Browsing by Author "Romain, Gabrielle"
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Item Beyond Autoantibodies: Biologic Roles of Human Autoreactive B Cells in Rheumatoid Arthritis Revealed by RNA?Sequencing(Arthritis and Rheumatism, 2017-06) Mahendra, Ankit; Yang, Xingyu; Abnouf, Shaza; Adolacion, Jay R.T.; Park, Daechan; Soomro, Sanam; Roszik, Jason; Coarfa, Cristian; Romain, Gabrielle; Wanzeck, Keith; Bridges, Louis Jr. S.; Aggarwal, Amita; Qiu, Peng; Agarwal, Sandeep K.; Mohan, Chandra; Varadarajan, NavinObjective:To obtain the comprehensive transcriptome profile of human citrulline?specific B cells from patients with rheumatoid arthritis (RA). Methods:Citrulline? and hemagglutinin?specific B cells were sorted by flow cytometry using peptide–streptavidin conjugates from the peripheral blood of RA patients and healthy individuals. The transcriptome profile of the sorted cells was obtained by RNA?sequencing, and expression of key protein molecules was evaluated by aptamer?based SOMAscan assay and flow cytometry. The ability of these proteins to effect differentiation of osteoclasts and proliferation and migration of synoviocytes was examined by in vitro functional assays. Results:Citrulline?specific B cells, in comparison to citrulline?negative B cells, from patients with RA differentially expressed the interleukin?15 receptor ? (IL?15R?) gene as well as genes related to protein citrullination and cyclic AMP signaling. In analyses of an independent cohort of cyclic citrullinated peptide–seropositive RA patients, the expression of IL?15R? protein was enriched in citrulline?specific B cells from the patients’ peripheral blood, and surprisingly, all B cells from RA patients were capable of producing the epidermal growth factor ligand amphiregulin (AREG). Production of AREG directly led to increased migration and proliferation of fibroblast?like synoviocytes, and, in combination with anti–citrullinated protein antibodies, led to the increased differentiation of osteoclasts. Conclusion:To the best of our knowledge, this is the first study to document the whole transcriptome profile of autoreactive B cells in any autoimmune disease. These data identify several genes and pathways that may be targeted by repurposing several US Food and Drug Administration–approved drugs, and could serve as the foundation for the comparative assessment of B cell profiles in other autoimmune diseases.Item Detection and isolation of auto-reactive human antibodies from primary B cells(Methods, 12/1/2014) Sendra, Victor G.; Lie, Anthony; Romain, Gabrielle; Agarwal, Sandeep K.; Varadarajan, NavinThe isolation of human monoclonal antibodies (hmAb) has emerged as a versatile platform in a wide variety of contexts ranging from vaccinology to therapeutics. In particular, the presence of high titers of circulating auto-antibodies is implicated in the pathology and outcome of autoimmune diseases. Therefore, the molecular characterization of these hmAb provides an avenue to understanding the pathogenesis of autoimmune diseases. Additionally, the phenotype of the auto-reactive B cells may have direct relevance for therapeutic intervention. In this report, we describe a high-throughput single-cell assay, microengraving, for the screening, characterization and isolation of anti-citrullinated protein antibodies (ACPA) from peripheral blood mononuclear cells (PBMC) of rheumatoid arthritis (RA) patients. Stimulated B cells are profiled at the single-cell level in a large array of sub-nanoliter nanowells (?105), assessing both the phenotype of the cells and their ability to secrete cyclic-citrullinated peptide (CCP)-specific antibodies. Single B cells secreting ACPA are retrieved by automated micromanipulation, and amplification of the immunoglobulin (Ig) heavy and light chains is performed prior to recombinant expression. The methodology offers a simple, rapid and low-cost platform for isolation of auto-reactive antibodies from low numbers of input cells and can be easily adapted for isolation and characterization of auto-reactive antibodies in other autoimmune diseases.Item IgG Fc Domains that Bind C1q but not Effector Fc? Receptors Delineate the Significance of Complement- Mediated Cell Cytotoxicity and Phagocytosis in Antibody Function(Nature Immunol, 2018-06) Lee, Chang-Han; Romain, Gabrielle; Yan, Wupeng; Watanabe, Makiko; Charab, Wissam; Todorova, Bilana; Lee, Jiwon; Triplett, Kendra; Donkor, Moses; Lungu, Oana I.; Lux, Anja; Marshall, Nicholas; Lindorfer, Margaret A.; Goff, Odile R.L.; Balbino, Bianca; Kang, Tae H.; Tanno, Hidetaka; Delidakis, George; Alford, Corrine; Taylor, Ronald P.; Nimmerjahn, Falk; Varadarajan, Navin; Bruhns, Pierre; Zhang, Jessie; Georgiou, GeorgeEngineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fc? receptor (Fc?R). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the Fc?R-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with Fc?R engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.Item Individual motile CD4+ T cells can participate in efficient multikilling through conjugation to multiple tumor cells(Cancer Immunology Research, 2016-05) Liadi, Ivan; Singh, Harjeet; Romain, Gabrielle; Rey-Villamizar, Nicolas; Merouane, Amine; Adolacion, Jay R.T.; Kebriaei, Partow; Huls, Helen; Qiu, Peng; Roysam, Badrinath; Cooper, Laurence J.N.; Varadarajan, NavinT cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multikilling via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that, although CAR4 cells can participate in killing and multikilling, they do so at slower rates, likely due to the lower granzyme B content. Significantly, in both sets of T cells, a minor subpopulation of individual T cells identified by their high motility demonstrated efficient killing of single tumor cells. A comparison of the multikiller and single-killer CAR+ T cells revealed that the propensity and kinetics of T-cell apoptosis were modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation, and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multikilling should be evaluated in the context of their ability to resist activation-induced cell death. We anticipate that TIMING may be used to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. Cancer Immunol Res; 3(5); 473–82. ©2015 AACR.Item Quantitative High-Throughput Single-Cell Cytotoxicity Assay for T cells(Journal of Visualized Experiments, 2013) Liadi, Ivan; Roszik, Jason; Romain, Gabrielle; Cooper, Laurence J.N.; Varadarajan, NavinCancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5. Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8. Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.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