CD134 Costimulation Couples the CD137 Pathway to Induce Production of Supereffector CD8 T Cells That Become IL-7 Dependent

The TNFR superfamily members 4-1BB (CD137) and OX40 (CD134) are costimulatory molecules that potently boost CD8 and CD4 T cell responses. Concomitant therapeutic administration of agonist anti-CD137 and -CD134 mAbs mediates rejection of established tumors and fosters powerful CD8 T cell responses. To reveal the mechanism, the role of CD137 expression by specific CD8 T cells was determined to be essential for optimal clonal expansion and accumulation of effector cells. Nonetheless, dual costimulation induced production of supereffector CD8 T cells when either the specific T cells or the host alone bore CD137. Perhaps surprisingly, the total absence of CD137 prevented anti-CD134 augmentation of supereffector differentiation demonstrating an unappreciated link between these related pathways. Ultimately, it was reasoned that these powerful dual costimulatory responses involved common gamma family members, and we show substantial increases of CD25 and IL-7Ralpha-chain expression by the specific CD8 T cells. To investigate this further, it was shown that IL-7 mediated T cell accumulation, but importantly, a gradual and preferential effect of survival was directed toward supereffector CD8 T cells. In fact, a clear enhancement of effector differentiation was demonstrated to be proportional to the increasing amount of IL-7Ralpha expression by the specific CD8 T cells. Therefore, dual costimulation through CD137 and CD134 drives production and survival of supereffector CD8 T cells through a distinct IL-7-dependent pathway.     

4-1BB and OX40 dual costimulation synergistically stimulate primary specific CD8 T cells for robust effector function

CD40, 4-1BB, and OX40 are costimulatory molecules belonging to the TNF/nerve growth factor superfamily of receptors. We examined whether simultaneous costimulation affected the responses of T cells using several different in vivo tracking models in mice. We show that enforced dual costimulation through 4-1BB and OX40, but not through CD40, induced profound specific CD8 T cell clonal expansion. In contrast, the response of specific CD4 T cells to dual costimulation was additive rather than synergistic. The synergistic response of the specific CD8 T cells persevered for several weeks, and the expanded effector cells resided throughout lymphoid and nonlymphoid tissue. Dual costimulation through 4-1BB and OX40 did not increase BrdU incorporation nor an increase in the number of rounds of T cell division in comparison to single costimulators, but rather enhanced accumulation in a cell-intrinsic manner. Mechanistically speaking, we show that CD8 T cell clonal expansion and effector function did not require T help, but accumulation in (non)lymphoid tissue was predominantly CD4 T cell dependent. To determine whether this approach would be useful in a physiological setting, we demonstrated that dual costimulation mediated rejection of an established murine sarcoma. Importantly, effector function directed toward established tumors was CD8 T cell dependent while being entirely CD4 T cell independent, and the timing of enforced dual costimulation was exquisitely regulated. Collectively, these data suggest that simultaneous dual costimulation through 4-1BB and OX40 induces a massive burst of CD8 T cell effector function sufficient to therapeutically treat established tumors even under immunocompromising conditions.     

OX40-mediated differentiation to effector function requires IL-2 receptor signaling but not CD28, CD40, IL-12Rbeta2, or T-bet

Ag-specific CD4 T cells transferred into unirradiated Ag-bearing recipients proliferate, but survival and accumulation of proliferating cells is not extensive and the donor cells do not acquire effector functions. We previously showed that a single costimulatory signal delivered by an agonist Ab to OX40 (CD134) promotes accumulation of proliferating cells and promotes differentiation to effector CD4 T cells capable of secreting IFN-gamma. In this study, we determined whether OX40 costimulation requires supporting costimulatory or differentiation signals to drive acquisition of effector T cell function. We report that OX40 engagement drives effector T cell differentiation in the absence of CD28 and CD40 signals. Two important regulators of Th1 differentiation, IL-12R and T-bet, also are not required for acquisition of effector function in CD4 T cells responsive to OX40 stimulation. Finally, we show that CD25-deficient CD4 T cells produce little IFN-gamma in the presence of OX40 costimulation compared with wild type, suggesting that IL-2R signaling is required for efficient OX40-mediated differentiation to IFN-gamma secretion.     

