PDF file - 308K, Phenotypic fidelity of transferred T cell subsets.

PDF file - 352K, Memory markers do not identify distinct subsets of T cells in vaccinated vs. unvaccinated donors.

PDF file - 139K, Reconstitution of endogenous CD8 T cells post-transplant.

PDF file - 111K, CpG/H11 vaccination but not CpG alone induces the expression of CD137 on FoxP3pos Tregs.

Abstract Adoptive immunotherapy with antitumor T cells is a promising novel approach for the treatment of cancer. However, T-cell therapy may be limited by the cotransfer of regulatory T cells (Treg). Here, we explored this hypothesis by using 2 cell surface markers, CD44 and CD137, to isolate antitumor CD4 T cells while excluding Tregs. In a murine model of B-cell lymphoma, only CD137negCD44hi CD4 T cells infiltrated tumor sites and provided protection. Conversely, the population of CD137posCD44hi CD4 T cells consisted primarily of activated Tregs. Notably, this CD137pos Treg population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, in vitro these CD137pos cells suppressed the proliferation of effector cells in a contact-dependent manner, and in vivo adding the CD137posCD44hi CD4 cells to CD137negCD44hi CD4 cells suppressed the antitumor immune response. Thus, CD137 expression on CD4 T cells defined a population of activated Tregs that greatly limited antitumor immune responses. Consistent with observations in the murine model, human lymphoma biopsies also contained a population of CD137pos CD4 T cells that were predominantly CD25posFoxP3pos Tregs. In conclusion, our findings identify 2 surface markers that can be used to facilitate the enrichment of antitumor CD4 T cells while depleting an inhibitory Treg population. Cancer Res; 72(5); 1239–47. ©2012 AACR.

Abstract Abstract 2093 Background: Adoptive immunotherapy is a promising novel approach to the treatment of cancer. However, clinical translation of adoptively transferred CD4 T cells is limited by cotransfer of an inhibitory population of regulatory CD4 T cells (Tregs). We identified a method of isolating viable antitumor CD4 T cells while excluding Tregs based on two surface markers—CD44 and CD137. Methods: We have developed a model for adoptive cell therapy of lymphoma whereby anti-tumor T cells are generated in vivo through vaccination with a CpG-loaded whole cell vaccine (CpG/H11). These vaccine-induced cells can protect from lethal tumor challenge when isolated and transferred into lethally irradiated, syngeneic recipient mice. We investigated the subsets of T cells involved in the anti-tumor response through a combination of in vitro and in vivo assays. Results: Adoptive transfer of CD137negCD44hi CD4 T cells, but not other sub-populations, provided protection from B cell lymphoma. We demonstrate that the population of CD137posCD44hi CD4 T cells consists primarily of activated Tregs. In vitro, these CD137pos cells suppressed the proliferation of effector cells in a contact-dependent manner. We observed that this CD137pos Treg population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, the addition of CD137posCD44hi CD4 cells to CD137negCD44hi CD4 cells suppressed the antitumor immune response. In the presence of CD137posCD44hi CD4 T cells, homing of other T cell populations to tumor sites was disrupted. These results suggest that CD137 expression on CD4 T cells defines a population of activated Tregs that prevent antitumor immune responses. Conclusions: Our findings identify two surface markers that can be used to facilitate the enrichment of anti-tumor CD4 T cells while depleting an inhibitory Treg population. This approach has direct applicability to clinical trials for patients with lymphoma. Disclosures: No relevant conflicts of interest to declare.

In this study, we demonstrate that the E3 ubiquitin ligase gene related to anergy in lymphocytes (GRAIL) is expressed in quiescent naive mouse and human CD4 T cells and has a functional role in inhibiting naive T cell proliferation. Following TCR engagement, CD28 costimulation results in the expression of IL-2 whose signaling through its receptor activates the Akt-mammalian target of rapamycin (mTOR) pathway. Activation of mTOR allows selective mRNA translation, including the epistatic regulator of GRAIL, Otubain-1 (Otub1), whose expression results in the degradation of GRAIL and allows T cell proliferation. The activation of mTOR appears to be the critical component of IL-2R signaling regulating GRAIL expression. CTLA4-Ig treatment blocks CD28 costimulation and resultant IL-2 expression, whereas rapamycin and anti-IL-2 treatment block mTOR activation downstream of IL-2R signaling. Thus, all three of these biotherapeutics inhibit mTOR-dependent translation of mRNA transcripts, resulting in blockade of Otub1 expression, maintenance of GRAIL, and inhibition of CD4 T cell proliferation. These observations provide a mechanistic pathway sequentially linking CD28 costimulation, IL-2R signaling, and mTOR activation as important requirements for naive CD4 T cell proliferation through the regulation of Otub1 and GRAIL expression. Our findings also extend the role of GRAIL beyond anergy induction and maintenance, suggesting that endogenous GRAIL regulates general cell cycle and proliferation of primary naive CD4 T cells.

