The Preclinical Development and Clinical Monitoring Facility (PDCMF) 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. In several ongoing ETIB clinical trials of autologous and allogeneic stem cell transplantation therapies (04-C-0055, 07-C-0195, 11-C-0016, 11-C-0136;PIs Daniel Fowler, Steven Pavletic, Claude Sportes, Ronald Gress and Kirsten Williams) we have assessed lymphodepletion during conditioning and immune reconstitution after transplant. We currently are using multiparameter flow cytometry to characterize lymphocyte repopulation, plasma ELISA to monitor cytokines and molecular assays of gene expression and T cell receptor repertoire diversity. We correlate the laboratory findings with clinical outcomes of relapse, infection, and chronic graft versus host disease (CGVHD). These parallel studies involve analysis of ongoing trials that cover the breadth of current transplant practices: non-myeloablative reduced intensity allogeneic transplantation for lymphoma using sibling and unrelated donors, autologous transplantation for myeloma, and myeloablative transplant for acute leukemias. We also support ongoing studies of lineage-specific immune reconstitution in patients transplanted for monogenic immune deficiencies involving GATA2 or DOCK8 (09-C-0096, 10-C-0174, PI: Dennis Hickstein) by utilizing the ETIB Flow Cytometry Facility (William Telford) to sort lymphocytes for subset-specific donor chimerism analysis. These immune monitoring studies 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 lymphoid malignancies (Salit et al, 2012;Salit et al, 2013), as well as reports on allogeneic transplant supported by adoptive transfer of T2-RAPA cells (Fowler et al, Blood 2013). Chronic graft vs host disease (CGVHD), the principle cause of non-relapse morbidity and mortality after allogeneic transplantation, is a major focus for research in the PDCMF core. We have supported the efforts of the multidisciplinary clinical team in an ongoing natural history protocol studying patients who have developed CGVHD (P.I. Steven Pavletic: 04-C-0281) by storing patient blood, biofluids and tissue for research. We have contributed to the research study by analyzing mechanisms of CGVHD pathogenesis. Furthermore, we have supported four therapeutic trials for CGVHD patient populations: (1) We are assessing leukotriene receptor (LTR) expression in leukocytes and in bronchial lavage cells to define the role of LTR in progressive fibrosis of lung airways in patients developing bronchiolitis obliterans, a severe complication of CGVHD (08-C-0097: P. I. Ronald Gress and Kirsten Williams:). (2) We have assessed the effect of Imatinib, a TGFbeta signaling inhibitor, on Th17 and Treg populations, as part of 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). (3) We are analyzing changes in inflammatory gene expression in a trial of Pomalidomide (12-C-0197;P.I. Steven Pavletic) to assess efficacy against fibrosis in CGVHD patients. (4) Finally we support tissue immunohistochemistry analyses and salivary cytokine tests in a trial of a mouthwash containing a potent steroid, Clobetasol, as a topical therapy for severe oral CGVHD (12-C-0068;P.I. Steven Pavletic/Jacqueline Mays). As part of our studies of the pathogenesis of CGVHD, we have characterized regulatory T cells in CGVHDthrough coordinated studies of blood and tissue. These studies determined that FoxP3+ regulatory T cells (Treg) increased in proportion to T effectors in tissue infiltrates in oral and cutaneous lichenoid cGVHD. Both T effector and FoxP3+ Treg cells expressed Tbet and the chemokine receptor CXCR3, consistent with a common mechanism of chemokine-mediated migration into tissue. Furthermore, functional markers (ICOS and CD39) and chemokine receptors (CXCR3) were both present in a higher proportion of FoxP3+ cells in tissues than in peripheral blood, consistent with recruitment and activation of Treg in cGVHD target tissues. Finally, the 'activated'CD45RA-FoxP3hi subset of Treg cells, which highly expresses functional markers, was found in comparable frequencies in cGVHD patients and normal controls, despite a significant deficit in naive 'resting'Treg. These findings are consistent with Treg functional capacity in cGVHD, and support efforts to expand Treg cells in vivo as therapy. (Imanguli et al, Leukemia 2014) In studies of CGVHD pathogenesis, we profiled gene expression of circulating monocytes in CGVHD patients, to use these cells as in situ reporter cells for systemic cytokine patterns in order to test the hypothesis that Interferon (IFN)-induced inflammatory processes may underlie many of the systemic processes in CGVHD (Imanguli et al, 2009;Hakim, 2010). Based on microarray analysis and confirmed by multiplex RNA assessments (supported by an NCI Staff Scientist Career Development Award), we determined that pathways induced by IFN were consistently upregulated across a broad spectrum of CGVHD patients, both those with severe inflammatory infiltrates in tissue and those with widespread sclerotic involvement. IFN-inducible genes, including ones specifically induced by type I IFN, were upregulated in parallel at the time of onset of CGVHD, and were reduced during treatment and after resolution of CGVHD symptoms. In addition, we found a consistent upregulation of receptor genes from the innate immune TLR/NLR/CLR pathways;these pathways are triggered by debris from dead cells to stimulate phagocytosis, induce IFNa production, and form inflammasomes. Many of these receptors are also inducible by IFN, consistent with a self-reinforcing inflammatory process sustaining CGVHD. Furthermore, through assessments of glucocorticoid-inducible genes, we discriminated the confounding effects of immunosuppressive steroids from disease-associated gene patterns. Finally, we corroborated the gene expression data through analyses of IFN-inducible factors in plasma and tissue. These findings substantiated a role for IFN in lymphocyte and dendritic cell trafficking into tissue, and, through upregulation of BAFF, an involvement of IFN in B cell activation in CGVHD. These interlocking assessments, performed on a broad spectrum of patients severely affected with CGVHD, support a new model for the initiation and persistence of CGVHD. This model defines CGVHD as a disorder of inflammation-driven immune dysregulation and these results support a new range of options for therapy of CGVHD through interdiction of interferon pathways.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Scientific Cores Intramural Research (ZIC)
Project #
1ZICBC010934-07
Application #
8938439
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Poe, Jonathan C; Jia, Wei; Su, Hsuan et al. (2017) An aberrant NOTCH2-BCR signaling axis in B cells from patients with chronic GVHD. Blood 130:2131-2145
Cooke, Kenneth R; Luznik, Leo; Sarantopoulos, Stefanie et al. (2017) The Biology of Chronic Graft-versus-Host Disease: A Task Force Report from the National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease. Biol Blood Marrow Transplant 23:211-234
Pirsl, Filip; Curtis, Lauren M; Steinberg, Seth M et al. (2016) Characterization and Risk Factor Analysis of Osteoporosis in a Large Cohort of Patients with Chronic Graft-versus-Host Disease. Biol Blood Marrow Transplant 22:1517-1524
Hakim, Frances T; Memon, Sarfraz; Jin, Ping et al. (2016) Upregulation of IFN-Inducible and Damage-Response Pathways in Chronic Graft-versus-Host Disease. J Immunol 197:3490-3503
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Grossman, Jennifer; Cuellar-Rodriguez, Jennifer; Gea-Banacloche, Juan et al. (2014) Nonmyeloablative allogeneic hematopoietic stem cell transplantation for GATA2 deficiency. Biol Blood Marrow Transplant 20:1940-8
Fowler, Daniel H; Mossoba, Miriam E; Steinberg, Seth M et al. (2013) Phase 2 clinical trial of rapamycin-resistant donor CD4+ Th2/Th1 (T-Rapa) cells after low-intensity allogeneic hematopoietic cell transplantation. Blood 121:2864-74

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