression of cytokine mRNAs. Thus, Rho GTPases acting through Rho kinase play a unique role in T cell activation during cellular immune responses by promoting structural rearrangements that are critical for T cell signaling.     

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.     

LFA-1-mediated costimulation of CD8+ T cell proliferation requires phosphatidylinositol 3-kinase activity

LFA-1 binding to ICAM-I provides a costimulatory signal for CD8(+) T cell activation that results in increased IL-2 mRNA levels and protein production to support proliferation. CD28 binding to its B7 ligands has the same effect, and the two costimulatory receptors activate some of the same intracellular signaling events, including up-regulation of phosphatidylinositol (PI) 3-kinase activity. However, costimulation by LFA-1 depends upon the activity of this enzyme, whereas costimulation by CD28 does not, as evidenced by differential effects of specific inhibitors of PI 3-kinase. When cells are costimulated with ICAM-1 in the presence of the inhibitors wortmannin or LY294002, proliferation is blocked, but increases in IL-2 mRNA levels and protein production are not. Costimulation also results in increased surface expression of CD25, which is essential for formation of an active IL-2R. This is blocked by the PI 3-kinase inhibitors when costimulation is via LFA-1 but not when it is via CD28. Finally, IL-2-driven proliferation is not blocked by the inhibitors once CD25 surface expression has increased. Thus, the PI 3-kinase-dependent step in CD8 T cell costimulation by LFA-1 is up-regulation of IL-2R expression. In contrast, CD28 engagement also increases IL-2R surface expression, but the up-regulation does not depend upon PI 3-kinase activity.     

Lipid raft assembly and Lck recruitment in TRAIL costimulation mediates NF-κB activation and T cell proliferation

The TNF-related apoptosis-inducing ligand was shown to provide a costimulatory signal that cooperates with the TCR/CD3 complex to induce T cell proliferation and cytokine production. Although a number of signaling pathways were linked to the TCR/CD3 complex, it is not known how these two receptors cooperate to induce T cell activation. In this study, we show that TRAIL-induced costimulation of T cells depends on activation of the NF-κB pathway. TRAIL induced the NF-κB pathway by phosphorylation of inhibitor of κB factor kinase and protein kinase C in conjunction with anti-CD3. Furthermore, we demonstrated that TRAIL costimulation induced phosphorylation of the upstream TCR-proximal tyrosine kinases, Lck and ZAP70. Ligation of the TRAIL by its soluble receptor, DR4-Fc, alone was able to induce the phosphorylation of Lck and ZAP70 and to activate the NF-κB pathway; however, it was insufficient to fully activate T cells to support T cell proliferation. In contrast, TRAIL engagement in conjunction with anti-CD3, but not TRAIL ligation alone, induced lipid raft assembly and recruitment of Lck and PKC. These results demonstrate that TRAIL costimulation mediates NF-κB activation and T cell proliferation by lipid raft assembly and recruitment of Lck. Our results suggest that in TRAIL costimulation, lipid raft recruitment of Lck integrates mitogenic NF-κB-dependent signals from the TCR and TRAIL in T lymphocytes.     

EC144, a synthetic inhibitor of heat shock protein 90, blocks innate and adaptive immune responses in models of inflammation and autoimmunity

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b(+) peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4(+) T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4(+) T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.     

Glucocorticoid-induced TNFR-related protein lowers the threshold of CD28 costimulation in CD8+ T cells

CD28 is well characterized as a costimulatory molecule in T cell activation. Recent evidences indicate that TNFR superfamily members, including glucocorticoid-induced TNFR-related protein (GITR), act as costimulatory molecules. In this study, the relationship between GITR and CD28 has been investigated in murine CD8(+) T cells. When suboptimal doses of anti-CD3 Ab were used, the absence of GITR lowered CD28-induced activation in these cells whereas the lack of CD28 did not affect the response of CD8(+) T cells to GITR costimulus. In fact, costimulation of CD28 in anti-CD3-activated GITR(-/-) CD8(+) T cells resulted in an impaired increase of proliferation, impaired protection from apoptosis, and an impaired rise of activation molecules such as IL-2R, IL-2, and IFN-gamma. Most notably, CD28-costimulated GITR(-/-) CD8(+) T cells revealed lower NF-kappaB activation. As a consequence, up-regulation of Bcl-x(L), one of the major target proteins of CD28-dependent NF-kappaB activation, was defective in costimulated GITR(-/-) CD8(+) T cells. What contributed to the response to CD28 ligation in CD8(+) T cells was the early up-regulation of GITR ligand on the same cells, the effect of which was blocked by the addition of a recombinant GITR-Fc protein. Our results indicate that GITR influences CD8(+) T cell response to CD28 costimulation, lowering the threshold of CD8(+) T cell activation.     

Effective proliferation of human regulatory T cells requires a strong costimulatory CD28 signal that cannot be substituted by IL-2

The strength of immune repression by regulatory T (Treg) cells is thought to depend on the efficiency of Treg cell activation. The stimuli and their individual strength required to activate resting human Treg cells, however, have so far not been elucidated in detail. We reveal here that induction of proliferation of human CD4(+)C25(+) Treg cells requires an extraordinary strong CD28 costimulatory signal in addition to TCR/CD3 engagement. CD28 costimulation, noteworthy, cannot be substituted by IL-2 to induce proliferation of Treg cells, which is in contrast to CD4(+)CD25(-) T cells. IL-2, in contrast, prevents spontaneous apoptosis of Treg cells, but does not initiate their amplification. IL-2 and CD28 costimulation clearly exhibit disparate effects on Treg cells which are in contrast to those on CD4(+)CD25(-) T cells. Moreover, the prerequisites for Treg cell proliferation differ strikingly from those for effector T cells, implying a balanced orchestration in initiating and limiting a T cell immune response. In addition, data are of relevance for the design of therapeutic strategies involving IL-2 administration and CD28 costimulation.     

Thy-1 signaling in the context of costimulation provided by dendritic cells provides signal 1 for T cell proliferation and cytotoxic effector molecule expression,but fails to trigger delivery of the lethal hit

Cross-linking of the GPI-anchored protein Thy-1 results in T cell proliferation and IL-2 synthesis. However, the exact function of Thy-1 in the process of T cell activation remains unknown, as does the effect of costimulation on Thy-1-driven T cell responses. In this study, we have investigated the ability of Thy-1 to substitute for traditional signal 1 in the context of costimulation provided by dendritic cells. Dendritic cells dramatically enhanced T cell proliferation and IL-2 synthesis in response to Thy-1 triggering by anti-Thy-1 mAb. This effect was not dependent on dendritic cell Fcgamma receptors, but was a result of B7-mediated costimulation (signal 2). T cells were also activated when microbeads coated with a combination of anti-Thy-1 and anti-CD28 mAbs were used to supply signals 1 and 2, respectively. Thy-1-stimulated T cells adhere to target cells and express perforin, granzyme B, and Fas ligand, but fail to kill target cells due to an inability to reorganize their secretion machinery. Moreover, in contrast to TCR signaling, Thy-1 triggering failed to induce cytotoxicity in redirected lysis assays. We conclude that Thy-1 triggering can partially substitute for signal 1, which, in combination with a strong signal 2, leads to robust T cell proliferation, IL-2 synthesis, and cytotoxic effector molecule expression, but does not induce cytolytic function. The block at the level of cytotoxic effector function that results when T cells are activated in the absence of a classical, Ag-specific signal 1 may constitute a mechanism to ensure the specificity of CTL responses and prevent potentially harmful promiscuous cytotoxicity.     

