Browsing by Author "Martinez-Paniagua, Melisa A."
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Item Multifaceted role of BTLA in the control of CD8+ T cell fate after antigen encounter(Clinical Cancer Research, 2018-10) Ritthipichai, Krit; Haymaker, Cara L.; Martinez-Paniagua, Melisa A.; Aschenbrenner, Andrew; Yi, Xiaohui; Zhang, Minying; Kale, Charuta; Vence, Luis M.; Roszik, Jason; Hailemichael, Yared; Overwijk, William W.; Varadarajan, Navin; Nurieva, Roza; Radvanyi, Laszlo G.; Hwu, Patrick; Bernatchez, ChantalePurpose: Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown an overall clinical response rate 40%–50% in metastatic melanoma patients. BTLA (B-and-T lymphocyte associated) expression on transferred CD8+ TILs was associated with better clinical outcome. The suppressive function of the ITIM and ITSM motifs of BTLA is well described. Here, we sought to determine the functional characteristics of the CD8+BTLA+TIL subset and define the contribution of the Grb2 motif of BTLA in T-cell costimulation. Experimental Design: We determined the functional role and downstream signal of BTLA in both human CD8+ TILs and mouse CD8+ T cells. Functional assays were used including single-cell analysis, reverse-phase protein array (RPPA), antigen-specific vaccination models with adoptively transferred TCR-transgenic T cells as well as patient-derived xenograft (PDX) model using immunodeficient NOD-scid IL2Rgammanull (NSG) tumor-bearing mice treated with autologous TILs. Results: CD8+BTLA? TILs could not control tumor growth in vivo as well as their BTLA+ counterpart and antigen-specific CD8+BTLA? T cells had impaired recall response to a vaccine. However, CD8+BTLA+ TILs displayed improved survival following the killing of a tumor target and heightened “serial killing” capacity. Using mutants of BTLA signaling motifs, we uncovered a costimulatory function mediated by Grb2 through enhancing the secretion of IL-2 and the activation of Src after TCR stimulation. Conclusions: Our data portrays BTLA as a molecule with the singular ability to provide both costimulatory and coinhibitory signals to activated CD8+ T cells, resulting in extended survival, improved tumor control, and the development of a functional recall response. Clin Cancer Res; 23(20); 6151–64. ©2017 AACR.Item Single-cell Profiling of Dynamic Cytokine Secretion and the Phenotype of Immune Cells(PLoS ONE, 2017-08) An, Xingyue; Sendra, Victor G.; Liadi, Ivan; Ramesh, Balakrishnan; Romain, Gabrielle; Haymaker, Cara L.; Martinez-Paniagua, Melisa A.; Lu, Yanbin; Radvanyi, Laszlo G.; Roysam, Badrinath; Varadarajan, NavinNatural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled ex vivo revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-?) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-? secretion. Furthermore, we observed that both the amount and rate of IFN-? secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner