Runx1-Cbfb Interacts with CHD7, a Chromatin Modifying Enzyme with a Potential Role in Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1300-1300
Author(s):  
Jingmei Hsu ◽  
Chung-Tsai Lee ◽  
Scott Gerber ◽  
Shuqian Yu ◽  
Nancy A. Speck
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Heart Defects ◽  
Genetic Alterations ◽  
Transactivation Domain ◽  
Hematopoietic Stem

Abstract Abstract 1300 Mutations in RUNX1 and CBFB are among the most common genetic alterations in hematologic malignancies, including acute myeloid and lymphoid leukemia (AML, ALL), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome and myeloproliferative neoplasms. Loss of Runx1-CBFb causes a failure of hematopoietic stem cell emergence during embryogenesis. Critical roles for Runx1-CBFb in adult hematopoiesis include hematopoietic stem and progenitor homeostasis, and lymphoid and megakaryocytic differentiation. We took an unbiased co-immunoprecipitation and mass spectrometry approach to identify Runx1-CBFb co-regulators in T cells, and identified chromodomain helicase binding protein 7 (CHD7) as a potential interacting partner. CHD7 is an ATP-dependent chromatin remodeling protein that primarily occupies enhancer and promoter regions. Autosomal dominant mutations in CHD7 cause CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). It was shown that CHD7 interacts with Sox2, and its occupancy correlates with H3K4me1/2 modifications and P300 binding at enhancer regions, and H3K4me3 marks at promoters. We confirmed the interaction of endogenous Runx1 and CHD7 in T cells. We demonstrate that the Runx1 transactivation domain, which is critical at all stages of hematopoiesis, is required for the CHD7 interaction. To elucidate an in vivo function for CHD7 in hematopoiesis, we generated a conditional pan-hematopoietic Chd7 deletion in mice using a floxed Chd7 allele and Vav1-Cre. Deletion of Chd7 in hematopoietic cells appears to cause no lineage specific defects. However, CHD7 deficient bone marrow cells had a competitive advantage in T cell reconstitution as compared to wild type cells, suggesting a role for CHD7 in restraining T cell numbers in the adult. Determining how CHD7 exerts its functions should shed light on underlying mechanisms in hematopoietic stem cell formation, T cell development, and hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Interruption of the IFN γR/CXCR3 Axis Results in Altered T Cell Trafficking In Vivo and Abrogation of GvHD While Maintaining a Robust Gvl Response

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2971-2971
Author(s):  
Jaebok Choi ◽  
Edward Dela Ziga ◽  
Julie Ritchey ◽  
Julie Prior ◽  
Lynne Collins ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell ◽  
Neutralizing Antibodies ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
Target Organs ◽  
Donor T Cells

