Expression data for naïve IL-2 and IL-12 primed Pmel-1 CD8+ T-cells
ABSTRACT: The expansion, trafficking and functional effectiveness of adoptively transferred CD8+ T-cells play a critical role in mediating effective anti-tumor immunity. However, the mechanisms which program the highly proliferative and functional state of CD8+ T-cells are not completely understood. We hypothesized that IL-12, a cytokine commonly induced by TLR activation, could enhance T-cell priming by altering responsiveness to antigen and cytokines. Priming of tumor specific CD8+ T-cells in the presence of IL-12 induced the acquisition of a 'polyfunctional' effector response and increased the generation of memory cells. Moreover, IL-12 priming also promoted high levels of the IL-2 receptor alpha-chain (CD25) and robust IL-2 mediated activation of STAT5. This sensitivity to IL-2 translated into enhanced in vivo proliferation of adoptively transferred CD8+ T-cells. Furthermore, real-time, in vivo imaging of T-cell trafficking confirmed the ability of IL-12 priming to drive in vivo proliferation. IL-12 priming enhanced the anti-tumor function of adoptively transferred cells by reducing established subcutaneous tumor burden, and significantly increasing survival in an established intracranial tumor model. Finally, IL-12 priming of human PBMCs generates tumor specific T-cells phenotypically and functionally similar to IL-12 primed Pmel-1 T-cells. These results highlight IL-12 as an important mediator of CD8+ T-cell effector function and anti-tumor immunity. We primed Pmel-1 TCR transgenic CD8+ T-cells with cognate antigen and either IL-2 or IL-12 and compared their gene expression profiles. This was used to identify pathways or genes necessary for anti-tumor activity in vivo. RNA was isolated from Pmel-1 T-cells primed with antigen and cytokine for 6 days and hybridized to Affymetrix arrays.
Project description:The expansion, trafficking and functional effectiveness of adoptively transferred CD8+ T-cells play a critical role in mediating effective anti-tumor immunity. However, the mechanisms which program the highly proliferative and functional state of CD8+ T-cells are not completely understood. We hypothesized that IL-12, a cytokine commonly induced by TLR activation, could enhance T-cell priming by altering responsiveness to antigen and cytokines. Priming of tumor specific CD8+ T-cells in the presence of IL-12 induced the acquisition of a 'polyfunctional' effector response and increased the generation of memory cells. Moreover, IL-12 priming also promoted high levels of the IL-2 receptor alpha-chain (CD25) and robust IL-2 mediated activation of STAT5. This sensitivity to IL-2 translated into enhanced in vivo proliferation of adoptively transferred CD8+ T-cells. Furthermore, real-time, in vivo imaging of T-cell trafficking confirmed the ability of IL-12 priming to drive in vivo proliferation. IL-12 priming enhanced the anti-tumor function of adoptively transferred cells by reducing established subcutaneous tumor burden, and significantly increasing survival in an established intracranial tumor model. Finally, IL-12 priming of human PBMCs generates tumor specific T-cells phenotypically and functionally similar to IL-12 primed Pmel-1 T-cells. These results highlight IL-12 as an important mediator of CD8+ T-cell effector function and anti-tumor immunity. Overall design: We primed Pmel-1 TCR transgenic CD8+ T-cells with cognate antigen and either IL-2 or IL-12 and compared their gene expression profiles. This was used to identify pathways or genes necessary for anti-tumor activity in vivo. RNA was isolated from Pmel-1 T-cells primed with antigen and cytokine for 6 days and hybridized to Affymetrix arrays.
Project description:The optimal expansion, trafficking, and function of adoptively transferred CD8(+) T cells are parameters that currently limit the effectiveness of antitumor immunity to established tumors. In this study, we addressed the mechanisms by which priming of self tumor-associated Ag-specific CD8(+) T cells influenced antitumor functionality in the presence of the inflammatory cytokine IL-12. In vitro priming of mouse tumor-specific CD8(+) T cells in the presence of IL-12 induced a diverse and rapid antitumor effector activity while still promoting the generation of memory cells. Importantly, IL-12-primed effector T cells dramatically reduced the growth of well-established s.c. tumors and significantly increased survival to highly immune resistant, established intracranial tumors. Control of tumor growth by CD8(+) T cells was dependent on IL-12-mediated upregulation of the high-affinity IL-2R (CD25) and a subsequent increase in the sensitivity to IL-2 stimulation. Finally, IL-12-primed human PBMCs generated tumor-specific T cells both phenotypically and functionally similar to IL-12-primed mouse tumor-specific T cells. These results highlight the ability of IL-12 to obviate the strict requirement for administering high levels of IL-2 during adoptive cell transfer-mediated antitumor responses. Furthermore, acquisition of a potent effector phenotype independent of cytokine support suggests that IL-12 could be added to adoptive cell transfer clinical strategies in cancer patients.