Ubiquitination of eukaryotic proteins regulates a broad range of cellular processes, including T cell activation and tolerance. We have previously demonstrated that GRAIL (gene related to anergy in lymphocytes), a transmembrane RING finger ubiquitin E3 ligase, initially described as induced during the induction of CD4 T cell anergy, is also expressed in resting CD4 T cells. In this study, we show that GRAIL can down-modulate the expression of CD83 (previously described as a cell surface marker for mature dendritic cells) on CD4 T cells. GRAIL-mediated down-modulation of CD83 is dependent on an intact GRAIL extracellular protease-associated domain and an enzymatically active cytosolic RING domain, and proceeds via the ubiquitin-dependent 26S proteosome pathway. Ubiquitin modification of lysine residues K168 and K183, but not K192, in the cytoplasmic domain of CD83 was shown to be necessary for GRAIL-mediated degradation of CD83. Reduced CD83 surface expression levels were seen both on anergized CD4 T cells and following GRAIL expression by retroviral transduction, whereas GRAIL knock-down by RNA interference in CD4 T cells resulted in elevated CD83 levels. Furthermore, CD83 expression on CD4 T cells contributes to T cell activation as a costimulatory molecule. This study supports the novel mechanism of ubiquitination by GRAIL, identifies CD83 as a substrate of GRAIL, and ascribes a role for CD83 in CD4 T cell activation.

Abstract Abstract 722 Immunomodulating monoclonal antibodies (mAb) directed against immune cell targets can be used to enhance antitumor immune responses. CD137 (4-1BB) is a costimulatory molecule expressed on a variety of activated immune cells, including T and NK cells. Anti-CD137 agonistic mAb has demonstrated antitumor activity in various tumor models and has now entered clinical trials for the treatment of solid tumors (BMS-663513). Here, we investigate the therapeutic potential of anti-CD137 mAb in lymphoma. Using microarray gene expression data, we compared CD137 mRNA expression across 17 different types of cancer. We found that lymphoma tumor specimens significantly over-expressed CD137 mRNA compared to other tumors (p<0.0001). Single cell analysis of primary lymphoma samples of various histologies (follicular lymphoma [n=29], mantle cell lymphoma [n=14], and diffuse large B cell lymphoma [n=11]) revealed that CD137 was expressed by a significant proportion of tumor-infiltrating T cells, particularly in the CD8 subset (mean[range]=6.1%[2.5-11.5], 15.4%[2.7-40.9] and 18.7%[6.7-51.7], respectively). In contrast, CD137 was not expressed by the tumor cells. These results suggest that the target of anti-CD137 mAb is present and selectively expressed on cells with potential antitumor activity in patients with lymphoma. Using a murine model, we then investigated the anti-lymphoma activity of anti-CD137 agonistic mAb in vivo. BALB/c mice, bearing large and established A20 lymphoma tumors, were treated with anti-CD137 mAb or other immunomodulating mAbs (anti-OX40, anti-CTLA4, anti-GITR) for comparison. The mAbs were given i.p. on days 5 and 10 post tumor inoculation. Anti-CD137 mAb demonstrated potent anti-lymphoma activity in vivo, and appeared to be significantly superior to the other immunomodulating mAbs tested (Figure 1). The antitumor effect of anti-CD137 mAb was not due to direct targeting of the malignant cells as A20 tumor cells do not express CD137. Depletion experiments revealed that anti-CD137 therapy required both NK and CD8 T cells. Analysis of tumor-bearing mice showed that anti-CD137 therapy increased the number of CD8 T cells and reduced the number of Tregs at the tumor site. Anti-CD137 therapy also induced long-lasting antitumor immunity as cured mice harbored antitumor IFN-g producing memory CD8 T cells and were protected from tumor re-challenge more than 100 days after initial therapy. In conclusion, this study demonstrates for the first time that anti-CD137 agonistic mAb has potent antitumor activity in lymphoma. These results support the evaluation of anti-CD137 mAb in clinical trials for patients with lymphoma. Disclosures: No relevant conflicts of interest to declare.

Despite the success of passive immunotherapy with monoclonal antibodies (mAbs), many lymphoma patients eventually relapse. Induction of an adaptive immune response may elicit active and long-lasting antitumor immunity, thereby preventing or delaying recurrence. Immunomodulating mAbs directed against immune cell targets can be used to enhance the immune response to achieve efficient antitumor immunity. Anti-CD137 agonistic mAb has demonstrated antitumor efficacy in various tumor models and has now entered clinical trials for the treatment of solid tumors. Here, we investigate the therapeutic potential of anti-CD137 mAb in lymphoma. We found that human primary lymphoma tumors are infiltrated with CD137+ T cells. We therefore hypothesized that lymphoma would be susceptible to treatment with anti-CD137 agonistic mAb. Using a mouse model, we demonstrate that anti-CD137 therapy has potent antilymphoma activity in vivo. The antitumor effect of anti-CD137 therapy was mediated by both natural killer (NK) and CD8 T cells and induced long-lasting immunity. Moreover, the antitumor activity of anti-CD137 mAb could be further enhanced by depletion of regulatory T cell (T(regs)). These results support the evaluation of anti-CD137 therapy in clinical trials for patients with lymphoma.

Activation of naive T lymphocytes is regulated through a series of discrete checkpoints that maintain unresponsiveness to self. During this multistep process, costimulatory interactions act as inducible signals that allow APCs to selectively mobilize T cells against foreign Ags. In this study, we provide evidence that the anergy-associated E3 ubiquitin ligase GRAIL (gene related to anergy in lymphocytes) regulates expression of the costimulatory molecule CD40L on CD4 T cells. Using its luminal protease-associated domain, GRAIL binds to the luminal/extracellular portion of CD40L and facilitates transfer of ubiquitin molecules from the intracellular GRAIL RING (really interesting new gene) finger to the small cytosolic portion of CD40L. Down-regulation of CD40L occurred following ectopic expression of GRAIL in naive T cells from CD40−/− mice, and expression of GRAIL in bone marrow chimeric mice was associated with diminished lymphoid follicle formation. These data provide a model for intrinsic T cell regulation of costimulatory molecules and a molecular framework for the initiation of clonal T cell anergy.