Evaluation of OX40 ligand as a costimulator of human antiviral memory CD8 T cell responses: comparison with B7.1 and 4-1BBL

CTL are important effectors of antiviral immunity. Designing adjuvants that can induce strong cytotoxic T cell responses in humans would greatly improve the effectiveness of an antiviral vaccination or therapeutic strategy. Recent evidence suggests that, in addition to its well-established role in costimulation of CD4 T cell responses, OX40L (CD134) can directly costimulate mouse CD8 T cells. In this study, we evaluated the role of OX40L in costimulation of human antiviral CD8 T cell responses and compared it with two other important costimulators, B7.1 (CD80) and 4-1BBL (CD137L). Delivery of OX40L to human monocytes using a recombinant replication-defective adenovirus led to greater expansion, up-regulation of perforin, enhanced cytolytic activity, and increased numbers of IFN-gamma- and TNF-alpha-producing antiviral memory CD8 T cells in cultures of total T cells. Synergistic or additive effects were observed when OX40L costimulation was combined with 4-1BBL (CD137L) or B7.1 (CD80) costimulation. In total T cell cultures, at low Ag dose, 4-1BBL provided the most potent costimulus for influenza-specific CD8 T cell expansion, followed by B7.1 (CD80) and then OX40L. For isolated CD8 T cells, 4-1BBL was also the most consistent costimulator, followed by B7.1. In contrast, OX40L showed efficacy in direct activation of memory CD8 T cells in only one of seven donors. Thus, OX40L costimulates human antiviral memory CD8 T cell responses largely through indirect effects and can enhance anti-influenza, anti-EBV, and anti-HIV responses, particularly in combination with 4-1BBL or B7.     

Polarization of naive CD4+ T cells toward the Th1 subset by CTLA-4 costimulation

In this study, we examined in vitro the role of CTLA-4 costimulation in the polarization of naive CD4+ T cells toward the Th1 subset. When CTLA-4 costimulation was blocked by the inclusion of anti-CTLA-4 Fab in cultures during priming of naive CD4+ T cells with anti-CD3 in the presence of splenic adherent cells, they were polarized toward the Th2 subset. Conversely, the engagement of CTLA-4 with immobilized anti-CTLA-4 or with CD80-P815 cells polarized naive CD4+ T cells costimulated with anti-CD3 and anti-CD28 toward the Th1 subset. The CTLA-4 costimulation during priming augmented TGF-beta1 mRNA accumulation in naive CD4+ T cells, and the inclusion of anti-TGF-beta in cultures for priming suppressed the effect of CTLA-4 costimulation on the Th1 polarization. The addition of low doses of TGF-beta1 in cultures for priming of naive CD4+ T cells enhanced the production of Th1 cytokines upon secondary stimulation, although Th2 cytokine production was not affected by the doses of TGF-beta1. The CTLA-4 costimulation was also shown to suppress IL-4 production of naive CD4+ T cells upon priming. These results indicate that the costimulation against CTLA-4 drives polarization of naive CD4+ T cells toward the Th1 subset independent of IL-12 through, at least in part, the enhancement of TGF-beta1 production, and it also hampers Th2 subset differentiation by affecting IL-4 production of naive CD4+ T cells.     

IL-21 promotes differentiation of naive CD8 T cells to a unique effector phenotype

