FALGUNI PATHAK HOUSTON PLUS

FALGUNI PATHAK HOUSTON PLUS

After 72В hours, CFSE dilution profile of CD8В T cells were obtained by flow cytometry B) Representative CFSE plots for unstimulated anti-CD3- and anti-CD28- stimulated splenocytes, plus control or TGFОІВЇ MDSC (left). Control or TGFОІ-MDSC were then co-cultured at different ratios with CFSE labelled naГЇve splenocytes (activated with soluble anti-CD3 and anti-CD28 antibodies). A) Histogram overlay and corresponding bar graphs (normalized to no treatment) showing mean fluorescent intensity (MFI) of iNOS, Nitric oxide (NO), Reactive oxygen species (ROS), and Arginase (ARG) in MDSC from no treatment, control or TGFОІ-MDSC (nВ =В 9). Control and TGFОІВЇMDSC were isolated and screened for functional markers. TGFОІ-MDSC lose immunosuppressive function, promote T cell proliferation and present antigen. Error bars indicate standard error of mean (SEM). Each graph summarizes data obtained from at least 3 experiments. Error bars indicate standard error of the mean (SEM). G) Summary of MHC Class II, CD86 and Fas-L expression in TGFОІВЇ MDSC generated in the presence or absence of 3В ВµM of SMAD-2 inhibitor, SB431542 (nВ =В 12). F) Representative flow cytometric histogram overlay (top) showing cell surface expression of MHC II, CD86, Flt-3 and Fas-L on MDSC generated from no treatment, control and TGF-ОІ conditioned tumor medium with summary bar graph below showing mean fluorescence intensity (nВ =В 5вЂ“9 per graph). E) Representative flow cytometric histogram overlay of p-SMAD2 expression of control and TGFОІ-MDSC (top), and bar graph showing cumulative data from atleast 3 experiments. Control MDSC samples are represented as red dots TGFОІ-MDSC samples as blue dots D) Heat map of proteins (nВ =В 16) most differentially-expressed between control and TGFОІ-MDSC (p-value В 1.5 or <В 0.8). x, y, and z axes represent three major principal components. C) Principal Component analysis (PCA) of the RPPA data. C-D) Protein lysates from control or TGF-ОІ MDSC were profiled with Reverse Phase Protein Assay (RPPA). A) Representative flow cytometry plots showing percent of total MDSC (nВ =В 16) along with their respective Giemsa images depicting morphology of CD11b+В cells B) Quantitation of different cellular fractions expressed as a stacked bar graph (nВ =В 8вЂ“12/group). After 5В days in culture MDSC were evaluated by flow cytometry or Giemsa staining. NaГЇve bone marrow cells cultured in medium only (no treatment) or bone marrow cells cultured with MEER tumor conditioned medium alone (Control) or in the presence of TGF-ОІ1 (TGF-ОІ1) were used to generate MDSC. TGFОІ-MDSC acquire more macrophage like phenotype and upregulate maturation markers. Thus, TGFОІ1 mediated programming of nascent MDSC leads to a potent anti-tumor phenotype potentially suitable for adoptive immunotherapy.Д¬D86 MDSC SMAD2 TGF-beta adoptive cellular therapy caspase-3 myeloid-derived suppressor cells (written out) radiotherapy tumor killing. TGFОІ-MDSC derived from human PBMC with tumor conditioned medium also lost immunosuppressive function and acquired tumor-killing activity. Radiation upregulated FAS expression on tumor cells, and the combination of radiotherapy and intratumoral injection of TGFОІ-MDSC strongly enhanced class I expression on tumor cells and induction of HPV E7 tetramer-positive CD8 + T cells, leading to clearance of established tumors and long-term survival. TGFОІ-MDSC also upregulated FAS-ligand expression, leading to FAS-dependent killing of murine human papillomavirus (HPV)-associated head and neck cancer cells and tumor spheroids in vitro and anti-tumor activity in vivo. Altered immune function was associated with SMAD-2 dependent upregulation of maturation and costimulatory molecules, and downregulation of inducible nitric oxide synthase (iNOS), an effector mechanism of immunosuppression. While MDSC derived ex vivo from murine bone marrow precursor cells with tumor-conditioned medium efficiently suppressed T cell proliferation, MDSC derived from conditioned medium in presence of TGF-ОІ1 (TGFОІ-MDSC) acquired a novel immune-stimulatory phenotype, losing the ability to inhibit T cell proliferation and acquiring enhanced antigen-presenting capability. MDSC programming or polarization has been proposed as a strategy for leveraging the developmental plasticity of myeloid cells to reverse MDSC immune suppressive functions, or cause them to acquire anti-tumor activity. Cancer-induced myeloid-derived suppressor cells (MDSC) play an important role in tumor immune evasion.

Falguni pathak houston