Abstract Abstract 2971 Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with relapsed/refractory leukemia, and marrow failure states such as myelodysplasia and aplastic anemia. However, allo-HSCT is complicated by allogeneic donor T cell-mediated graft-versus-host disease (GvHD) which can be life-threatening especially in recipients of unrelated or HLA-mismatched hematopoietic stem cell products. These same alloreactive donor T cells also mediate a beneficial graft-versus-leukemia (GvL) effect. Thus, the clinical goal in allo-HSCT is to minimize GvHD while maintaining GvL. Recent studies have suggested that this might be achieved by infusing regulatory T cells (Tregs) which in some preclinical models suppress GvHD-causing alloreactive donor T cells but have only limited effects on GvL-promoting alloreactive donor T cells. Unfortunately, Tregs exist in low frequency in the peripheral blood, are costly to purify and expand, and after expansion are difficult to isolate due to the lack of cell surface markers, all of which prevent their routine use in the clinic. Thus, alternative therapeutic approaches that do not require Tregs are needed. Using a MHC-mismatched GvHD model, B6 (H-2b) → Balb/c (H-2d), we demonstrated that infusion of IFN γR deficient allogeneic donor T cells induce significantly less GvHD, compared to WT T cells, determined by survival (74% vs. 0 % in overall survival; p =0.0004), weight and percentages of B220+ B cells (12.4% vs. 3.8%; p =0.0205), CD3+ T cells (14.3% vs. 4.3%; p =0.0025) in blood. Of note was that the IFN γR deficient donor T cells maintained a beneficial GvL effect, which was examined in both a systemic leukemia and a solid tumor model using luciferase-expressing A20 cells derived from Balb/c. We found that IFN γR deficient donor T cells responded normally to allogeneic antigens as measured by in vitro mixed lymphocyte reaction analyses, and express similar levels of granzyme B, compared to WT T cells. However, IFN γR deficient T cells trafficked predominantly to the spleen while WT T cells trafficked to gastrointestinal tract and peripheral lymph nodes, which are major GvHD target organs, based on in vivo bioluminescence imaging. All of these findings suggest that the reduced GvHD was not due to reduced function, altered subsets or relative deficiency of allogeneic donor T cells but from modification of in vivo trafficking of IFN γR deficient donor T cells compared to WT T cells. We further demonstrated that the IFN γR-mediated signaling in alloreactive donor T cells was required for expression of CXCR3 which has been implicated in trafficking of T cells to areas of inflammation and target organs, commonly known to be the sites of GvHD. CXCR3−/− T cells demonstrated a reduction in GvHD while maintenance of the same robust GvL effect using the same MHC mismatched transplant model. Thus, the IFN γR-CXCR3 axis represents a promising therapeutic target for future efforts to mitigate GvHD while maintaining GvL after allo-HSCT. Current studies are focused on 1) whether forced expression of CXCR3 rescues the GvHD-inducing potential of IFN γR deficient donor T cells and 2) if inhibition of IFN γR signaling (IFN γR, JAK1 and/or JAK2, CXCR3 and STAT1) using both neutralizing antibodies and small molecule inhibitors can recapitulate the anti-GvHD and pro-GvL effects seen in IFN γR−/− and CXCR3−/− T cells. Disclosures: No relevant conflicts of interest to declare.

Recruitment Of T Cells and Macrophages To The Eyes In Recipients Of Allogeneic Hematopoietic Stem Cell Transplants Correlate With Inflammatory Cytokine Presence In Ocular Gvhd

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2012-2012
Author(s):  
Samantha Herretes ◽  
Juan C. Murillo ◽  
Duncan Ross ◽  
Tan Yaohong ◽  
Henry Barreras ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell ◽  
Ocular Surface ◽  
Conflicts Of Interest ◽  
Fluorescent Microscopy ◽  
Clinical Signs ◽  
Corneal Tissue ◽  
Hematopoietic Stem

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) has become the standard of care for the treatment of several life-threatening hematologic malignancies as well as certain immunodeficiency disorders. Unfortunately, as the survival rate of patients with these diseases is improved, the quality of life is negatively impacted by the development of Graft vs. Host Disease (GVHD). GVHD is a complex, multi-organ disorder arising from an immunological attack by donor allo-reactive T cells that results in damage to vital organs including the liver, skin, hematopoietic compartment and the ocular surface of the eye. Ocular GVHD occurs in >60% of these patients and is characterized by dry eye, conjunctiva damage, punctate keratopathy, corneal ulceration and perforation. Despite the high frequency of eye involvement in patients undergoing GVHD, little is known regarding the underlying immune mechanisms responsible for ocular GVHD, limiting the ophthalmic care of these patients to palliative therapies and global anti-inflammatory drugs. In this study, we examined the ocular and immunological changes occurring in recipients of MHC-matched, minor antigen mis-matched donor HSCT. C3H.SW (H-2b, Ly9.1+) mice transplanted with EGFP+ B6 (H-2b, Ly9.1-) T cell depleted bone marrow cells (TCD-BM) supplemented with T cells: a) underwent weight loss and began exhibiting clinical signs of GVHD ∼3wks post-HSCT, b) contained damaged thymuses, c) expressed an inverted CD4/CD8 ratio in the peripheral lymphoid compartments, d) contained activated effector cells and e) low CD19 levels. Importantly, these mice also developed ocular surface disease evidenced by progression of ocular surface damage characterized by increased corneal fluorescein staining and ulceration by week 6 (Figure). Furthermore, histological analyses demonstrated that only mice that developed systemic GVHD exhibited corneal thickening and epithelial irregularity. Ocular pathology was also associated with conjunctiva involvement indicated by significant goblet cell destruction as well as dense inflammatory cell infiltrates identified by intra-vital fluorescent microscopy (Figure). IHC and flow analyses demonstrated donor EGFP+ Ly9.1- CD4+ and CD8+ T cells. Notably, significant levels of Ly9.1- EGFP-CD11b+ macrophages had also infiltrated the ocular surface. In contrast to the systemic CD8>CD4 GVHD associated phenotype, the T cell infiltrate in the ocular compartment was reversed; i.e. CD8<CD4. We detected IFNγ and TNFα mRNA from corneal tissue, which is consistent with Th1 effector allo-reactive cells and M1 inflammatory macrophages involvement in ocular GVHD. In total, the present findings have identified alterations and pathology in the eye and adnexa reflective of ocular GVHD and unequivocally demonstrate the presence of donor T cells in the ocular surface. We hypothesize that T cell–macrophage interactions underlie the pathology detected in this pre-clinical model and studies are underway to develop local therapeutic modalities targeting these infiltrative populations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Direct evidence for new T-cell generation by patients after either T-cell–depleted or unmodified allogeneic hematopoietic stem cell transplantations

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2235-2242 ◽  
Author(s):  
Sharon R. Lewin ◽  
Glenn Heller ◽  
Linqi Zhang ◽  
Elaine Rodrigues ◽  
Eva Skulsky ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell ◽  
Opportunistic Infections ◽  
Cell Receptor ◽  
Hematopoietic Stem ◽  
Cell Immunity ◽  
The Difference ◽  
T Cell Phenotypes

Abstract Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution of normal T-cell immunity. Recipient thymic activity, biologic features of the allograft, and preparative regimens all contribute to immune reconstitution. We evaluated circulating T-cell phenotypes and T-cell receptor rearrangement excision circles (TRECs) in 331 blood samples from 158 patients who had undergone allogeneic HSCTs. All patients had received myeloablative conditioning regimens and were full donor chimeras in remission. Younger patients exhibited more rapid recovery and higher TRECs (P = .02). Recipients of T-cell–depleted allografts initially had lower TRECs than unmodified allograft recipients (P < .01), but the difference abated beyond 9 months. TREC level disparities did not achieve significance among adults with respect to type of allograft. Measurable, albeit low, TREC values correlated strongly with severe opportunistic infections (P < .01). This finding was most notable during the first 6 months after transplantation, when patients are at greatest risk but before cytofluorography can detect circulating CD45RA+ T cells. Low TRECs also correlated strongly with extensive chronic graft-versus-host disease (P < .01). Recipients of all ages of either unmodified or T-cell–depleted allografts therefore actively generate new T cells. This generation is most notable among adult recipients of T-cell–depleted allografts, most of whom had also received antithymocyte globulin for rejection prophylaxis. Low TREC values are significantly associated with morbidity and mortality after transplantation. T-cell neogenesis, appropriate to age but delayed in adult recipients of T-cell– depleted allografts, justifies interventions to hasten this process and to stimulate desirable cellular immune responses.

Berkeley Sickle Mice Demonstrate CD8- and NK1.1-Dependent Increased Rejection of Allogeneic Hematopoietic Stem Cell Transplants.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2332-2332
Author(s):  
Leslie Kean ◽  
Kelly Hamby ◽  
Jennifer Perry ◽  
Christian Larsen ◽  
David Archerq
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Sickle Cell Disease ◽  
Hematopoietic Stem Cell ◽  
Sickle Cell ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Cell Immunity ◽  
Increased Risk

Abstract While hematopoietic stem cell transplantation (HSCT) represents the only curative therapy for sickle cell disease, sickle patients undergoing HSCT face many complications, including an increased risk of graft rejection compared to non-sickle patients. We have used the Berkeley sickle mouse model to study the potential mechanisms underlying this increased risk of rejection. Using a CD28/CD40 costimulation-blockade-based non-myeloablative HSCT regimen, we transplanted Berkeley sickle mice with fully allogeneic SJL bone marrow. While the vast majority (&gt;85%, n=25) of control C57BL/6 animals became stably chimeric and immunologically donor-tolerant with this transplant regimen, sickle mice were much more prone to reject the transplant (~20% graft acceptance, n=25). Both CD8+ cells and NK1.1+ cells were found to contribute to this rejection, as depletion of either of these cell populations led to a marked increase in the percent of engrafted mice (&gt;85% graft acceptance, n=15–25), while depletion of CD4+ cells led to the opposite effect, with 0% (n=25) animals engrafted in this depletion cohort. The increased propensity of HSCT rejection in the Berkeley sickle mice may, in part, be explained by the presence of increased numbers of donor-reactive T cells (5–10-fold compared to C57BL/6 controls) in naïve sickle mice, despite their lack of exposure to donor antigens, and their housing in a Specific-Pathogen-Free environment. We speculate that these increased numbers of anti-donor T cells may occur as a result of heightened inflammation in the context of active sickle cell disease, which could lead to increased expansion and persistence of a T cell repertoire containing anti-donor heterologous T cell immunity. This heterologous immunity may have a profound effect on the success of HSCT for sickle cell disease, especially when non-myeloablative regimens are employed.