IL-21, the most recently described member of the common gamma-chain cytokine family, is produced by activated CD4 T cells, whereas CD8 T cells express the IL-21 receptor. To investigate a possible role for IL-21 in the priming of naive CD8 T cells, we examined responses of highly purified naive OT-I CD8 T cells to artificial APCs displaying Ag and B7-1 on their surface. We found that IL-21 enhanced OT-I clonal expansion and supported development of cytotoxic effector function. High levels of IL-2 did not support development of effector functions, but IL-2 was required for optimal responses in the presence of IL-21. IL-12 and IFN-alpha have previously been shown to support naive CD8 T cell differentiation and acquisition of effector functions through a STAT4-dependent mechanism. Here, we show that IL-21 does not require STAT4 to stimulate development of cytolytic activity. Furthermore, IL-21 fails to induce IFN-gamma or IL-4 production and can partially block IL-12 induction of IFN-gamma production. CD8 T cells that differentiate in response to IL-21 have a distinct surface marker expression pattern and are characterized as CD44(high), PD-1(low), CD25(low), CD134(low), and CD137(low). Thus, IL-21 can provide a signal required by naive CD8 T cells to differentiate in response to Ag and costimulation, and the resulting effector cells represent a unique effector phenotype with highly effective cytolytic activity, but deficient capacity to secrete IFN-gamma.     

Induction of CD137 expression by viral genes reduces T cell costimulation

Intracellular pathogens are subject to elimination by a cellular immune response, and were therefore under evolutionary pressure to develop mechanisms that allow them to inhibit especially this arm of immunity. CD137, a T cell costimulatory molecule, and its ligand, CD137 ligand (CD137L), which is expressed on antigen presenting cells (APC), are potent drivers of cellular cytotoxic immune responses. Here, we report that different viruses usurp a negative feedback mechanism for the CD137–CD137L system that weakens cellular immune responses. Latent membrane protein (LMP)-1 and Tax, oncogenes of Epstein-Barr virus (EBV), and human T-cell lymphotropic virus (HTLV)-1, respectively, induce the expression of CD137. CD137 is transferred by trogocytosis to CD137L-expressing APC, and the CD137–CD137L complex is internalized and degraded, resulting in a reduced CD137-mediated T cell costimulation and a weakened cellular immune response which may facilitate the escape of the virus from immune surveillance. These data identify the usurpation of a CD137-based negative feedback mechanism by intracellular pathogens that enables them to reduce T cell costimulation.     

Differentiation of naive CD4<Superscript>+</Superscript> T cells towards T helper 2 cells is not impaired in rheumatoid arthritis patients

An impaired differentiation of naive CD4+ T cells towards Th2 cells may contribute to the chronic tissue-destructive T-cell activity in rheumatoid arthritis (RA). The differentiation of naive CD4+ T cells into memory Th2 cells by IL-7 in comparison with that by IL-4 was studied in RA patients and in healthy controls. Naive CD4+ T cells from peripheral blood were differentiated by CD3/CD28 costimulation in the absence of or in the presence of IL-7 and/or IL-4. The production of IFN-gamma and IL-4 was measured by ELISA and by single-cell FACS analysis to indicate Th1 and Th2 cell activity. CD3/CD28 costimulation and IL-7 were early inducers of IL-4 production, but primarily stimulated IFN-gamma production. In contrast, in short-term cultures exogenously added IL-4 did not prime for IL-4 production but suppressed IL-7-induced IFN-gamma production. Upon long-term stimulation of naive CD4+ T cells, IFN-gamma production was differentially regulated by IL-7 and IL-4, but IL-4 production was increased by both IL-7 and IL-4. IL-7 and IL-4 additively induced polarization towards a Th2 phenotype. This susceptibility of naive CD4+ T cells to become Th2 cells upon culture with IL-7 and IL-4 was increased in RA patients compared with that in healthy controls. These findings demonstrate that, in RA patients, differentiation of naive CD4+ T cells towards a Th2 phenotype by CD3/CD28 costimulation, IL-7 and IL-4 is not impaired. The perpetuation of arthritogenic T-cell activity in RA therefore seems not to be the result of intrinsic defects of naive CD4+ T cells to develop towards suppressive memory Th2 cells.     

