Thioredoxin-1 Alters Anti-Tumor T Cell Immuno-Metabolic Phenotype
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ABSTRACT: ABSTRACT Adoptive transfer of tumor epitope reactive T cells is a promising strategy to control tumor growth. However, chronically stimulated T cells expanded for adoptive cell transfer (ACT) are susceptible to cells death in an oxidative tumor microenvironment. Since oxidation of cell surface thiols (c-SH) also alters functionality of proteins, we hypothesized that increased level of thioredoxin, an anti-oxidant molecule that facilitates reduction of proteins by cysteine thioldisulfide exchange, in T cells will result in sustained anti-tumor function. Using Pmel-Trx1 transgenic mouse derived splenic T cells we observed higher c-SH expression that inversely correlated with ROS, and susceptibility to TCR restimulation or oxidation mediated cell death. These Trx1 overexpressing T cells showed CD62Lhi central memory-like (Tcm) phenotype with reduced glucose uptake (2-NBDGlo) and effector function (IFNγlo). Further, using tumor reactive T cells cultured in presence of recombinant Trx resulted in increased dependence of T cells on mitochondrial metabolism and led to improved tumor control. Thus, strategies to increase antioxidant capacity of anti-tumor T cells modulate its immune-metabolic phenotype leading to improved immunotherapeutic control of established tumors.
Project description:Fibroblast growth factor receptors (FGFRs) can act as driving oncoproteins in certain cancers due to mutation, over-expression or activating gene fusions and are therefore attractive drug targets. Here we have characterized tumour cell responses to three new inhibitors of FGFR1-3, AZ12576089, AZ12908010 and the clinical candidate AZD4547, making comparisons with the well-characterized FGFR inhibitor PD173074. Using a panel of 16 human tumour cell lines we show that the anti-proliferative activity of AZ12908010 and AZD4547 is strongly linked to the presence of de-regulated FGFR signalling. In contrast, AZ12576089 was also able to inhibit proliferation of cells lacking de-regulated FGFR, suggesting off-target effects. Acquired resistance to targeted tyrosine kinase inhibitors (TKIs) is a growing problem in the clinic. To assess how FGFR-dependent tumour cells may adapt to long-term exposure to FGFR inhibitors we generated a derivative of the KMS-11 myeloma cell line (FGFRY373C) with acquired resistance to AZ12908010 (KMS-11R cells). Basal P-FGFR3, P-FRS2 and P-ERK1/2 and D-type cyclins were all inhibited by AZ12908010 in parental KMS-11 cells whereas these markers were constitutively elevated and refractory to drug in KMS-11R cells. Sequencing of FGFR3 in KMS-11R cells revealed the presence of a heterozygous mutation at the gatekeeper residue, encoding FGFR3V555M. Consistent with this KMS-11R cells were cross-resistant to AZD4547 and PD173074 but remained fully sensitive to AZ12576089, confirming that the anti-proliferative effects of AZ12576089 are not related to FGFR inhibition. These results define the selectivity and efficacy of two new FGFR inhibitors and identify a secondary gatekeeper mutation as a mechanism of acquired resistance to FGFR inhibitors that should be anticipated as clinical evaluation proceeds.
Project description:Pancreas and spleen were microdissected from wild type (C57B6/SV129 background) E14.5 embryos and P0 newborn pups. Pools of 30-60 tissues were used. Tissues were digested for 1 hour at 37°C in HBSS/0.1% collagenase A/20 µg/ml DNase I, followed by dissociation of the cell clusters into single cells using a non-enzymatic dissociation medium (Sigma). Single cell suspensions were blocked with mouse IgGs and anti-CD16/32 Abs and immunostained with biotin-anti-CD11b (Biolegend) and RPE-conjugated goat anti-CCR2 (R&D Systems), followed by Cy5.5 conjugated streptavidin. CD11b+CCR2+ cell were then isolated by FACS sorting. mRNA from purified cells was prepared using the RNAeasy kit (Qiagen) and run on a MouseWG-6 v2.0 Expression BeadChip.
