CpG vaccine strategies induce tumor-reactive T cells for adoptive therapy of lymphoma [electronic resource]

Despite the success of passive immunotherapy with monoclonal antibodies (mAb) directed against tumor cells (e.g. anti-CD20, rituximab), many lymphoma patients eventually relapse. Active immunotherapy for the treatment of lymphoma aims to induce an adaptive and long-lasting antitumor immune response to prevent or prolong time to recurrence. Although antitumor immune cells can be found in cancer patients, these cells may be rendered ineffective in eradicating cancer due to tumor-induced immunosuppression. One approach to overcome this—adoptive cell therapy—involves the isolation of tumor-specific T cells followed by their re-administration to a patient after a myeloablative conditioning regimen. The work discussed in this dissertation describes our studies on a novel approach to adoptive cell therapy for lymphoma. Specifically, we have investigated vaccination methods for generating tumor-reactive T cells in vivo and demonstrate a strategy to isolate this specific population. Prior work from our lab has shown that a combination of chemotherapy (CTX) plus intra-tumor injection of CpG cures a majority of tumors in the A20 mouse B cell lymphoma model. In these studies we found that it was necessary to inject CpG directly into the tumor. We concluded that CpG can have an immunostimulatory effect on either the tumor B cell or on the host APC to enhance uptake and presentation of tumor antigens thereby leading to a cytotoxic CD8 antitumor T cell response. We posited that the effectiveness of the CTX + CpG vaccination maneuver may be limited by endogenous regulatory factors of the immune system. Myeloablation eliminates many of these factors and creates an environment that is conducive to the adoptive transfer of anti-tumor lymphocytes. Transferred cells respond to homeostatic proliferation signals and repopulate 'empty' lymphocyte niches. We utilized CTX + CpG active immunization to generate anti-tumor T cells in vivo and transferred these T cells into lymphodepleted recipient mice. We refer to the preparation of these cells and subsequent transfer as 'immunotransplant'. Transferred T cells cured large and metastatic tumors. We demonstrated that tumor rejection was mediated by donor CD8 T cells. These transferred tumor-specific Teffector cells preferentially expanded, increasing the Teffector:Treg ratio in recipients. This work demonstrates that in situ vaccination is an efficient and effective means to generate T cells for adoptive therapy. The second phase of our work focused on designing an alternative strategy for generating antitumor T cells in vivo. We designed a CpG-loaded tumor cell vaccine made up of irradiated-tumor cells (a rich source of tumor antigens) loaded with CpG. The T cells induced by this vaccine could mediate antitumor immune responses and were more effective when adoptively transferred into lymphodepleted mice. CpG-loaded tumor cells were phagocytosed delivering both tumor antigen and the immunostimulatory CpG molecule to APCs. These APCs then expressed increased levels of costimulatory molecules and induced T cell immunity. TLR9 was required in the APC but not in the CpG-loaded tumor cell. We demonstrate that T cells induced by this vaccine were effective in adoptive cellular therapy for lymphoma and led to regression of large and established tumors. Interestingly, this therapeutic effect could be transferred by CD4 but not by CD8 T cells. This CpG-loaded whole cell vaccination has strong potential for translation to the clinical setting. We were surprised that our CpG-loaded tumor cell vaccine induced an antitumor CD4 T cell response. To date, the field of adoptive cell therapy has focused primarily on CD8 CTLs and our early work with CTX + CpG vaccination supported this paradigm. However, the concept of CD4 T cells coordinating broad, antitumor responses is important for the field of adoptive therapy. CD4 cells play central roles in nearly all aspects of the adaptive immune response including the recruitment of other immune cell types as well as the activation of B cells and APCs. However, clinical translation of using CD4 T cells for adoptive therapy is limited by potential to co-transfer regulatory CD4 T cells (Tregs). In the third phase of our work, we identified a method for isolating viable antitumor CD4 T cells while excluding Tregs based on two surface markers—CD44 and CD137. 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. Moreover, in vivo the addition of CD137posCD44hi CD4 cells to CD137negCD44hi CD4 cells suppressed the antitumor immune response. These results suggest that CD137 expression on CD4 T cells defines a population of activated Tregs that prevent antitumor immune responses. Similar to observations in the murine model, human lymphoma biopsies also contain a population of CD137pos CD4 T cells that are predominantly CD25posFoxP3pos Tregs. In conclusion, 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. Together, these findings define a T cell-based therapy for lymphoma. We have established two methods of vaccination that are effective in generating antitumor T cells and show that these cells can reject established and metastatic tumors. T cell responses differ based on the route of vaccination, however we show that both vaccine-induced CD4 and CD8 T cells can mediate tumor rejection. Finally, we have described two surface molecules that could facilitate isolation of tumor-reactive CD4 T cells while removing tumor-reactive regulatory T cells. This work has direct implications for clinical therapy and a proof-of-concept clinical trial of adoptive immunotherapy for mantle cell lymphoma is ongoing.

Advisor: Levy Ronald
Davis Mark M. advisor. Thesis advisor
Negrin Robert S. advisor. Thesis advisor
Steinman Lawrence advisor. Thesis advisor