Arvind Chhabra

CD4+CD25− T Cells Transduced to Express MHC Class I-Restricted Epitope-Specific TCR Synthesize Th1 Cytokines and Exhibit MHC Class I-Restricted Cytolytic Effector Function in a Human Melanoma Model

Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4+ T cells. Considering the difficulties in simultaneously engaging CD4+ and CD8+ T cells in tumor immunotherapy, especially in an Ag-specific manner, redirecting CD4+ T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope-specific TCRs in CD4+ T cells has emerged as a strategic consideration. Such TCR-engineered CD4+ T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have conducted a critical examination of functional characteristics of CD4+ T cells engineered to express the α- and β-chains of a high functional avidity TCR specific for the melanoma epitope, MART-127–35, as a prototypic human tumor Ag system. We found that unpolarized CD4+CD25− T cells engineered to express the MART-127–35 TCR selectively synthesize Th1 cytokines and exhibit a potent Ag-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8+ CTL. Such TCR engineered CD4+ T cells, therefore, might be useful in clinical immunotherapy.

Effect of CD4+CD25+ and CD4+CD25− T Regulatory Cells on the Generation of Cytolytic T Cell Response to a Self but Human Tumor-Associated Epitope In Vitro

CD4+ T cells naturally expressing CD25 molecules (natural T regulatory cells (Tregs)) have a role in maintaining self tolerance and in regulating responses to infectious agents, transplantation Ags, and tumor Ags. CD4+ Tregs induced from CD4+CD25− precursors (induced Tregs) also regulate immune responses in the periphery. However, which of these Tregs is a major impediment in generating antitumor CTL responses is not clear. We show that although the CD4+CD25+ subsets isolated from peripheral blood-derived lymphocytes do suppress the proliferation of CD4+CD25− effector T cells, they do not suppress the activation and expansion of the self but melanoma-associated, melanoma Ag-reactive T cell 1 (MART-1)27–35-specific CD8+ T cells stimulated by the respective peptide-loaded matured dendritic cells in vitro. The CD4+CD25− counterparts, in contrast, lead to the generation of CD25+ glucocorticoid-inducible TNFR+-Forkhead/winged helix transcription factor+ populations and efficiently suppress the activation and expansion of the MART-127–35 epitope-specific CTLs. Our data suggest that when CTL precursors are optimally stimulated, natural Tregs are not a formidable constraint toward generating a robust antitumor CTL response, but induced Tregs could be.

Silencing of endogenous IL-10 in human dendritic cells leads to the generation of an improved CTL response against human melanoma associated antigenic epitope, MART-127-35

Dendritic cells (DC) present antigenic epitopes to and activate T cells. They also polarize the ensuing T cell response to Th1 or Th2 type response, depending on their cytokine production profile. For example, IL-12 producing DC generate Th1 type T cell response whereas IL-10 producing DC is usually tolerogenic. Different strategies--such as the use of cytokines and anti-cytokine antibodies, dominant negative forms of protein, anti-sense RNA etc.--have been employed to influence the cytokine synthetic profile of DC as well as to make DC more immunogenic. Utilizing GFP expressing recombinant adenoviruses in association with lipid-mediated transfection of siRNA, we have silenced the endogenous IL-10 gene in DC. We show that IL-10 gene silenced DC produces more IL-12 and also generates a better cytolytic T cell response against the human melanoma associated epitope, MART-1(27-35), in vitro. We also show that the GFP expressing adenoviral vector can be used to optimize the parameters for siRNA delivery in primary cells and show that RNA interference methodology can efficiently knock down virus encoded genes transcribed at very high multiplicity of infection in DC.

Rescuing Melanoma Epitope-Specific Cytolytic T Lymphocytes from Activation-Induced Cell Death, by SP600125, an Inhibitor of JNK: Implications in Cancer Immunotherapy