Interruption of IFNγR Signaling Results in Altered T Cell Trafficking in Vivo and Abrogation of GvHD While Maintaining a Robust Gvl Response

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 455-455
Author(s):  
Jaebok Choi ◽  
Edward Dela Ziga ◽  
Julie Ritchey ◽  
Lynne Collins ◽  
Julie Prior ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Trafficking ◽  
Hematopoietic Stem ◽  
T Cell Trafficking ◽  
Target Organs ◽  
Donor T Cells

Abstract Abstract 455 Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with relapsed/refractory leukemia, and marrow failure states such as myelodysplasia and aplastic anemia. However, allo-HSCT is complicated by allogeneic donor T cell-mediated graft-versus-host disease (GvHD) which can be life-threatening especially in recipients of unrelated or HLA-mismatched hematopoietic stem cell products. These same alloreactive donor T cells also mediate a beneficial graft-versus-leukemia (GvL) effect. Thus, the clinical goal in allo-HSCT is to minimize GvHD while maintaining GvL. Recent studies have suggested that this might be achieved by infusing regulatory T cells (Tregs) which in some preclinical models suppress GvHD-causing alloreactive donor T cells but have only limited effects on GvL-promoting alloreactive donor T cells. Unfortunately, Tregs exist in low frequency in the peripheral blood, are costly to purify and expand, and after expansion are difficult to isolate due to the lack of cell surface markers, all of which prevent their routine use in the clinic. Thus, alternative therapeutic approaches that do not require Tregs are needed. We have found that interferon gamma receptor deficient (IFNγR−/−) allogeneic donor T cells induce significantly less GvHD in both a MHC fully-mismatched (B6 (H-2b) → Balb/c (H-2d)) and a minor-mismatched (B6 (H-2b) → B6×129(H-2b)) allo-HSCT models compared to WT T cells. In addition, IFNγR−/− donor T cells maintain a beneficial GvL effect, which has been examined in both systemic leukemia and solid tumor models using luciferase-expressing A20 cells derived from Balb/c. We find that IFNγR−/− T cells migrate primarily to the spleen while WT T cells to GI tract and peripheral lymph nodes (LNs) using bioluminescence imaging (BLI), suggesting that altered T cell trafficking of IFNγR−/− T cells to GvHD target organs might be the major reason for the reduced GvHD. We further demonstrate that the IFNγR-mediated signaling in alloreactive donor T cells is required for expression of CXCR3 which has been implicated in trafficking of T cells to areas of inflammation and target organs, commonly known to be the sites of GvHD. Indeed, CXCR3−/− T cells recapitulate the reduced GvHD potential of IFNγR−/− T cells. In addition, forced overexpression of CXCR3 in IFNγR−/− T cells via retroviral transduction partially rescues the GvHD defect observed in IFNγR−/− T cells. We next examine if inhibition of IFNγR signaling using a small molecule inhibitor can recapitulate the anti-GVHD effects seen in IFNγR−/− T cells. We find that INCB018424, an inhibitor of JAK1/JAK2 which are the mediators of IFNγR signaling, blocks CXCR3 expression in vitro. Most importantly, in vivo administration of INCB018424 after allo-HSCT alters T cell trafficking and significantly reduces GvHD. Thus, the IFNγR signaling pathway represents a promising therapeutic target for future efforts to mitigate GvHD while maintaining GvL after allo-HSCT. Moreover, this pathway can be exploited in other diseases besides GvHD such as those from organ transplantation, chronic inflammatory diseases and autoimmune diseases. Disclosures: DiPersio: genzyme: Honoraria.

