Project description:The goal was to determine how IL-12 affects gene expression by murine CTL. Experiment Overall Design: Following RBC lysis, splenocytes from OT-1 TCR transgenic mice (5 million/ml) were cultured in six well-plates (5-6ml/well) in IMDM supplemented with 10% FCS and 55μM 2-ME with 1μM SIINFEKL. After 4 days, the cells were harvested by purification with Ficoll-Paque PLUS and placed in fresh medium containing 0 or 20ng/ml rmIL-12. Twenty-four hours later the cells were harvested and used in experiments. The live cells harvested were >98% CD8+Vα2+ (OT-1).
Project description:Gene expression analysis of WT and IL-2Ra-deficient CTL (P14) isolated 8 days after inffection with LCMV. The goals of the study are to assess the impact of IL-2 signals on effector and memory CTL differentiation.
Project description:Engineered cytokine-based approaches for immunotherapy of cancer are poised to enter the clinic, with IL-12 being at the forefront. However, little is known about potential mechanisms of resistance to cytokine therapies. We found that orthotopic murine lung tumors were resistant to systemically delivered IL-12 fused to murine serum albumin (MSA, IL12-MSA) due to low IL-12R expression on tumor-reactive CD8+ T cells. IL2-MSA increased binding of IL12-MSA by tumor-reactive CD8+ T cells, and combined administration of IL12-MSA and IL2-MSA led to enhanced tumor-reactive CD8+ T cell effector differentiation, decreased numbers of tumor-infiltrating CD4+ regulatory T (Treg) cells, and increased survival of lung tumor-bearing mice. Predictably, the combination of IL-2 and IL-12 at therapeutic doses led to significant dose-limiting toxicity. Administering IL-12 and IL-2 analogs with preferential binding to cells expressing IL12rb1 and CD25, respectively, led to a significant extension of survival in mice with lung tumors while abrogating dose-limiting toxicity. These findings suggest that IL-12 and IL-2 represent a rational approach to combination cytokine therapy whose dose-limiting toxicity can be overcome with engineered cytokine variants.
Project description:CD8+ cytotoxic T lymphocytes (CTLs) play a major role in defense against intracellular pathogens, and their functions are specified by antigen recognition and innate cytokines. While effector CTLs eliminate the infection, a small population of memory cells are retained that yields more rapid and robust response upon re-infection. Antigen presenting cells secrete an array of innate cytokines including IL-12 and IFN-α after recognition of pathogens. Both IL-12 and IFN-α have been shown to act as the third signal regulating the development of CTLs. We have shown that these two cytokines have a non-redundant effect in generation of human effector CTL. IL-12 alone is sufficient for effector CTL genesis marked by IFN-γ and TNF-α production, as well as increased cytolytic activity. Even in the presence of IFN-α, IL-12 programs CTLs that express the chemokine receptor CXCR3 and effector cytokines. Using microarray analysis we have investigated how IL-12 and IFN-α differentially regulate the genetic programming pathways that give rise to effector CTLs among multiple human donors. We have also analyzed the gene expression patterns of cells sorted from healthy human peripheral blood that display surface markers of effector memory CTL (designated as ex vivo) samples.
Project description:CD8+ cytotoxic T lymphocytes (CTLs) play a major role in defense against intracellular pathogens, and their functions are specified by antigen recognition and innate cytokines. While effector CTLs eliminate the infection, a small population of memory cells are retained that yields more rapid and robust response upon re-infection. Antigen presenting cells secrete an array of innate cytokines including IL-12 and IFN-α after recognition of pathogens. Both IL-12 and IFN-α have been shown to act as the third signal regulating the development of CTLs. We have shown that these two cytokines have a non-redundant effect in generation of human effector CTL. IL-12 alone is sufficient for effector CTL genesis marked by IFN-γ and TNF-α production, as well as increased cytolytic activity. Even in the presence of IFN-α, IL-12 programs CTLs that express the chemokine receptor CXCR3 and effector cytokines. Using microarray analysis we have investigated how IL-12 and IFN-α differentially regulate the genetic programming pathways that give rise to effector CTLs among multiple human donors. We have also analyzed the gene expression patterns of cells sorted from healthy human peripheral blood that display surface markers of effector memory CTL (designated as ex vivo) samples. 5 healthy human donor samples were used for the in vitro cultures. For each donor the CFSE labeled cells (CD8+CD45RA+) were cultured in the presence of neutralized, IL-12, IFN-a, and IL-12+IFN-a conditions and plate-bound anti-CD3+anti-CD28 for 3.5 days. Total RNA from CFSEhi (Undiv) and CFSElo (Div) sorted cells were used for Illumina Bead Array. 4 healthy human donor samples were used for the ex vivo samples. Total RNA was collected from FACS sorted CD8+CCR7hiCXCR3lo and CD8+CCR7loCXCR3hi cells without any stimulation.
Project description:Goal of this study was to compare transcriptional changes in CTL cells compared to Tc17 cells We used microarray to detail the global programme of gene expression underlying CTL and Tc17 cell differentiation and identified distinct classes of upregulated genes thereby
Project description:In this model, TLR2-TLR6 mediates MyD88 pathway gets activated, which activates IL-12 production and induces iNOS expression when the Macrophage is infected with Leishmania parasite. The early induction of IL-10 takes place which leads to the induction of NFIL3, HDAC3, and SHP-1. They inhibit IL-12 production and thus hamper IL-12 induced IFN-gamma mediated; Nitric oxide production. A key transcription factor NFAT5 connects IL-12 and IL-10 pathways. It upregulates IL-12 and downregulates IL-10. In this model, NFAT5 is downregulated which is also inhibiting IL-12.
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.
Project description:Comparison of transcriptional profile of CD8 cytotoxic T lymphocytes terated with the mTORC1 inhibitor rapamycin or the mTOR inhibitor KU-0063794 and comparison with proteomic analysis. Abstract: High resolution mass spectrometry maps the cytotoxic T lymphocyte (CTL) proteome and the impact of mammalian target of rapamycin complex 1 (mTORC1) on CTL. We show that the CTL proteome is dominated by metabolic regulators and granzymes and that mTORC1 selectively represses and promotes expression of a protein subset (~10%) including key CTL effector molecules and signaling proteins. mTORC1 also controlled flux through a subset of metabolic pathways rather than acting as an on/off switch for global CTL metabolism. Proteomic data highlighted the potential for mTORC1 negative control of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production in CTL. Further work revealed that mTORC1 represses PIP3 production and determines the mTORC2 requirement for activation of the serine/threonine kinase AKT. Unbiased proteomic analysis thus provides a comprehensive understanding of CTL identity and mTORC1 control of CTL function.