Activation-induced cell death (AICD) as well as programmed cell death (PCD) serve to control the expansion of activated T cells to limit untoward side effects of continued effector responses by T cells and to maintain homeostasis. AICD of T cells in tumor immunotherapy can be counterproductive particularly if the activated T cells undergo apoptotic death after the very first secondary encounter of the specific epitope. We examined the extent to which tumor epitope-specific CTLs that are activated and expanded in an in vitro-matured dendritic cell-based primary stimulation protocol undergo AICD following their first secondary encounter of the cognate epitope. Using the MART-127–35 epitope as a prototype vaccine epitope, we also examined whether these CTLs could be rescued from AICD. Our results demonstrate that a substantial fraction of MART-127–35 epitope-specific primary CTLs undergo AICD upon the very first secondary encounter of the cognate epitope. The AICD in these CTLs is neither caspase dependent nor is it triggered by the extrinsic death signaling pathways (Fas, TNFR, etc.). These CTLs, interestingly, could be rescued from AICD by the JNK inhibitor, SP600125. We also found that SP600125 interferes with their IFN-γ response but does not block their cytolytic function. The rescued CTLs, however, regain their capacity to synthesize IFN-γ if continued in culture without the inhibitor. These observations have implications in tumor immunotherapy and in further studies for regulation of AICD in CTLs.

Activation-induced cell death of human melanoma specific cytotoxic T lymphocytes is mediated by apoptosis-inducing factor

Activation‐induced cell death (AICD) of T cells can be an impediment towards achieving a robust and long‐lived cytolytic T lymphocyte (CTL) response from active specific immunization or after adoptive cell transfer in cancer immunotherapy. The mechanism of AICD in primary CTL, however, remains poorly understood. It is widely believed that AICD is driven by signals from death receptors (DR) and that the cell death takes place in a caspase‐dependent manner, although it has been shown that AICD of T cells can be induced by internal triggers and that death takes place in a caspase‐independent manner. We show here that AICD in human melanoma epitope‐specific primary CTL involves selective mitochondrio‐nuclear translocation of the apoptosis inducing factor (AIF) without cytochrome c release, caspase‐3 and caspase‐8 activation, and results from large‐scale DNA fragmentation. The c‐jun‐N terminal kinase (JNK) inhibitor, SP600125, blocks the mitochondrio‐nuclear translocation of AIF and prevents AICD in these CTL. These findings suggest that the AICD in human melanoma epitope specific primary CTL is mediated by mitochondrial AIF release and JNK is involved in regulation of this death process.

Cross-presentation of a human tumor antigen delivered to dendritic cells by HSV VP22-mediated protein translocation.

Dendritic cells (DC) capture antigens from apoptotic and/or necrotic tumor cells and cross‐present them to T cells, and various ways of delivering tumor antigens to DC in vitro and in vivo are being pursued. Since fusions of antigenic proteins with the HSV integument protein VP22 are capable of intercellular trafficking, this approach has been exploited for delivery of antigens to antigen‐presenting cells. Adenoviral vectors were used to express the tumor‐associated‐but‐self‐antigen MART‐1 fused to HSV VP22 in MART‐1‐negative A375 melanoma cells and in DC. When expressed in A375 cells and allowed to spread to DC across a transwell barrier, the VP22‐MART‐1 fusion protein localized to both early and late endosomal structures of the DC. The DC loaded with the VP22‐MART‐1 fusion by intercellular trafficking efficiently presented the MART‐127–35 epitope to MART‐127–35‐specific CTL. Furthermore, transloaded DC were capable of expanding the population of MART‐127–35‐specific CTL. Thus, a tumor antigen acquired by intercellular trafficking can be cross‐presented by DC. This experimental approach should therefore be useful not only for studying the mechanism of cross‐presentation but also for vaccine development.

Presence of Low Dose of Fludarabine in Cultures Blocks Regulatory T Cell Expansion and Maintains Tumor-Specific Cytotoxic T Lymphocyte Activity Generated with Peripheral Blood Lymphocytes

Background: For tumor vaccine-based immunotherapy of cancer, the expansion of tumor antigen-specific cytotoxic T lymphocytes (CTL) in the patients by blocking induced regulatory T (Treg) cells is the most important objective now. Fludarabine (FLU), a known anticancer drug, has been shown to downregulate Treg cells in vivo in chronic leukemia patients. Melanoma tumor antigen Mart-127–35-specific CD8+ CTLs generated in vitrowith total peripheral blood lymphocytes (PBL) lose their activity within 14–21 days with concomitant expansion of Treg cells. When CD4+ cells are removed from PBL and CTL are generated with purified CD8+ cells, the CTL survive and maintain their activity for a significantly longer period. Methods: We used a low dose of FLU in the cultures in Mart-1-specific CTL generation assays with total PBL. Blood samples were taken from HLA-A2-positive melanoma patients and normal donors. Autologous matured dendritic cells pulsed with Mart-127–35 peptide were used to generate CTL responses using purified CD8+ cells or total PBL. Results: The presence of FLU in the cultures with PBL helped to generate a significantly higher number of antigen-specific CTLs as detected by Mart-1 HLA-A2 tetramer staining. Specificity of such CTLs was determined by IFN-γ secretion or by cytotoxicity against the target cells bearing the specific antigen. The presence of FLU stopped the expansion of IL-10-producing CD4+ Treg cells in the cultures with PBL. Analyses of expanded CD4+ cells isolated from PBL in vitro cocultures with FLU showed a Th1 type of function. Those cells secreted higher amounts of IFN-γ and very low levels of IL-10, or no IL-10 at all, upon restimulation. Conclusion: The observations of the study are as important for adaptive immunotherapy of cancer as they are for vaccine-based approaches.

