ALBERT

Description: Abstract 578 Autologous stem cell transplantation (ASCT) for multiple myeloma (MM) offers a unique setting to explore the role of immunotherapeutic strategies in eradicating residual disease. A fundamental challenge to developing an effective anti-tumor immune response is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Key elements contributing to tumor-mediated immune suppression are the increased presence of regulatory T cells in patients with malignancy, and upregulation of the PD-1/PDL1 pathway. Tumor expression of PD-L1 promotes T cell tolerance by binding PD-1 on activated T cells and suppressing their capacity to secrete stimulatory cytokines. In addition, the PD-1/PDL-1 pathway has been shown to inhibit T cell-mediated lysis of tumor cells, potentially preventing a clinically meaningful immunologic response to tumor vaccines. We are conducting a clinical trial in which patients with MM are treated with an anti-PD1 antibody (CT-011) alone (Cohort 1) and in combination with a dendritic cell/myeloma fusion cell vaccine (Cohort 2) following ASCT. To date, 27 patients have been enrolled into Cohort 1, in which patients receive three infusions of CT-011 at doses of 3mg/kg given at 6 week intervals beginning 1–3 months following ASCT. Mean age of the patients is 57 years; 61% are male. 11 patients have received at least two infusions of CT-011. The remaining patients are undergoing pre-transplant therapy/transplant. CT-011 has been well tolerated, with possibly related adverse events consisting of transient grade 1–2 leukopenia, diarrhea, fatigue, arthralgia, rash, and peri-orbital edema. One patient developed grade 3 neutropenia, which resolved after two days without growth factor. Immunologic response was determined by quantifying circulating tumor reactive T cells prior to each dose of CT-011 and at 1, 3, 6 months following the last infusion, as defined by the percentage of T cells expressing IFNg in response to ex vivo exposure to autologous tumor lysate. 4 patients have completed 6 months of follow up after the third dose of CT-011, and are evaluable for immune response. CT-011 therapy was associated with the dramatic expansion of myeloma specific T cells. Mean percentage of circulating tumor reactive CD4+ and CD8+ T cells increased from 1.5 and 1.96 respectively prior to the first infusion of CT-011, to 4.26 and 8.28 respectively 1 month following the third infusion. As determined by tetramer staining in the subset of patients who are HLA A2.1, infusion of CT-011 resulted in a mean 9 fold expansion of T cells specific to the MUC1 antigen, which is aberrantly expressed by myeloma cells. Notably, immunologic response to CT-011 persists at 6 months following completion of therapy. Clinical response, as determined by time to disease progression, will be determined with longer follow up, as the median time from transplant is presently 8 months. We are initiating enrollment to Cohort 2, in which patients will be vaccinated with an autologous DC/myeloma fusion vaccine 1 week prior to each dose of CT-011. These data demonstrate that CT-011 results in the expansion of tumor reactive lymphocytes in the early post-transplant period, providing an ideal platform for combination with a tumor vaccine. Disclosures: Rosenblatt: CureTech Ltd.: Research Funding. Schickler:CurTech Ltd.: Employment, Research Funding. Rotem-Yehudar:CureTech Ltd: Employment, Research Funding. Avigan:CureTech Ltd: Research Funding.

Description: Quiescent T cells express Tob, an APRO gene family member, which functions as a transcriptional regulator. Subtractive hybridization identified Twisted gastrulation (Tsg) as one of the genes suppressed by Tob. Tsg is a secreted protein that interacts with Drosophila decapentaplegic (Dpp) and its vertebrate orthologs BMP2/4 and regulates morphogenetic effects in embryos. Here, we report the expression and function of Tsg in human T cells. Tsg mRNA was almost undetectable in unstimulated T cells and was up-regulated after activation by TCR/CD3 and either CD28, IL-2, or PMA. Tsg protein had no effect on responses of primary T cells to TCR/CD3 stimulation but had a potent inhibitory effect on proliferation and cytokine production of primed alloreactive CD4+ cells. Surprisingly, Tsg did not affect phosphorylation of the BMP-specific Smad1 but induced phosphorylation of the TGF-β–specific Smad2 and mediated DNA binding on Smad3/4 consensus-binding sites, suggesting that it acted downstream of TGF-β. In vitro association assays revealed a direct interaction of Tsg and TGF-β proteins. Thus, Tsg functions as an agonist synergizing with TGF-β to inhibit T-cell activation. Modulation of Tsg signaling may represent a novel target for molecular intervention toward control of aberrant T-cell responses during ongoing graft-versus-host disease (GVHD) and autoimmune diseases.