Intercellular adhesion molecule 1 is critical for activation of CD28-deficient T cells

Presentation of Ag to T lymphocytes in the absence of the requisite costimulatory signals leads to an Ag-specific unresponsiveness termed anergy, whereas Ag presentation in conjunction with costimulation leads to clonal expansion. B7/CD28 signaling has been shown to provide this critical costimulatory signal and blockade of this pathway may inhibit in vitro and in vivo immune responses. Although T cells from CD28-deficient mice are lacking in a variety of responses, they nonetheless are capable of various primary and secondary responses without the induction of anergy expected in the absence of costimulation. This suggests that there may be alternative costimulatory pathways that can replace CD28 signaling under certain circumstances. In this paper, we show that ICAM-1becomes a dominant costimulatory molecule for CD28-deficient T cells. ICAM-1 costimulates anti-CD3-mediated T cell proliferation and IL-2 secretion in CD28-deficient murine T cells. Furthermore, splenocytes from ICAM-1-deficient mice could not activate CD28-deficient T cells and splenocytes lacking both ICAM and CD28 fail to proliferate in response to anti-CD3-induced T cell signals. This confirms that not only can ICAM-1 act as a CD28-independent costimulator, but it is the dominant, requisite costimulatory molecule for the activation of T cells in the absence of B7/CD28 costimulation.     

CD99 costimulation up-regulates T cell receptor-mediated activation of JNK and AP-1

Although CD28 is the principal T cell costimulatory molecule for the T cell receptor, a number of other cell surface proteins have costimulatory functions and perform specific roles in different contexts. Here we analyzed the mechanism of CD99 costimulation of the T cell receptor. Cooperation of CD99 engagement with suboptimal TCR/CD3 signals resulted in greatly enhanced CD4+ T cell proliferation. CD99 costimulation also led to elevated expression of CD25 and GM1 on the CD4+ T cell surface within 3 days. In Jurkat TAg cells, CD99 costimulation led to increased apoptosis compared to stimulation with CD3 or CD99 alone. CD99 costimulation also augmented activation of MAP kinases, especially of JNK, and increased AP-1 activation was also observed using a luciferase reporter assay. These results show that CD99 has a costimulatory function for T cells and acts by a mechanism distinct from CD28.     

Differential requirements of na?ve and memory T cells for CD28 costimulation in autoimmune pathogenesis.

Experimental autoimmune encephalomyelitis (EAE) is the most extensively studied animal model of the human disease multiple sclerosis (MS). In EAE, CNS demyelination is induced by immunization with myelin proteins or adoptive transfer of myelin-reactive CD4+ T cells. Since the antigen specificity of the immune response believed to be responsible for the pathology of MS is not well defined, therapies that target aspects of T cell activation that are not antigen specific may be more applicable to the treatment of MS. As a result, understanding the role of costimulatory molecules in the activation of na?ve and memory T cells has become an area of extensive investigation. Na?ve T cells require two signals for activation. Signal one is provided by engagement of the T cell receptor (TCR) with MHC/peptide complexes and provides antigen specificity to the immune response. The second signal, termed costimulation, is usually provided by B7 molecules on APC to CD28 molecules expressed on T cells and is antigen-independent. This review will discuss our current understanding of costimulation in the induction and perpetuation of EAE, as well as the potential of costimulation blockade in the treatment of MS.     

Functional analysis of B and T lymphocyte attenuator engagement on CD4+ and CD8+ T cells

