We have recently reported that CD8 ؉ T-cell memory maintenance after immunization with recombinant human adenovirus type 5 (rHuAd5) is dependent upon persistent transgene expression beyond the peak of the response. In this report, we have further investigated the location and nature of the cell populations responsible for this sustained response. The draining lymph nodes were found to be important for primary expansion but not for memory maintenance, suggesting that antigen presentation through a nonlymphoid source was required. Using bone marrow chimeric mice, we determined that antigen presentation by nonhematopoietic antigenpresenting cells (APCs) was sufficient for maintenance of CD8 ؉ T-cell numbers. However, antigen presentation by this mechanism alone yielded a memory population that displayed alterations in phenotype, cytokine production and protective capacity, indicating that antigen presentation through both hematopoietic and nonhematopoietic APCs ultimately defines the memory CD8 ؉ T-cell response produced by rHuAd5. These results shed new light on the immunobiology of rHuAd5 vectors and provide evidence for a mechanism of CD8 ؉ T-cell expansion and memory maintenance that relies upon both hematopoietic and nonhematopoietic APCs. (Blood. 2011;117(4):1146-1155) IntroductionRecombinant adenovirus vectors have proven to be robust immunogens for eliciting T-cell immunity. 1-3 Vaccines derived from recombinant human adenovirus serotype 5 (rHuAd5) have displayed remarkable potency in various models prompting further investigation. To better understand the immunobiology of rHuAd5, we have been studying both the nature of the CD8 ϩ T cells elicited by these vaccines and the mechanisms of CD8 ϩ T-cell priming and memory maintenance. Immunization with rHuAd5 typically produces a sustained memory population with a protracted contraction phase, 4-8 although these kinetics may be influenced by vector configuration and route of administration. 9 The memory CD8 ϩ T-cell population is composed primarily of effector T cells (T EFF ) and effector memory T cells (T EM ), 5,6 which is indicative of a persistent viral infection. Indeed, we have recently determined that sustained, low-level antigen expression from the rHuAd5 vector plays a key role in maintaining the CD8 ϩ T-cell memory population. 10 Premature extinction of transgene expression causes pronounced CD8 ϩ T-cell contraction, but only modestly affects phenotype, suggesting that memory maintenance and phenotype may be regulated by distinct mechanisms.The relationship between memory CD8 ϩ T-cell phenotype and protective immunity remains to be fully established. T EM provide optimal immune protection against certain agents [11][12][13] ; therefore, understanding the nature of the antigen-presenting cells (APCs) involved in the generation and maintenance of CD8 ϩ T EM will provide important information for vaccine design. Dendritic cells (DCs) are thought to be critical for the induction of antiviral CD8 ϩ T-cell responses. At least 7 distinct DC populations have...
Despite clear evidence of immunogenicity, cancer vaccines only provide a modest clinical benefit. To evaluate the mechanisms that limit tumor regression following vaccination, we have investigated the weak efficacy of a highly immunogenic experimental vaccine using a murine melanoma model. We discovered that the tumor adapts rapidly to the immune attack instigated by tumor-specific CD8+ T cells in the first few days following vaccination, resulting in the upregulation of a complex set of biological networks, including multiple immunosuppressive processes. This rapid adaptation acts to prevent sustained local immune attack, despite continued infiltration by increasing numbers of tumor-specific T cells. Combining vaccination with adoptive transfer of tumor-specific T cells produced complete regression of the treated tumors but did not prevent the adaptive immunosuppression. In fact, the adaptive immunosuppressive pathways were more highly induced in regressing tumors, commensurate with the enhanced level of immune attack. Examination of tumor infiltrating T-cell functionality revealed that the adaptive immunosuppression leads to a progressive loss in T-cell function, even in tumors that are regressing. These novel observations that T cells produced by therapeutic intervention can instigate a rapid adaptive immunosuppressive response within the tumor have important implications for clinical implementation of immunotherapies.
BackgroundAdoptive cell transfer of tumor-specific T lymphocytes (T cells) is proving to be an effective strategy for treating established tumors in cancer patients. One method of generating these cells is accomplished through engineering bulk T cell populations to express chimeric antigen receptors (CARs), which are specific for tumor antigens. Traditionally, these CARs are targeted against tumor antigens using single-chain antibodies (scFv). Here we describe the use of a designed ankyrin repeat protein (DARPin) as the tumor-antigen targeting domain.MethodsWe prepared second generation anti-HER2 CARs that were targeted to the tumor antigen by either a DARPin or scFv. The CARs were engineered into human and murine T cells. We then compared the ability of CARs to trigger cytokine production, degranulation and cytotoxicity.ResultsThe DARPin CARs displayed reduced surface expression relative to scFv CARs in murine cells but both CARs were expressed equally well on human T cells, suggesting that there may be a processing issue with the murine variants. In both the murine and human systems, the DARPin CARs were found to be highly functional, triggering cytokine and cytotoxic responses that were similar to those triggered by the scFv CARs.ConclusionsThese findings demonstrate the utility of DARPins as CAR-targeting agents and open up an avenue for the generation of CARs with novel antigen binding attributes.
Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution.
The use of engineered T cells in adoptive transfer therapies has shown significant promise in treating hematological cancers. However, successes treating solid tumors are much less prevalent. Oncolytic viruses (OVs) have the capacity to induce specific lysis of tumor cells and indirectly impact tumor growth via vascular shutdown. These viruses bear natural abilities to associate with lymphocytes upon systemic administration, but therapeutic doses must be very high in order to evade antibodies and other components of the immune system. As T cells readily circulate through the body, using these cells to deliver OVs directly to tumors may provide an ideal combination. Our studies demonstrate that loading chimeric antigen receptor–engineered T cells with low doses of virus does not impact receptor expression or function in either murine or human T cells. Engineered T cells can deposit virus onto a variety of tumor targets, which can enhance the tumoricidal activity of the combination treatment. This concept appears to be broadly applicable, as we observed similar results using murine or human T cells, loaded with either RNA or DNA viruses. Overall, loading of engineered T cells with OVs represents a novel combination therapy that may increase the efficacy of both treatments.
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