Description: The treatment of hematological malignancies with umbilical cord blood transplantation (UCBT) is rapidly increasing for adult patients. Disadvantages of UCBT include insufficient cell numbers for adult patient reconstitution, a lack of antigen experienced cells, and deficits in T cell signal transduction mechanisms. Consequently, UCBT is frequently associated with impaired immune function and high infection-related mortality. To counter these difficulties, transplantation with two UCB units has been employed to improve immune reconstitution in adult patients. We evaluated both the quantitative and functional reconstitution of cellular immunity in a group of adult patients undergoing UCBT. Thirty-two patients with a median age of 50 years with hematopoietic malignancies were treated with reduced intensity conditioning (Flu/Mel/rATG) followed by infusion with two sequential UCB grafts and GvHD prophylaxis with tacrolimus and sirolimus. The grafts were at least a 4/6 match with each other and the recipient. Here we report the results of 27 patients who have completed at least one year of follow up. Assessments were done prior to transplant and at various time intervals until 12 months post UCBT. Neutrophil and platelet engraftment occurred at a median of 21 days and 42 days, respectively. CD3+ populations remained severely depressed until 8 wks post-transplant when they gradually began to re-emerge. However the CD4+ and CD8+ populations demonstrated distinctly different reconstitution kinetics. At 6 months the median value of absolute numbers of CD4+ lymphocytes was 35% of pre-transplant levels increasing to 42% at 1 yr post-transplant, a median value far below the normal range for adults. In contrast, at 6 months post-transplant CD8+ lymphocytes remained severely depressed to 12% of pre-transplant levels, but dramatically increased and reached normal levels by 1 yr after UCBT. Interestingly, both the CD14+ monocyte and the CD16+CD56+ NK cell populations expanded dramatically at 4 wks post-transplant and reached pre-transplant levels and were within the normal range by 6 months. CD20+ B cell repopulation began at 8 wks post-transplant, displayed a striking expansion leading to a 17-fold increase in the median value for B cell numbers over pre-transplant values at 1 year and resulting in a median value of absolute numbers near the top of the normal range. To evaluate functional T cell immune reconstitution in vivo, we performed IFN-γ ELISpot analysis on CMV stimulated PBLs and compared the results to a PCR-based assay for CMV viremia. Additionally, we assessed the reconstitution of thymopoiesis with the T cell receptor excision circle (TREC) assay and real-time PCR. 16/27 patients and 26/52 UCB products were CMV seropositive prior to transplant. In the post-UCB period, development of CMV-specific effectors as determined by ELISpot did not always correlate with clearance of CMV viremia. Specifically, prior to 8 weeks post-UCBT, 8 out of 12 (67%) patients with CMV-positive ELISpot displayed CMV viremia, between 8 weeks and 100 days post-UCBT 4 out of 11 (36%) patients with CMV-positive ELISpot displayed CMV viremia and after 100 days post-UCBT only 3 out of 10 patients (30%) who developed CMV-positive ELISpot remained positive for CMV viremia. Identification of functional CMV effectors was only associated with the numbers of CD8+CD45RA+ cells (p=0.01) and the development of high TREC concentrations (p=0.01) that were detected after 6 months of UCBT, and was independent of GvHD or mixed chimerism. Taken together these results indicate that reconstitution of T cell immunity after UCBT is characterized by delayed recovery of CD4+ and CD8+ T cells and correlates with reconstitution of thymopoiesis and increase of naïve CD8+CD45RA+ T cells that can develop into efficient pathogen-specific effectors.