Evaluation Of Immunocompentence In Tolerant Chimeric Recipients Of Hematopoietic Stem Cell/Renal Transplants

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4483-4483
Author(s):  
Joseph Leventhal ◽  
Larry D Bozulic ◽  
Mark D. Badder ◽  
Mary Jane Elliott ◽  
Michael N Issa ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell ◽  
Peripheral Blood ◽  
High Frequency ◽  
Blood Type ◽  
Phase 2 ◽  
Hematopoietic Stem ◽  
Tcr Repertoire

A phase 2 protocol was developed to attempt to induce donor-specific tolerance to renal allografts in related and unrelated donor/recipient combinations (IND 13947 phase 2). Subjects were conditioned with fludarabine (days -5, -4, -3), cyclophosphamide (50 mg/ky days -3 and +3), and 200 cGy total body irradiation. The kidney transplant was performed day 0. G-CSF mobilized peripheral blood stem cells processed to remove GVHD-producing cells and retain graft facilitating cells (FC) was administered on day +1. The conditioning was well tolerated and the subjects were managed as outpatients after post-operative day 2. This has resulted in high levels of durable chimerism and immunosuppression-free graft survival without GVHD or engraftment syndrome in mismatched related and unrelated recipients of living donor FC/hematopoietic stem cell/kidney allografts. Nine subjects are completely off immunosuppression from 2 months to 3 years. A number of others are in the process of tapering. In the present study, we have prospectively analyzed recovery of immune function, persistence of vaccination memory, and response to vaccination in subjects who exhibit high levels of chimerism. Chimerism testing was performed using molecular short tandem report (sensitivity ±5%). Three of four subjects who had been vaccinated to hepatitis B prior to transplantation and whose donors had not been vaccinated retained their immunity following transplantation. All subjects tested exhibited memory for varicella and the majority did as well for measles (9/11), mumps (8/11), and rubella (5/10). A blood group disparity was present in 9 chimeric donor/recipient pairs. One chimeric subject converted to donor blood type, 3 exhibited mixed donor/host RBC chimerism, and 5 retained their own blood type. Six chimeric subjects have been immunized with pneumococcal vaccine after transplantation. All generated an immune response to vaccination, confirming immunocompetence to generate an antibody response to antigen. Notably, recovery of CD8+ and CD4+ central memory, naïve, and effector memory T cells occurred within one year post-transplantation to levels that were not significantly different from pre-transplantation. In addition, CD31+/CD45RA+ CD8+ and CD4+ T cells representative of recent thymic emigrants were present by 3 months, demonstrating de novo thymic production of T cells after transplantation. Four patients were randomly selected for study of Tcell repertoire (TCR) generation after transplantation. Two had achieved durable full donor chimerism and the other two did not have durable chimerism. Peripheral blood samples freshly obtained from donors and recipients and T cell subsets were isolated using MACs microbead system. DNA was extracted and sequenced by ImmunoSeq (Adaptive Biotech, Seattle, WA) to evaluate TCR clonal repertoires in recipients. Although clonal diversity in TCR repertoire was reduced in post-Tx recipients (0.9 ± 0.05 pre-Tx vs. 0.79 ± 0.09 post-Tx), the repertoire was diverse enough to suggest recovery of immune competence. Interestingly, at least 97% of the unique sequences observed in post-Tx recipient were not present in either donor or recipient pre-Tx. Within the pool of shared sequences, full chimerism correlated with a shift towards homology with the donor, while loss of chimerism correlated with recipient pre-Tx. In addition, the chimeric patients also exhibited reduced diversity of TCR sequences and increased clonality. Top 20 “high frequency” clones are most stably expressed. CD8+ cells had the highest number of “high frequency” clones. Notably, the pattern of “low frequency” clones was highest in the CD127-CD4+CD25+ regulatory T cell subset, indicating an extensive and rapidly changing TCR repertoire. Taken together, these data suggest that immunologic recovery is robust in these nonmyeloablatively conditioned tolerant chimeric subjects. Disclosures: Bozulic: Regenerex, LLC: Employment. Badder:Regenerex, LLC: Employment. Ildstad:Regenerex, LLC: Equity Ownership.

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