Project description:We designed a novel intratumoral (IT) IL-12 mRNA therapy to promote local IL-12 tumor production whilst mitigating systemic effects. A single IT dose of mouse (m)IL-12 mRNA induced IFNγ and CD8+ T cell-dependent tumor regression in multiple syngeneic mouse models including MC38-R, and animals with a complete response demonstrated immunity to re-challenge. In order to further investigate the impact of mIL-12 mRNA on the MC38-R TME, we performed tumor transcriptomic evaluation 24h and 7 days after a single IT dose of 0.5 micrograms IL-12 or control mRNA. mRNA was isolated from snap-frozen syngeneic tumors using an RNeasy mini kit (Qiagen) and quantified on the Affymetrix mouse 430 2.0 microarray.
Project description:We conducted immune- and RNA-sequencing of HLA-A24-restricted CMVpp65-specific CTLs to better understand the immune reconstitution of CMV-CTLs after allo-HCT. To the best of our knowledge, this is the first report on the features of TCRβ-CDR3, diversity, and GEP of HLA-A24 CMV-CTLs according to the CMV-reactivation pattern among recipients after allo-HCT. In addition, we further sought to demonstrate homogeneity or heterogeneity according to individual CTL clones using single-cell RNA-sequencing technology.
Project description:TGFb is a pleiotropic cytokine which can exert its regulatory effects on differentiation of cytotoxic T lymphocytes (CTLs) and alter their cell fate. Canonical TGFb signaling inhibits the formation of KLRG1+ effector cells and circulating memory cells while inducing the formation of resident memory cells. However, SMAD4 functions contradictory to TGFb where it is required for the formation of KLRG1+ effector cells and circulating memroy cells and actively inhibit the formation of resident memory cells. Using, RNA-Sequencing we identified that Smad4 alters the gene expression profile of pathogen-specific CTLs after intranasal infection with Listeria monocytogenes expressing chicken ovalbumin.
Project description:Celiac disease is an intestinal inflammatory disorder induced by dietary gluten in genetically susceptible individuals. The mechanisms underlying the massive expansion of interferon g–producing intraepithelial cytotoxic T lymphocytes (CTLs) and the destruction of the epithelial cells lining the small intestine of celiac patients have remained elusive. We report massive oligoclonal expansions of intraepithelial CTLs that exhibit a profound genetic reprogramming of natural killer (NK) functions. These CTLs aberrantly expressed cytolytic NK lineage receptors, such as NKG2C, NKp44, and NKp46, which associate with adaptor molecules bearing immunoreceptor tyrosine-based activation motifs and induce ZAP-70 phosphorylation, cytokine secretion, and proliferation independently of T cell receptor signaling. This NK transformation of CTLs may underlie both the self-perpetuating, gluten-independent tissue damage and the uncontrolled CTL expansion leading to malignant lymphomas in severe forms of celiac disease. Because similar changes were detected in a subset of CTLs from cytomegalovirus-seropositive patients, we suggest that a stepwise transformation of CTLs into NK-like cells may underlie immunopathology in various chronic infectious and inflammatory diseases. Keywords: NKG2C; LAK; CTL; NK receptor; IEL; Mucosal Immunity; Celiac Disease
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:Bach2 codes for a transcriptional regulator exerting major influences on T cell mediated immune regulation. Effector CTLs derived from in vitro activation of murine CD8+ T cells showed increased proliferative and cytolytic capacity in the absence of BACH2. Before activation, BACH2-deficient CD8+ T cells had a higher abundance of memory and reduced abundance of naïve cells compared to wild-type. CTLs derived from central memory T cells were more potently cytotoxic than those derived from naïve T cells, but even within separated subsets, BACH2-deficiency conferred a cytotoxic advantage. Immunofluorescence and electron microscopy revealed larger granules in BACH2-deficient compared to wild-type CTLs, and proteomic analysis showed an increase in granule content, including perforin and granzymes. Thus, the enhanced cytotoxicity observed in effector CTLs lacking BACH2 arises not only from differences in their initial differentiation state but also inherent production of enlarged cytolytic granules. These results demonstrate how a single gene deletion can produce a CTL super-killer.
Project description:Copy number analyses of regionally separated intratumoral biopsies of prostate cancers. Intratumoral heterogeneity (ITH) leads to regional biases of the mutational landscape in a single tumor and may influence the single biopsy-based clinical diagnosis and treatment decision. To evaluate the extent of ITH in unifocal prostate cancers (PCAs) that had not been sought, we analyzed multiple regional biopsies from three PCAs using DNA copy number analyses. DNA copy number showed ITH including regional biases in the presentation of a well-known driver of TMPRSS2-ERG fusion. Our analyses identified a substantial level of genetic ITH in unifocal PCAs at the genomic levels, which should be taken into account for the curation of biomarkers in the clinical setting. Four intratumoral biopsies were obtained per tumor for three prostate cancers. Radical prostatectomy tissue from three patients with prostate cancers were obtained. Board-certified pathologists reviewed the hematoxylin&eosin stained sections and identified tumor-rich regions (> 80% purity). We selected four different areas for biopsy that were at least 5mm apart and were comprised of the most common Gleason pattern (the most common histologic patterns with minimal histologic differences). Copy number profiling was performed using Agilent 180K platform according to the manufacturer's protocol.