Inhibition of c-Jun N-terminal kinase rescues influenza epitope-specific human cytolytic T lymphocytes from activation-induced cell death

Cytolytic T lymphocytes (CTL) play an important role in defense against viral infections. Following clonal expansion and effector functions, a vast majority of the antigen‐specific CTL undergoes programmed cell death to maintain homeostasis. We have shown earlier that melanoma epitope‐specific CTL are quite sensitive to activation‐induced cell death (AICD) even on the secondary encounter of the antigen. Excessive sensitivity of viral antigen‐specific CTL to AICD, however, would be counterproductive. It might be argued that although CTL for a “self” epitope might be more prone to AICD for maintaining self‐tolerance, viral antigen‐specific CTL are likely to be less sensitive to AICD. We show here that influenza matrix protein‐derived MP58–66 epitope‐specific CTL, activated in vitro and bearing a memory phenotype, are just as sensitive to AICD. The AICD in these CTL is not blocked by the pan‐caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp (OMe)‐fluoromethylketone or by soluble Ig‐Fc chimeras of the death receptors [Fas, TNF receptor (TNF‐R), TRAIL‐RI, TRAIL‐RII]. However, the MP58–66‐specific CTL can be rescued from AICD by the c‐jun‐N‐terminal kinase (JNK) inhibitor SP600125. These results have implications for immunotherapeutic intervention in rescuing viral epitope‐specific CTL from AICD.

Antigen presentation by MART-1 adenovirus-transduced interleukin-10-polarized human monocyte-derived dendritic cells.

Dendritic cells (DC) play critical roles in generating an immune response and in inducing tolerance. Diverse microenvironmental factors can ‘polarize’ DC toward an immunogenic or non‐immunogenic phenotype. Among the various microenvironmental factors, interleukin‐10 (IL‐10) exhibits a potent immunosuppressive effect on antigen‐presenting cells (APC). Here, we show that monocyte‐derived DC generated in the presence of IL‐10 exhibit a profound down‐regulation of many genes that are associated with immune activation and show that the IL‐10‐grown DC are poor stimulators of CD8+ T cells in a strictly autologous and major histocompatibility complex (MHC) class I‐restricted melanoma antigen recognized by T cells (MART‐1) epitope presentation system. However, these IL‐10‐grown DC can efficiently activate the epitope‐specific CD8+ T cells when they are made to present the epitope following transduction with an adenoviral vector expressing the MART‐1 antigen. In addition, we show that the MART‐1 protein colocalizes with the MHC class I protein, equally well, in the iDC and in the DC cultured in presence of IL‐10 when both DC types are infected with the viral vector. We also show that the vector transduced DC present the MART‐127−35 epitope for a sustained period compared to the peptide pulsed DC. These data suggest that although DCs generated in the presence of IL‐10 tend to be non‐immunogenic, they are capable of processing and presenting an antigen when the antigen is synthesized within the DC.

Mitochondria-Centric Activation Induced Cell Death of Cytolytic T Lymphocytes and its Implications for Cancer Immunotherapy

Premature death of the adoptively transferred cytolytic T lymphocytes (CTL) by means of activation induced cell death (AICD) represents one of the major constraints in devising an effective anti-cancer immune intervention strategy. Understanding the mechanism of AICD is, therefore, critical for developing methods to interfere with this death process. Although the existing paradigm on AICD centers around the initiation of the cascade of events originating from the engagement of death receptors leading to the activation of effector caspases and eventually resulting in cell death, recent findings have questioned the universal role of caspases as the cell death executioners. We here review our current understanding of the contribution of caspase-dependent and caspase-independent death executioners in AICD of T cells. We will also discuss the involvement of mitochondria-centric death pathway in AICD of human tumor associated antigen-specific primary CTL and its implications in cancer immunotherapy.