Description: Hematological malignancies demonstrate susceptibility to T cell mediated killing as evidenced by the potent graft versus disease effect mediated by alloreactive lymphocytes following allogeneic transplantation. A major area of investigation concerns the development of adoptive immunotherapy that would more selectively target and eliminate tumor cells. This strategy is dependent on the expansion of tumor reactive effector cells while minimizing the presence of regulatory T cells and other contributing elements to tumor mediate immunosuppression. Prior studies have demonstrated that ligation of the CD3/CD28 complex results in expansion of T cells ex vivo with phenotypic characteristics that are dependent on the immunologic milieu at the time of stimulation. We have developed a promising cancer vaccine in which autologous tumor cells are fused with dendritic cells (DCs) resulting in the presentation of a wide spectrum of tumor antigens in the context of DC mediated costimulation. We postulated that stimulation with DC/tumor fusions followed by anti-CD3/CD28 would result in the expansion of activated T cells targeting tumor antigens. Tumor cells were obtained from peripheral blood of patients with AML, or bone marrow aspirates of patients with AML and multiple myeloma. DCs were generated from adherent mononuclear cells cultured with rhIL-4, GM-CSF and TNF and fused with tumor cells by coculture in 50% solution of polyethylene glycol. T cells were stimulated by DC/tumor fusions prior to or following exposure to antiCD3/CD28 antibody coated plates for 48 hours. Stimulation by fusions followed by anti-CD3/CD28 resulted in the synergistic expansion of T cells as manifested by the stimulation index (SI) in excess of that seen when cells were stimulated by either pathway alone or first stimulated by anti-CD3/CD28 then fusions. DC/AML fusions induced autologous T cell proliferation with an SI of 3.3 with memory effector cells (CD45RO+) comprising 10% of the total T cell population. In contrast, sequential stimulation with DC/AML fusions followed by anti-CD3/CD28 resulted in a rise in T cell proliferation (SI 8.2) of which 39% of the resultant populations expressed CD45RO. Similarly a rise in CD4+/CD25+ cells was observed following sequential stimulation with DC/AML fusions followed by anti-CD3/CD28 (9.3% vs. 2.7% following stimulation with DC/AML fusions alone). In addition, an increased percentage of CD4+/CD25+ cells expressed IFNγ when exposed to anti-CD3/CD28 following coculture with fusion cells (7% compared to 2% with fusions alone). A significant rise in the percent of Foxp3+ cells was not seen. Expression of granzyme B is up regulated in activated cytolytic CD8+ T cells that confer perforinmediated killing of target cells. As compared to un-stimulated T cells, stimulation with DC/tumor fusions or anti-CD3/CD28 alone resulted in a 3.3 and 3.8 fold increase in CD8+ T cells expressing granzyme B, respectively. In contrast, sequential stimulation with DC/tumor fusions and anti-CD3/CD28 induced a 19-fold expansion of granzyme B+ cells consistent with their enhanced cytolytic capacity. In addition, sequential stimulation with DC/tumor fusions and anti-CD3/CD28 results in heightened capacity to lyse autologous tumor targets as compared to T cells stimulated by fusions alone in a cytotoxicity assay. In spectratyping analysis, T cells undergoing sequential stimulation with DC/tumor fusion cells and anti-CD3CD28 demonstrate greater skewing of CDR3-size usage in the T cell receptor as compared to T cells stimulated by fusions or anti-CD3/CD28 alone consistent with the expansion of a defined clonal population. The pattern of gene expression in T cells stimulated sequentially by DC/AML fusions and anti-CD3CD28 is being assessed by gene arrays to further define the unique nature of this population. In conclusion, we have demonstrated that stimulation of T cells by DC/tumor fusions followed by exposure to anti-CD3/CD28 antibodies results in the expansion of tumor reactive activated T cell populations. A clinical trial evaluating the safety and efficacy of adoptive immunotherapy using T cells generated by sequential stimulation with DC/tumor fusions and anti- CD3CD28 for patients with multiple myeloma following autologous transplantation is planned.