Enhancement of HIV-specific CD8 T cell responses by dual costimulation with CD80 and CD137L

HIV-specific CD8 T cell responses are defective in chronic HIV infection. In this study, we report that costimulation with either CD137L (4-1BBL) or CD80 (B7.1) enhanced the Ag-specific expansion and acquisition of effector function by HIV-specific memory CD8 T cells. Ag-specific T cells from recently infected donors showed maximal expansion with single costimulatory molecules. Dual costimulation of T cells from recently infected donors or from healthy donors responding to influenza epitopes led to enhanced responses when the accumulation of cytokines was measured. However, accumulation of regulatory cytokines, particularly IFN-gamma, led to inhibition of further Ag-specific CD8 T cell expansion in the cultures. This inhibition was relieved by neutralization of IFN-gamma or of IFN-gamma, TNF, and IL-10. Thus, strong costimulation of T cells in vitro can lead to induction of regulatory cytokines at levels that limit further T cell expansion. In marked contrast, T cells from long-term (>4 years) infected HIV+ donors exhibited reduced Ag-specific CD8 T cell expansion, reduced CD4 T cell responses, and minimal cytokine accumulation. Dual costimulation with both 4-1BBL and B7.1 enhanced responses of T cells from long-term infected subjects to a level similar to that obtained with T cells from early in HIV infection. Experiments with purified CD8 T cells showed that B7.1 and 4-1BBL could act directly and synergistically on CD8 T cells. Taken together, these data suggest that 4-1BBL and B7.1 have additive or synergistic effects on HIV-specific CD8 T cell responses and represent a promising combination for therapeutic vaccination for HIV.     

Inhibition of IL-4 responses after T cell priming in the context of LFA-1 costimulation is not reversed by restimulation in the presence of CD28 costimulation

Costimulation is one of several factors that influence the differentiation of CD4+ Th cell responses. Previously, we have shown that Ag presentation in the context of LFA-1 costimulation by fibroblasts transfected with class II and ICAM-1 (ProAd-ICAM) can drive naive CD4-positive T cells into cell cycle, but these T cells die by apoptosis 4-5 days after stimulation. In this report we show that the death of these cells can be prevented by the addition of exogenous IL-2 (20 U/ml) or by restimulation with Ag presented in the context of CD28 costimulation. Under these conditions, T cells go through extensive cell division and normal cell expansion. However, when T cells that have been primed by Ag presented in the context of LFA-1 costimulation are restimulated, they secrete IL-2 and IFN-gamma, but little or no IL-4. The inability of ProAd-ICAM-primed T cells to produce IL-4 was restored by the addition of IL-4 to the priming culture. However, IL-4 responses were not restored by representation of Ag in the context of CD28 costimulation, even as early as 24 h after priming with Ag presented by ProAd-ICAM cells. These findings suggest that differential expression of B7-1 and ICAM-1 by APCs during the initiation of immune responses may alter the differentiation of Th populations.     

4-1BB ligand, a member of the TNF family, is important for the generation of antiviral CD8 T cell responses.

4-1BB (CD137) is a costimulatory molecule expressed on activated T cells and interacts with 4-1BB ligand (4-1BBL) on APCs. To investigate the role of 4-1BB costimulation for the development of primary immune responses, 4-1BBL-deficient (4-1BBL-/-) mice were infected with lymphocytic choriomeningitis virus (LCMV). 4-1BBL-/- mice were able to generate CTL and eliminate acute LCMV infection with normal kinetics, but CD8 T cell expansion was 2- to 3-fold lower than in wild-type (+/+) mice. In the same mice, virus-specific CD4 Th and B cell responses were minimally affected, indicating that 4-1BB costimulation preferentially affects CD8 T cell responses. This result contrasts with our earlier work with CD40L-deficient (CD40L-/-) mice, in which the CD8 T cell response was unaffected and the CD4 T cell response was markedly impaired. When both 4-1BBL- and B7-dependent signals were absent, CD8 T cell expansion was further reduced, resulting in lower CTL activity and impairing their ability to clear LCMV. Altogether, these results indicate that T cells have distinct costimulatory requirements: optimal CD8 responses require 4-1BBL-dependent interactions, whereas CD4 responses are minimally affected by 4-1BB costimulation, but require CD40-CD40L and B7-dependent interactions.     