T cell activation can be profoundly altered by coinhibitory and costimulatory molecules. B and T lymphocyte attenuator (BTLA) is a recently identified inhibitory Ig superfamily cell surface protein found on lymphocytes and APC. In this study we analyze the effects of an agonistic anti-BTLA mAb, PK18, on TCR-mediated T cell activation. Unlike many other allele-specific anti-BTLA mAb we have generated, PK18 inhibits anti-CD3-mediated CD4+ T cell proliferation. This inhibition is not dependent on regulatory T cells, nor does the Ab induce apoptosis. Inhibition of T cell proliferation correlates with a profound reduction in IL-2 secretion, although this is not the sole cause of the block of cell proliferation. In contrast, PK18 has no effect on induction of the early activation marker CD69. PK18 also significantly inhibits, but does not ablate, IL-2 secretion in the presence of costimulation as well as reduces T cell proliferation under limiting conditions of activation in the presence of costimulation. Similarly, PK18 inhibits Ag-specific T cell responses in culture. Interestingly, PK18 is capable of delivering an inhibitory signal as late as 16 h after the initiation of T cell activation. CD8+ T cells are significantly less sensitive to the inhibitory effects of PK18. Overall, BTLA adds to the growing list of cell surface proteins that are potential targets to down-modulate T cell function.     

Costimulation of resting B lymphocytes alters the IL-4-activated IRS2 signaling pathway in a STAT6 independent manner: implications for cell survival and proliferation

INTRODUCTIONThe inappropriate enhancement of lymphocytesurviVal due to a block in programmed cell deathand/or an enhancement of entry into the cell cyclecan contribute to the abnormal expansion of clonesresulting in tumorigenesis or the breakdown of pe-ripheral self-toleranced, 2]. Proper lymphocytehomeostasis is critical for normal immune functionand is maintained by a complex series of cellularinteractions and the action of secreted cytokines.Illterleukin-4 (IL-4), a cytokine produced …     

Costimulation of resting B lymphocytes alters the IL－4－activated IRS2 signaling pathway in a STAT6 independent manner：implications for cell survival and proliferation

IL-4 is an important B cell survival and growth factor. IL-4 induced the tyrosine phosphorylation of IRS2 in resting B lymphocytes and in LPS- or CD40L-activated blasts. Phosphorylated IRS2 coprecipitated with the p85 subunit of PI 3' kinase in both resting and activated cells. By contrast, association of phosphorylated IRS2 with GRB2 was not detected in resting B cells after IL-4 treatment although both proteins were expressed. However, IL-4 induced association of IRS2 with GRB2 in B cell blasts. The pattern of IL-4-induced recruitment of p85 and GRB2 to IRS2 observed in B cells derived from STAT6 null mice was identical to that observed for normal mice. While IL-4 alone does not induce activation of MEK, a MEK1 inhibitor suppressed the IL-4-induced proliferative response of LPS-activated B cell blasts. These results demonstrate that costimulation of splenic B cells alters IL-4-induced signal transduction independent of STAT6 leading to proliferation. Furthermore, proliferation induced by IL-4 in LPS-activated blasts is dependent upon the MAP kinase pathway.     

Lipopolysaccharide-induced NF-kappa B activation in mouse 70Z/3 pre-B lymphocytes is inhibited by mevinolin and 5''-methylthioadenosine: roles of protein isoprenylation and carboxyl methylation reactions.

We show that both the lipopolysaccharide (LPS)-induced activation of NF-kappa DNA binding and kappa gene expression are blocked by treating murine pre-B lymphocyte 70Z/3 cells with 5'-methylthioadenosine (MTA), an inhibitor of several S-adenosylmethionine-dependent methylation reactions. We further show that the LPS-induced incorporation of radioactivity from [methyl-3H]methionine into methyl ester-like linkages on a group of membrane polypeptides is also inhibited by MTA treatment, suggesting the involvement of protein methylation reactions in the LPS signal transduction pathway. We also find that NF-kappa B and kappa gene activation in LPS-treated 70Z/3 cells is blocked by mevinolin, an inhibitor that prevents protein isoprenylation. Interestingly, mevinolin-treated cells also exhibited a marked reduction in the methylation of membrane proteins. Neither MTA nor mevinolin significantly inhibited NF-kappa B activation by phorbol myristate acetate, suggesting that these agents act early in signal transduction. These results provide the first evidence that carboxyl methylated and/or isoprenylated proteins play an essential role in the LPS-signaling pathway.