Description: We have previously determined that quiescent T cells express Tob, a member of the novel APRO gene family, which functions to repress T cell activation. Tob is as a transcriptional regulator and may regulate mRNA transcription. To identify genes that are regulated by Tob, Jurkat T cells were transfected with Tob cDNA or empty vector. Differential gene expression was determined by suppression subtractive hybridization. Twisted Gastrulation (Tsg) was one of the genes suppressed by Tob. Tsg is an evolutionarily conserved secreted protein that interacts with Drosophila Decapentaplegic (Dpp) and its vertebrate orthologs BMP2/4, and also the extracellular Dpp/BMP inhibitors short gastrulation (sog) and chordin. As a result, Tsg affects the binding of Dpp/BMP2/4 to their receptors and subsequent downstream signaling. BMPs belong to a family of secreted signaling molecules the founder of which, TGF-b, is essential for immune homeostasis and maintenance of T cell quiescence. In the thymus, Tsg functions as a regulator of thymocyte development by inhibiting BMP2/4 signaling which arrests thymocyte differentiation. Here, we examined Tsg expression and function in mature CD4+ human T cells. Tsg mRNA was expressed at very low levels in unstimulated T cells and was highly upregulated after activation by TCR/CD3 and either CD28, IL-2 or PMA. To determine the function of Tsg, we generated recombinant human Tsg tagged with V5 epitope. Based on its effects on thymocytes, we hypothesized that Tsg may regulate BMP2/4-mediated effects on mature T cells. Culture of CD4+ T cells with various concentrations of BMP2 or BMP4 in the presence or absence of anti-CD3 mAb did not affect T cell proliferation or cytokine production. Addition of Tsg to cultures containing BMP2/4 did not alter T cell responses. Because our results showed that Tsg mRNA was induced after activation, we tested pre-activated, polyclonal CD4+ alloreactive T cell lines. Neither BMP2 nor BMP4 had any effect on proliferation of alloreactive T cell lines in the presence or absence of anti-CD3 mAb. Strikingly, Tsg induced a significant inhibitory effect on CD3-mediated proliferation and cytokine production, including IL-2, IL-4, IFN-g and IL-10. This effect was not altered by the presence of BMP2 or BMP4. In contrast, Tsg enhanced the inhibitory effect of TGF-b1 suggesting that Tsg regulates TGF-b and not BMP downstream signaling in pre-activated, mature CD4+ T cells. Consistent with this hypothesis, Tsg did not affect phosphorylation of the BMP-specific Smad1, but induced phosphorylation of the TGF-b-specific Smad2 and mediated DNA binding on Smad3/4 consensus sites. In vitro association assays using recombinant Tsg-V5 and TGF-b revealed a direct interaction of these proteins as determined by co-immunoprecipitation followed by western blot with V5-specific and TGF-b-specific antibodies. Moreover, soluble anti-TGF-b receptor reversed the inhibitory effect of Tsg on pre-activated T cells either in the presence or in the absence of TGF-b, providing functional evidence for the biological significance of the Tsg/TGF-b interaction. Our results show that Tsg is a potent agonist of TGF-b downstream signaling in human CD4+ T cells and suggest that enhancement of TGF-b mediating signaling by Tsg may represent a novel target for molecular intervention for induction of transplantation tolerance.

Description: Elucidating the mechanisms that regulate T cell activation and tolerance in vivo will provide insights into the maintenance of physiologic homeostasis and will facilitate development novel strategies for induction of transplantation tolerance. Transient activation of the small GTPase Rap1 is one of the physiologic consequences of TCR ligation and is mandatory for β1 and β2 integrin-mediated adhesion. In contrast, sustained increase of active Rap1 inhibits T cell activation and IL-2 transcription in vitro. In order to understand the role of Rap1 in the immune responses of the intact host we generated transgenic (Tg) mice, which express the active Rap1 mutant Rap1E63 in T cells. Rap1E63-Tg mice had no defects in thymocyte development or maturation. Rap1E63-Tg thymocytes were capable of activating Ras and Erk1/2 and, compared to wild type (WT) thymocytes, displayed enhanced LFA-1:ICAM-1-mediated adhesion and increased proliferation in response to anti-CD3. Surprisingly, although lymph node and splenic CD4+ cells from the Rap1E63-Tg mice also displayed increased LFA-1:ICAM-1-mediated adhesion, they had significantly impaired activation of Erk1/2 and dramatically reduced proliferation and IL-2 production in response to anti-CD3 and WT antigen presenting cells (APC). The defective responses of CD4+ T cells suggest that Rap1E63-Tg mice may have impaired helper function in vivo. To address this issue we immunized Rap1E63-Tg and WT mice with TNP-OVA, a T-cell dependent antigen. Total IgG, IgG1 and IgG2a were dramatically reduced, indicating that Rap1E63-Tg mice had a defect in immunoglobulin class switching, consistent with defective helper T cell-dependent B cell activation. Because these results suggest that Rap1E63-Tg CD4+ cells may have an anergic phenotype, we tested rechallenge responses. We immunized Rap1E63-Tg and WT mice with TNP-OVA in vivo and subsequently we rechallenged T cells in vitro with WT APC pulsed with OVA. Compared with WT, Rap1E63-Tg T cells had dramatically reduced proliferation, IFN- γ and IL-2 production on rechallenge, findings consistent with T cell anergy. Using suppression subtraction hybridization we determined that Rap1E63 induced mRNA expression of CD103, a marker that defines a potent subset of regulatory T cells (Treg). Strikingly, Rap1E63-Tg mice had a 5-fold increase of CD103+CD25+CD4+ Treg compared to WT mice. Rap1E63-Tg CD103+CD25+CD4+ Treg expressed the highest level of Foxp3 among all T cell subsets and had the most potent inhibitory effect on proliferation and IL-2 production when added into cultures of WT CD4+CD25− cells. Importantly, removal of the CD103+ cells significantly restored Erk1/2 activation, proliferation and IL-2 production of Rap1E63-Tg CD4+ T cells. Generation of CD103+ Treg occurs after thymic development and requires encounter of peripheral autoantigen. Consistent with this, differences in CD103+ Treg were detected only between lymph node and splenic cells and not between thymocytes from Rap1E63-Tg and WT mice. Since generation of CD103+ Treg depends on the strength of TCR signal, these results suggest that by enhancing adhesion, active Rap1 regulates the generation of Treg. Moreover, these results provide evidence that active Rap1 is a potent negative regulator of immune responses in vivo and have significant implications for the development of immune-based therapies geared towards tolerance induction.