Regulation of follicular dendritic cell networks by activated T cells: the role of CD137 signaling

B cells, but not T cells, are considered to be important for the formation of follicular dendritic cell (FDC) clusters. Stimulation with agonist mAbs against CD137 (4-1BB), a TNFR family member primarily expressed on activated T cells, was effective in promoting T cell responses, but paradoxically suppressed T-dependent humoral immunity and autoantibody production in autoimmune disease models. Our present study shows that agonistic anti-CD137 treatment activates T cells, resulting in diminished FDC networks in B cell follicles, which are important components in T-dependent humoral immune responses both before and after the initiation of an immune response. Pretreatment with anti-CD137 before the secondary immunization inhibited memory Ab responses. Interestingly, CD137 costimulation-induced diminishment of FDC is T cell dependent. In addition, both CD4(+) and CD8(+) T cells are recruited into FDC area and are able to regulate FDCs by CD137 costimulation through a direct or indirect mechanism. These studies have revealed a previously unappreciated role of T cells in the regulation of FDC networks.     

CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation.

The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4Ralpha-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.     

CD86 has sustained costimulatory effects on CD8 T cells

CD80 and CD86 both costimulate T cell activation. Their individual effects in vivo are difficult to study as they are coordinately up-regulated on APCs. We have studied mice expressing rat insulin promoter (RIP)-CD80 and RIP-CD86 on the NOD and NOD.scid genetic background to generate in vivo models, using diabetes as a readout for cytotoxic T cell activation. Accelerated spontaneous diabetes onset was observed in NOD-RIP-CD80 mice and the transfer of diabetes from 6-wk-old NOD mice to NOD.scid-RIP-CD80 mice was greater compared with NOD-RIP-CD86 and NOD.scid-RIP-CD86 mice, respectively. However, the secondary in vivo response was maintained if T cells were activated through CD86 costimulation compared with CD80. This was demonstrated by greater ability to cause recurrent diabetes in NOD-RIP-CD86 diabetic mice transplanted with 6-wk-old NOD islets and adoptively transferred diabetes from diabetic NOD-RIP-CD86 mice to NOD.scid mice. In vitro, CD80 costimulation enhanced cytotoxicity, proliferation, and cytokine secretion in activated CD8 T cells compared with CD86 costimulation. We demonstrated increased CTLA-4 and programmed death-1 inhibitory molecule expression following costimulation by both CD80 and CD86 (CD80 > CD86). Furthermore, T cells stimulated by CD80 were more susceptible to inhibition by CD4(+)CD25(+) T cells. Overall, while CD86 does not stimulate an initial response as strongly as CD80, there is greater sustained activity that is seen even in the absence of continued costimulation. These functions have implications for the engineered use of costimulatory molecules in altering immune responses in a therapeutic setting.     

Critical synergy of CD30 and OX40 signals in CD4 T cell homeostasis and Th1 immunity to Salmonella

CD30 and OX40 (CD134) are members of the TNFR superfamily expressed on activated CD4 T cells, and mice deficient in both these molecules harbor a striking defect in the capacity to mount CD4 T cell-dependent memory Ab responses. This article shows that these mice also fail to control Salmonella infection because both CD30 and OX40 signals are required for the survival but not commitment of CD4 Th1 cells. These signals are also needed for the survival of CD4 T cells activated in a lymphopenic environment. Finally, Salmonella and lymphopenia are shown to act synergistically in selectively depleting CD4 T cells deficient in OX40 and CD30. Collectively these findings identify a novel mechanism by which Th1 responses are sustained.     

Differentiation of regulatory T cells 1 is induced by CD2 costimulation.

Induction and maintenance of peripheral tolerance is an important phenomenon for the control of homeostasis in the immune system. There is now compelling evidence for CD4(+) T cells that prevent immune pathology, both in autoimmunity and in transplantation. However, the mechanisms involved in the specific differentiation of these T cells are unknown. We had previously shown that repetitive stimulations of naive T cells in the presence of IL-10 induce the differentiation of T regulatory cells 1. We further dissected the mechanism of IL-10 function and demonstrated that IL-10 acts by the down-regulation of most costimulatory molecules without modifying the expression of CD58. Using artificial APCs expressing various costimulatory molecules, we demonstrated that, in contrast to other costimulation patterns, costimulation via CD2 alone, in the absence of costimulations through CD28- or LFA-1, induced T cell anergy in an IL-10-independent pathway along with the differentiation of Ag-specific regulatory T cells. T regulatory cell-1 differentiation via CD2 was very efficient as both high IL-10 secretion and regulatory function were observed after the first stimulation of naive T cells with CD32-CD58 L cells. The possibility to rapidly induce the differentiation of Ag-specific regulatory T cells will certainly accelerate their characterization and their potential use as regulators of T cell-mediated diseases.     