Project description:Because cytokine-priming signals direct CD8(+) T cells to acquire unique profiles that affect their ability to mediate specific immune responses, here we generated IL-9-skewed CD8(+) T (Tc9) cells by priming with Th9-polarized condition. Compared with type-I CD8(+) cytotoxic T (Tc1) cells, Tc9 secreted different cytokines and were less cytolytic in vitro but surprisingly elicited greater antitumor responses against advanced tumors in OT-I/B16-OVA and Pmel-1/B16 melanoma models. After adoptive transfer, Tc9 cells persisted longer and differentiated into IFN-?- and granzyme-B (GrzB)-producing cytolytic Tc1-like effector cells. Phenotypic analysis revealed that adoptively transferred Tc9 cells secreted IL-2 and were KLRG-1(low) and IL-7R?(high), suggesting that they acquired a signature of "younger" phenotype or became long-term lived cells with capacity of self-renewal. Our results also revealed that Tc9-mediated therapeutic effect critically depended on IL-9 production in vivo. These findings have clinical implications for the improvement of CD8(+) T-cell-based adoptive immunotherapy of cancers.
Project description:Determining how tumor immunity is regulated requires understanding the extent to which the anti-tumor immune response "functions" in vivo without therapeutic intervention. To better understand this question, we developed advanced multimodal reflectance confocal/two photon fluorescence intra-vital imaging techniques to use in combination with traditional ex vivo analysis of tumor specific T cells. By transferring small numbers of melanoma-specific CD8+ T cells (Pmel-1), in an attempt to mimic physiologic conditions, we found that B16 tumor growth alone was sufficient to induce naive Pmel-1 T cell proliferation and acquisition of effector phenotype. Tumor -primed Pmel-1 T cells, are capable of killing target cells in the periphery and secrete IFN?, but are unable to mediate tumor regression. Within the tumor, Pmel-1 T cells have highly confined mobility, displaying long term interactions with tumor cells. In contrast, adoptively transferred non tumor-specific OT-I T cells show neither confined mobility, nor long term interaction with B16 tumor cells, suggesting that intra-tumor recognition of cognate self antigen by Pmel-1 T cells occurs during tumor growth. Together, these data indicate that lack of anti-tumor efficacy is not solely due to ignorance of self antigen in the tumor microenvironment but rather to active immunosuppressive influences preventing a protective immune response.
Project description:We have shown that DC vaccine is superior to peptide vaccine in terms of priming and expansion of antigen-specific CD8+ T cells. DC vaccine-primed pmel-1 cells displayed better effecter functions than cells by peptide-primed cells in terms of cytokine production and externalization of cytotoxic granules. Furthermore DC vaccine-primed cells were metabolically distinct from peptide-primed cells. To confirm these findings, we performed a microarray analysis using splenic pmel-1 T cells from mice immunized with hgp100 peptide vaccine or DC vaccine. We also used splenic naïve pmel-1 T cells as a control. Overall design: C57BL/6 mice transferred with pmel-1 splenocytes were treated with hgp100 peptide vaccine or DC vaccine twice on days 0 and 14. On day 28, pmel-1 CD8+ T cells were sorted from harvested splenocytes of immunized mice. Gene expression was measured in naïve pmel-1 CD8+ T cells and sorted pmel-1 CD8+ T cells
Project description:Engineering CD8? T cells to deliver interleukin 12 (IL-12) to the tumor site can lead to striking improvements in the ability of adoptively transferred T cells to induce the regression of established murine cancers. We have recently shown that IL-12 triggers an acute inflammatory environment that reverses dysfunctional antigen presentation by myeloid-derived cells within tumors and leads to an increase in the infiltration of adoptively transferred antigen-specific CD8? T cells. Here, we find that local delivery of IL-12 increased the expression of Fas within tumor-infiltrating macrophages, dendritic cells, and myeloid-derived suppressor cells (MDSC), and that these changes were abrogated in mice deficient in IL-12-receptor signaling. Importantly, upregulation of Fas in host mice played a critical role in the proliferation and antitumor activity of adoptively transferred IL-12-modified CD8? T cells. We also observed higher percentages of myeloid-derived cell populations within tumors in Fas-deficient mice, indicating that tumor stromal destruction was dependent on the Fas death receptor. Taken together, these results describe the likely requirement for costimulatory reverse signaling through Fasl on T cells that successfully infiltrate tumors, a mechanism triggered by the induction of Fas expression on myeloid-derived cells by IL-12 and the subsequent collapse of the tumor stroma.