Description: Autologous stem cell transplantation results in tumor cytoreduction and improved disease outcomes in patients with multiple myeloma (MM), but patients ultimately relapse from persistent disease. A promising area of investigation is the development of cancer vaccines that educate host immunity to target and eliminate myeloma cells and can be used to eradicate residual disease following autologous stem cell transplantation. The early post-transplant period is characterized by a transient reversal of tumor mediated tolerance due to the reduction in disease bulk, the depletion of regulatory T cells. We have developed a cancer vaccine model in which DCs are fused to autologous MM cells resulting in the presentation of multiple tumor antigens with the capacity to elicit a broad anti-tumor response. We are conducting a study in which patients with MM undergo stem cell transplantation followed by post-transplant vaccination with 3 doses of DC/MM fusions. DCs were generated from adherent mononuclear cells cultured with GM-CSF and IL-4 for 5–7 days and matured with TNFα. DCs strongly expressed costimulatory and maturation markers. Myeloma cells were isolated from bone marrow aspirates and were identified by their expression of CD38, CD138, and/or MUC1. DC and MM cells were fused with polyethylene glycol and fusion cells were quantified by determining the percentage of cells that coexpress unique DC and myeloma antigens. To date, 26 patients have been enrolled. All patients have undergone successful vaccine generation. Mean yield of the DC and myeloma preparations was 171×106 and 70×106 cells, respectively. Mean fusion efficiency was 40% and the mean cell dose generated was 4×106 fusion cells. Mean viability of the DC, myeloma, and fusion preparations was 88%, 86%, and 78%, respectively. As a measure of their potency as antigen presenting cells, fusion cells potently stimulated allogeneic T cell proliferation in vitro. Mean stimulation indexes were 12, 57, 31 for T cells stimulated by myeloma cells, DCs, and fusion cell preparations at an APC: T cell ratio of 1:10. Adverse events judged to be potentially vaccine related were mild, and included injection site reactions, pruritis, myalgias, fever, chills, headache, fatigue and tachycardia. To date 14 patients have completed vaccinations and initial follow up of which 8 have achieved a complete remission and 6 a partial remission. Of note, 4 patients achieved complete remission only after undergoing post-transplant vaccination. We are examining the effect of transplant and vaccination on measures of cellular immunity, antitumor immunity and levels of activated as compared to regulatory T cells. T cell responses to PHA mitogen and tetanus toxoid were transiently depressed post-transplant. Similarly, DTH responses to candida antigen were absent post-transplant in all but 1 patient. In contrast, a significant increase was noted post-transplant in circulating tumor reactive lymphocytes as determined by T cell expression of IFNγ by CD4 and CD8 cells following ex vivo coculture with autologous myeloma cell lysate (Mean percentage of tumor reactive CD8 cells was 0.9 and 11 pre and post-transplant, respectively p=0.01; mean percentage of CD4 cells was 0.7 and 2.7; p=0.02). A further amplification of tumor reactive lymphocytes was seen with vaccination in a subset of patients (mean percentage of CD4 and CD8 tumor reactive T cells was 4.9 and 15, respectively). A decrease in the median levels of circulating regulatory T cells and a relative increase in the ratio of activated (CD4/CD25low)/regulatory (CD4/CD25high) cells was observed following transplantation. This finding suggests that although nonspecific T cell responses are muted in the early posttransplant period, there is a greater capacity to recognize tumor antigens, potentially due to the depletion of regulatory T cells and the decline in tumor mediated immune suppression. In summary, fusion cell vaccination in conjunction with stem cell transplantation was well tolerated, induced anti-tumor immunity and clinical responses in patients with MM. The post-transplant period is characterized by increased levels of activated as compared to regulatory T cells and enhanced levels of T cells with the capacity to respond to myeloma cells. The increase in tumor reactive T cells post-transplant is further amplified following exposure to the DC/MM fusion vaccine.