Tumor-specific T cell activation by recombinant immunoreceptors: CD3 zeta signaling and CD28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3 zeta signaling receptor molecule.

Recombinant immunoreceptors with specificity for the carcinoembryonic Ag (CEA) can redirect grafted T cells to a MHC/Ag-independent antitumor response. To analyze receptor-mediated cellular activation in the context of CD28 costimulation, we generated: 1) CEA+ colorectal tumor cells that express simultaneously B7-1 and B7-2, and 2) CEA-specific immunoreceptors that harbor intracellularly the signaling moities either of CD28 (BW431/26-scFv-Fc-CD28), CD3zeta (BW431/26-scFv-Fc-CD3zeta), or FcepsilonRIgamma (BW431/26-scFv-Fc-gamma). By retroviral gene transfer, we grafted activated T cells from the peripheral blood with these immunoreceptors. T cells that express the FcepsilonRIgamma or CD3zeta signaling receptor lysed specifically CEA+ tumor cells and secreted high amounts of IFN-gamma upon receptor cross-linking, whereas anti-CEA-CD28 receptor-grafted T cells did not, indicating that CD28 signaling alone is not sufficient for efficient T cell activation. CD28 costimulation did not affect cytolysis by T cells equipped with gamma- or zeta-signaling receptors, but enhanced both IFN-gamma secretion and proliferation. CD28 costimulation, however, was required for efficient IL-2 secretion of anti-CEA-gamma receptor-grafted T cells. Both purified CD4+ and CD8+ T cells grafted with immunoreceptors required CD28 costimulation for complete T cell activation. We integrated both CD28 and CD3zeta signaling domains into one combined immunoreceptor molecule (BW431/26-scFv-Fc-CD28/CD3zeta) with dual signaling properties. T cells grafted with the combined CD28/CD3zeta signaling receptor secreted high amounts of IL-2 upon Ag binding without exogenous B7/CD28 costimulation, demonstrating that both MHC-independent cellular activation and CD28 costimulation for complete T cell activation can be delivered by one recombinant receptor molecule.     

Role of CD4 T cell help and costimulation in CD8 T cell responses during Listeria monocytogenes infection

CD4 T cells are known to assist the CD8 T cell response by activating APC via CD40-CD40 ligand (L) interactions. However, recent data have shown that bacterial products can directly activate APC through Toll-like receptors, resulting in up-regulation of costimulatory molecules necessary for the efficient priming of naive T cells. It remains unclear what role CD4 T cell help and various costimulation pathways play in the development of CD8 T cell responses during bacterial infection. In this study, we examined these questions using an intracellular bacterium, Listeria monocytogenes, as a model of infection. In CD4 T cell-depleted, CD4(-/-), and MHC class II(-/-) mice, L. monocytogenes infection induced CD8 T cell activation and primed epitope-specific CD8 T cells to levels commensurate with those in normal C57BL/6 mice. Furthermore, these epitope-specific CD8 T cells established long-term memory in CD4(-/-) mice that was capable of mounting a protective recall response. In vitro analysis showed that L. monocytogenes directly stimulated the activation and maturation of murine dendritic cells. The CD8 T cell response to L. monocytogenes was normal in CD40L(-/-) mice but defective in CD28(-/-) and CD137L(-/-) mice. These data show that in situations where infectious agents or immunogens can directly activate APC, CD8 T cell responses are less dependent on CD4 T cell help via the CD40-CD40L pathway but involve costimulation through CD137-CD137L and B7-CD28 interactions.     

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.