Project description:Myeloid-derived cells comprising the tumor stroma represent a heterogeneous population of cells critical to the structure, function and growth of established cancers. We have recently found that engineering tumor-specific CD8+ T cells to secrete IL-12 (IL-12TD) can lead to striking improvements in T-cell activity against established melanomas in murine models. Surprisingly, IL-12-dependent enhancement of CD8+ T-cell anti-tumor function did not occur through direct ligation of receptors on lymphocytes or NK cells. Instead, IL-12 sensitized host bone marrow-derived tumor-stromal cells, partly through interferon-gamma, to indirectly enhance the effects of adoptively-transferred T cells. Direct presentation of antigen by tumor was not necessary, but MHC class I expression on endogenous cells was essential for IL-12 mediated anti-tumor enhancements. Upon successful treatment with IL-12TD cells, we observed the selective elimination of tumor-infiltrating CD11b+ F4/80+ macrophages, CD11b+/ClassII+/CD11c+ dendritic cells and CD11b+/Ly6C+/Ly6G- but not CD11b+/Ly6C+/Ly6G+ myeloid-derived suppressor cells within regressing lesions. These results are consistent with a model whereby IL-12 triggers the maturation of myeloid-derived cells into competent antigen cross-presenting cells. Licensed recognition of these antigens by effector T cells may in turn trigger the collapse of the tumor stroma and aid in the regression of large vascularized lesions. Samples were collected at 3 days and 7 days from tumors treated in-vivo with no treatment, Mock pmel-1 CD8+ cell treatment, or IL-12 pmel-1 CD8+ cell treatment. There were 4 biological replicates of each sample type. There were a total of 24 samples.
Project description:Cardiac antigen-specific CD8(+) T cells are involved in the autoimmune component of human myocarditis. Here, we studied the differentiation and migration of pathogenic CD8(+) T cell effector cells in a new mouse model of autoimmune myocarditis. A transgenic mouse line was derived that expresses cardiac myocyte restricted membrane-bound ovalbumin (CMy-mOva). The endogenous adaptive immune system of CMy-mOva mice displays tolerance to ovalbumin. Adoptive transfer of naive CD8(+) T cells from the ovalbumin-specific T cell receptor-transgenic (TCR-transgenic) OT-I strain induces myocarditis in CMy-mOva mice only after subsequent inoculation with ovalbumin-expressing vesicular stomatitis virus (VSV-Ova). OT-I effector T cells derived in vitro in the presence or absence of IL-12 were adoptively transferred into CMy-mOva mice, and the consequences were compared. Although IL-12 was not required for the generation of cytolytic and IFN-gamma-producing effector T cells, only effectors primed in the presence of IL-12 infiltrated CMy-mOva hearts in significant numbers, causing lethal myocarditis. Furthermore, analysis of OT-I effectors collected from a mediastinal draining lymph node indicated that only effectors primed in vitro in the presence of IL-12 proliferated in vivo. These data demonstrate the importance of IL-12 in the differentiation of pathogenic CD8(+) T cells that can cause myocarditis.
Project description:It is commonly believed that T cells have difficulty reaching tumors located in the brain due to the presumed "immune privilege" of the central nervous system (CNS). Therefore, we studied the biodistribution and anti-tumor activity of adoptively transferred T cells specific for an endogenous tumor-associated antigen (TAA), gp100, expressed by tumors implanted in the brain. Mice with pre-established intracranial (i.c.) tumors underwent total body irradiation (TBI) to induce transient lymphopenia, followed by the adoptive transfer of gp100(25-33)-specific CD8+ T cells (Pmel-1). Pmel-1 cells were transduced to express the bioluminescent imaging (BLI) gene luciferase. Following adoptive transfer, recipient mice were vaccinated with hgp100(25-33) peptide-pulsed dendritic cells (hgp100(25-33)/DC) and systemic interleukin 2 (IL-2). This treatment regimen resulted in significant reduction in tumor size and extended survival. Imaging of T cell trafficking demonstrated early accumulation of transduced T cells in lymph nodes draining the hgp100(25-33)/DC vaccination sites, the spleen and the cervical lymph nodes draining the CNS tumor. Subsequently, transduced T cells accumulated in the bone marrow and brain tumor. BLI could also detect significant differences in the expansion of gp100-specific CD8+ T cells in the treatment group compared with mice that did not receive either DC vaccination or IL-2. These differences in BLI correlated with the differences seen both in survival and tumor infiltrating lymphocytes (TIL). These studies demonstrate that peripheral tolerance to endogenous TAA can be overcome to treat tumors in the brain and suggest a novel trafficking paradigm for the homing of tumor-specific T cells that target CNS tumors.
Project description:Concurrent helminth infection potently inhibits T cell immunity; however, whether helminthes prevent T cell priming or skew clonal recruitment and effector differentiation is not known. Using coinfection with two natural mouse pathogens, Heligmosomoides polygyrus and Toxoplasma gondii, to investigate the negative impact of helminthes on the CD8 T cell response, we demonstrate helminth-induced suppression of IL-12-dependent differentiation of killer-like receptor G1+ effector CD8 T cells and IFN-? production. Nevertheless, reversal of helminth suppression of the innate IL-12 response of CD8?+ dendritic cells, which occurred in STAT6-deficient mice, was not sufficient to normalize CD8 T cell differentiation. Instead, a combined deficiency in IL-4 and IL-10 was required to reverse the negative effects of helminth coinfection on the CD8 T cell response. Monoclonal T. gondii-specific CD8 T cells adoptively transferred into coinfected mice recapitulated the spectrum of helminth-induced effects on the polyclonal CD8 T response, indicating the lack of requirement for clonal skewing.