The Preclinical Service Core (PSC) projects have developed from transplantation protocols implemented by the clinical staff of ETIB. Using peripheral blood and marrow, and tumor and CGVHD tissue biopsies, we have evaluated lymphocyte subsets, cytokine content, T cell receptor repertoire diversity and thymopoietic activity. All data are incorporated into protocol-specific spreadsheets, linking samples to protocol arms and transplant time points, and are accessible by branch clinicians over secure NIH networks.Effects of IL-7 administration: In a Phase I clinical trial of IL-7 administration (P. I. Claude Sportes: 03-C-0152), we determined that IL-7 significantly expanded naive and central memory CD4 and CD8 T cells, as compared to effector cells, resulting in a significant increase in the diversity of the overall T cell receptor repertoire and identifying a concurrent effect on B lymphopoiesis (Sportes et al 2009;Sportes et al 2010). The PSC supports a newly initiated vaccine trial (11-C-0146;P.I. Claude Sportes), that assesses the effect of IL-7 on vaccine responses in older patients, by assessing effects of a new IL-7 formulation on T and B cell populations and T cell receptor repertoire.Cytokine and monokine production following based transplant regimens incorporating ex vivo expanded cytokine defined T effectors: An ongoing project has assessed lymphokine and monokine production capacity post transplant, focusing on allogeneic transplants incorporating immune therapy with Th2.rapa (P. I. Dan Fowler lymphoma and renal carcinoma: 04-C-0055, 08-C-0088) or T1/T2 donor-derived cells (P.I. Michael Bishop metastatic breast cancer: 04-C-0131). In the early post-transplant period, the cytokine and monokine production capacity of peripheral blood cells has been determined by generation of anti CD3/anti CD28-stimulated stimulated supernatants and by the assessment of the frequencies of cytokine producing T cells by flow cytometry. These assays support these protocols by evaluating the extent and durability of the cytokine shift resulting from the infusion of T1/T2 or Th2-rapa cells (Hardy et al, 2011). Immune reconstitution following lymphodepletion: In several ongoing ETIB clinical trials of allogeneic stem cell transplantation therapies (04-C-0055, 07-C-0195, 09-C-0210;09-C-0096;PIs Daniel Fowler, Michael Bishop, Steven Pavletic and Dennis Hickstein) the process of immune reconstitution has been assessed, focusing on the factors contributing to individual variation in recovery. These data have contributed to a report on the efficacy of EPOCH-F, a novel non-myeloablative induction regimen, in allogeneic stem cell transplantation in patients with multiple myeloma and lymphophoid malignancies(Jamshed et al., 2011;Salit et al, 2012). We currently are using 10-color flow cytometery to characterize lymphocyte repopulation and correlate this with thymic recovery, relapse, GVHD and reactivation of chronic latent viruses. These parallel studies involve analysis of three ongoing trials (07-C-0195, 11-C-0016, 11-C-0136) that cover the breadth of current transplant practices: allogeneic transplantation with unrelated donors, autologous transplantation with addition of T1-Rapa adoptive T cell therapy, and myeloablative transplant for acute hematologic malignancies. In addition, when assessment of lineage-specific repopulation is important, we use the ETIB Flow Cytometry Core facility (William Telford) to sort lymphocytes for subset-specific donor chimerism analysis. These assays include ongoing studies on patients transplanted for monogenic immune deficiency involving GATA2 or DOCK8 (09-C-0096, PI: Dennis Hickstein) and on patients receiving dual donor umbilical cord blood (09-C-0210, PI: Michael Bishop). Finally, we have collaborated with the Hematology Branch of the NHLBI to provide analyses of T cell receptor repertoire diversity in long term recipients of allogeneic stem cell transplantation, demonstrating that recipients develop a diverse repertoire after 10 years that is comparable to the donor repertoire (Le et al., 2011).Immune populations and dysfunction in chronic graft vs host disease (CGVHD): In an ongoing natural history protocol (P.I. Steven Pavletic: 04-C-0281), patients who have developed CGVHD following allogeneic transplantation have been evaluated by a multidisciplinary clinical team. The PCS core has supported the study by contributing to the identification of CGVHD biomarkers and the understanding of CGVHD pathogenesis. Two studies of factors contributing to the development of sclerotic skin disease in CGVHD have been accepted (Martires et al.,2011;Martires et al.2011b) as well as a study of correlations of clinical laboratory tests with CGVHD prognosis (Grkovic et al., 2012). Furthermore, we have supported a therapeutic trial for bronchiolitis obliterans, a severe complication of CGVHD (P. I. Ronald Gress and Kirsten Williams: 08-C-0097), by assessing leukotriene receptor (LTR) expression in leukocytes and in bronchial lavage cells to define the role of LTR in progressive fibrosis of lung airways. We support a collaborative trial testing Imatinib therapy on sclerotic cutantaneous CGVHD (Dermatology and Pediatric Branch, protocol 08-C-0148, P.I. Edward Cowen and Kristin Baird), by assessing the effect of this TGFbeta signaling inhibitor on Th17 and Treg populations. We also support a trial testing the efficacy of a mouthwash containing a potent steroid, Clobetasol, as a topical therapy for oral CGVHD (12-C-0068;P.I. Steven Pavletic). Finally, CGVHD has become a significant focus for research in the PCS core. Three PCS projects have been initiated from this study. First, we have determined that plasma levels of the cytokine BAFF are elevated in CGVHD. This increase is significantly correlated both with elevated plasma levels of Interferon (IFN)-induced inflammatory cytokines and with reduced levels of circulating B cells. Second, we have initiated studies into the role of regulatory T cells (Treg) in CGVHD by characterizing Treg in the circulation and in affected tissues. Finally we are using tissue immunohistochemistry, plasma cytokine ELISA and assays of gene expression to test the hypothesis that IFN-induced inflammatory processes may underlie many of the systemic processes in CGVHD (Imanguli et al, 2009;Hakim, 2010). These studies formed the basis for an application for a Staff Scientist Career Development Award, funded in January 2010, This project, detailed under the Innovation Award section, used circulating monocytes as reporter cells for determining the cytokine and chemokine pathways underlying the development of the autoimmune-like symptoms of CGVHD. In coordinated studies, we have assessed plasma levels of cytokines and chemokines and have measured the gene expression patterns of sorted monocytes by microarray and quantitative multiplex RNA measurements. We determined that Type I IFN-driven processes are elevated in CGVHD patients, as compared with normal donors and with non-GVHD transplant recipients. The work may support the development of diagnostic or prognostic assays of CGVHD and contribute to new therapeutic approaches.Relevant cancer sites: Hodgkins Disease/Lymphoma, Non-Hodgkins Lymphoma, Leukemia, Multiple Myeloma. Relevant Research Areas: Immunology, Bone Marrow Transplantation, Autoimmune Disease, SLE/Lupus, Interferon, Hematology/Lymph, Aging, Stem Cell Research, Clinical Research.
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