Project description:To understand why cancer vaccine-induced T cells often fail to eradicate tumors, we studied immune responses in mice vaccinated with gp100 peptide emulsified in incomplete Freund's adjuvant (IFA), commonly used in clinical cancer vaccine trials. After gp100 peptide/IFA vaccination, tumor-specific CD8+ T cells (adoptively transferred from gp100-specific TCR-transgenic pmel-1 mice) accumulated not in tumors but at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, IFN-γ and FasL-mediated apoptosis, resulting in systemic hyporesponsiveness to subsequent vaccination. Provision of anti-CD40 antibody, TLR7 agonist and interleukin-2 (covax) reduced T cell apoptosis but did not prevent vaccination site sequestration. A non-persisting vaccine formulation shifted T cell localization towards tumors, inducing superior anti-tumor activity. Short-lived formulation also reduced systemic T cell dysfunction and promoted memory formation, as shown by gene expression profiling and other measures. Persisting peptide/IFA vaccine depots, currently used to vaccinate cancer patients, can induce specific T cell sequestration at vaccination sites followed by dysfunction and deletion; short-lived depot formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines. To study the fate of melanoma-specific CD8+ T cells after peptide vaccination, we tracked T cell receptor-transgenic pmel-1 T cells in mice vaccinated with heteroclitic gp100_25-33 peptide emulsified in IFA. Splenic pmel-1 CD8+ T cells were purified at 6 and 21 days after vaccination with either gp100/IFA/covax or gp100/saline/covax, and then their total RNA was extracted and used for comparison by gene expression profiling.
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:Spleens from the B6 mice were isolated and single cell suspension was made. CD4 T cells were purified from the splenocytes using magnetic bead separation. Briefly, Splenocytes were incubated with biotinylated antibody cocktail consisting of antibodies (Biolegend) to CD19, B220, CD11b, CD11c, NK1.1, Gr1, CD25 CD8. After a wash step, cells were incubated with streptavidin conjugated magnetic particles (BD Biosciences). After washing, CD4 T cells were isolated by applying a magnetic field and removing the untouched cells. Purified CD4 T cells were then activated with plate-bound anti-CD3 plus anti-CD28 in presence of either Th1 or Th17 or Th1/17 polarizing condition for 3 days. Total RNA from the 3 days differentiated Th1, Th17 and Th1/17 cells was isolated using mirVana miRNA isolation Kit (Invitrogen).
Project description:Effector cells for adoptive immunotherapy can be generated by in vitro stimulation of naïve or memory subsets of CD8+ T cells. While the characteristics of CD8+ T cell subsets are well defined, the heritable influence of those populations on their effector cell progeny is not well understood. We studied effector cells generated from naïve or central memory CD8+ T cells and found that they retained distinct gene expression signatures and developmental programs. Effector cells derived from central memory cells tended to retain their CD62L+ phenotype, but also to acquire KLRG1, an indicator of cellular senescence. In contrast, the effector cell progeny of naïve cells displayed reduced terminal differentiation, and, following infusion, they displayed greater expansion, cytokine production, and tumor destruction. These data indicate that effector cells retain a gene expression imprint conferred by their naïve or central memory progenitors, and they suggest a strategy for enhancing cancer immunotherapy. Experiment Overall Design: Effector cells were generated from naive or central memory CD8+ T cells. The cells were then rested (unstimulated) or restimulated (stimulated). This experimental design resulted in 4 groups (Naïve-derived/stimulated, Naïve-derived/unstimulated, Central memory-derived/stimulated, Central memory-derived/unstimulated). Three replicates from independent experiments were analyzed.
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. 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:To gain insights into the genetic program activated within the distinct vascular and inflammatory transplant microenvironments in Id1/Id3 deficient and WT mice, we performed a series of gene screening experiments using RNA from total graft tissue as well as from myeloid and endothelial (i.e., CD31+ and ISB4+) cells isolated from the grafts at 1 week post-transplantation.
Project description:We found a candidate region (with 55 known or predicted genes) that was found to linkage to the MACS syndrome. Because it is transmitted in an autosomal recessive fashion, and given the fact most recessive disorders are caused by loss-of-function mutations often resulting in decreased mRNA levels, we hypothesized that screening the expression of the various genes located within the disease interval may point to candidate genes of interest. We therefore established fibroblast cell lines from punch skin biopsies obtained from 2 patients and 4 ethnically matched healthy controls. We then compared global gene expression using microarrays in the 6 cell lines (all genes contained within the disease interval were represented on the array).
Project description:Sensitivity to temozolomide (TMZ) is restricted to a subset of glioblastoma patients, with the major determinant of resistance a lack of promoter methylation of the gene encoding the DNA methyltransferase MGMT, although other mechanisms are thought to be active. In a genome-wide screen of paediatric and adult glioma cells, we identified a co-ordinated upregulation of HOX gene expression in the MGMT-independent cell line KNS42. As a recent study has proposed a mechanism for this observation whereby transcriptional activation of the HOXA cluster is reversible by a PI3-kinase inhibitor through an epigenetic mechanism involving histone H3K27 trimethylation, we sought to investigate whether this was active in our system. We thus treated KNS42 cells for 24 hours with the dual PI3-kinase / mTOR inhibitor PI-103 at 5x IC50 and carried out gene expression profiling using Illumina HT-12 microarrays.