Description: Key PointsThe CHAMPION-1 study is the first clinical trial to investigate carfilzomib on a once-weekly dosing schedule with dexamethasone. Once-weekly carfilzomib (30-minute infusion; 20 and 70 mg/m2) with dexamethasone is feasible and effective in relapsed/refractory MM.

Description: Ligation of the T cell receptor (TCR) and costimulatory receptors leads to cytokine secretion and clonal expansion, whereas ligation of TCR alone leads to anergy. We have previously determined that anergic cells express Tob, a member of the novel APRO gene family, which inhibits T cell activation. The precise molecular mechanisms via which Tob mediates its effects in T cells are not fully understood. Tob functions as transcriptional coactivator and enhances DNA binding of Smads. Therefore, Tob may regulate de novo mRNA synthesis or gene transcription. To identify genes that are induced by Tob, Jurkat T cells that lack endogenous Tob, were transfected with Tob cDNA or empty vector and differential gene expression was determined by suppression subtractive hybridization. TRIM36 was one of the genes induced by Tob. TRIM36 is a RING finger E3 ubiquitin ligase. It belongs to a recently identified tripartite motif (TRIM) gene family which also includes Pyrin/Marenosrtin, MID1, MUL, PML, RFP and TIF1, proteins implicated in familial human diseases and cancer. E3 proteins confer substrate specificity to the ubiquitin system. Previous studies have shown that the trancriptional profile of anergic cells includes the E3 ubiquitin ligases Cbl-b, GRAIL and Itch. Therefore, the finding that Tob, a transcriptional regulator expressed in anergic cells, induces expression of TRIM36 E3 ubiquitin ligase is very intriguing. Northern blot analysis confirmed that TRIM36 mRNA was selectively upregulated in anergic T cells. To determine the role of TRIM36 on IL-2 gene transcription, Jurkat T cells were transfected with full-length TRIM36 cDNA along with the IL-2 promoter/enhancer cDNA (2kb) linked to the luciferase gene. TRIM36 inhibited CD3+CD28-mediated IL-2 transcription by 90%. Interestingly, when cells were stimulated with PMA+Ionomycin, which bypass the TCR proximal signals, IL-2 transcription was almost unaffected. These results prompted us to search for candidate ubiquitination substrates among signaling molecules that have a critical role on TCR-mediated T cell activation and IL-2 transcription. Previous studies have shown that among T cell signaling molecules, TCRζ, ZAP70, PLC-γ1 and PKC-? undergo ubiquitin-targeted degradation. For this reason, we investigated whether any of these proteins might be substrates for TRIM36-mediated ubiquitination. V5-tagged TRIM36 or empty vector was expressed in Jurkat T cells followed by stimulation with anti-CD3+anti-CD28 mAbs in the presence of ubiquitin aldehyde that prevents substrate deubiquitination. Immunoblot with antibodies specific for TCR ζ, ZAP70, PLC-γ1 and PKC-? showed that expression of PLC-γ1 and PKC-? was selectively reduced in the presence of TRIM36. Immunoprecipitation with V5 mAb followed by immunoblot with substrate-specific antibodies revealed that PLC- γ1 and PKC-? coprecipitated with TRIM36. Immunoblot with ubiquitin-specific antibody revealed that PLC-γ1 and PKC- ? were substrates for ubiquitination by TRIM36. Our results show that at least one molecular mechanism via which Tob mediates its inhibitory effect on T cell activation involves the induction of TRIM36 ubiquitin ligase, which mediates degradation of two key signaling proteins, PLC- γ1 and PKC-?. Moreover, these results suggest that TRIM36 may represent a novel target of molecular intervention for induction of transplantation tolerance.