Decreased Numbers of Tregs in Aplastic Anemia Is Detected by Immunohistochemistry and Flow Cytometry.

Abstract Idiopathic aplastic anemia (AA) is characterized by immune-mediated destruction of hematopoietic stem cells, leading to peripheral pancytopenia. Immune pathogenesis in AA is supported by experimental data, as well as clinical observations and may be related to the breach of peripheral or central tolerance. Regulatory T cells (Treg) constitute one of the most important mechanisms of central tolerance engaged in the down-modulation of autoreactive T cells. Tregs have been found to be reduced in several autoimmune diseases and decreased frequencies of Tregs were also reported in AA and MDS. Overexpression of the high affinity IL-2 receptor alpha chain (CD25) and the forkhead family transcription factor P3 (FoxP3), required for the development and function of Tregs, serve as phenotypic markers for Tregs. We investigated Treg levels in a cohort of AA patients (N=21) and healthy individuals (N=15); flow cytometric quantification of Treg was carried out after surface/intracellular staining of whole blood for Treg markers (CD3, CD4, CD25, FoxP3). After proper gating (light scatter properties, CD3, CD4, CD25), CD4+ T cells were subdivided into CD25−, CD25int and CD25hi populations, and the co-expression of CD25hi and Foxp3 was analyzed. In comparison to controls, AA patients (N=12) show not only lower frequencies of CD4+CD25hi+ T cells within the total lymphocyte population (median 0.07% vs. 0.21%; p=.03), but also absolute lower absolute numbers (1.31/uL vs. 5.78/uL, p=.0002). Similarly, CD4+CD25hi+FoxP3+ T cells were found to be depressed in untreated AA patients in comparison to controls (median 0.07% vs. 0.21% and 1.06/uL vs. 4.76/uL; p=.03 and p=.003). While Tregs were lower in patients with active disease unresponsive to immunosuppressive treatment (responder 0.1% vs non responder 0.07%, CD4+CD25hi Tcells, p=.02), serial testing performed in 6 patients treated with ATG/CsA did not reveal correlation between hematologic improvement and recovery of Treg numbers. When double immunohistochemical staining for CD3 and Foxp3 was performed in pre-treatment bone marrow core biopsies of AA patients (N=3) and controls (N=2) a mean of 3 CD3+Foxp3+ cells/10 high power fields (hpf) were counted (vs. mean 28/10 hpf, p<.05 in controls), suggesting that lower numbers of Tregs were also present in the bone marrow of AA patients. In conclusion, our results suggest that Tregs are decreased in blood and marrow of patients with idiopathic AA, consistent with the breach of peripheral tolerance in AA. In addition to flow cytometry, immunohistochemical staining of histologic specimens can be used for the quantitative analysis of Tregs in bone marrow failure syndromes and other immune-mediated conditions such as GvHD.

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Inhibition of Notch Signaling in T Cells Prevents Immune-Mediated Bone Marrow Failure.

Blood ◽

10.1182/blood.v114.22.180.180 ◽

2009 ◽

Vol 114 (22) ◽

pp. 180-180

Author(s):

Gloria T Shan ◽

Ivy Tran ◽

Ashley R Sandy ◽

Ann Friedman ◽

Yi Zhang ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Notch Signaling ◽

Aplastic Anemia ◽

Research Funding ◽

Bone Marrow Failure ◽

Comprehensive Cancer Center ◽

Cancer Center ◽

Immune Mediated

Abstract Abstract 180 Aplastic anemia is a severe bone marrow disorder characterized by the loss of hematopoietic stem cells (HSC). HSC destruction is thought to be T cell-mediated in a majority of patients with aplastic anemia. Global immunosuppression and HSC transplantation can induce disease remission, but these treatments are not effective in all patients and can promote life-threatening complications. Thus, novel immunomodulatory approaches are needed in this disorder. Notch is a conserved cell-cell communication pathway that can regulate T cell differentiation and function with context-dependent effects. To study the role of Notch signaling in pathogenic T cells causing immune-mediated bone marrow failure, we inhibited canonical Notch signaling in mature T cells through conditional expression of the pan-Notch inhibitor DNMAML (ROSA-DNMAMLf × Cd4-Cre mice). We used two complementary mouse models of immune-mediated bone marrow failure that mimic features of aplastic anemia: administration of C57BL/6 (B6) T cells into sublethally irradiated (500 rads) minor histocompatibility antigen mismatched BALB/b recipients (Chen et al., J Immunol 2007; 178:4159), or infusion of B6 lymphocytes into unirradiated MHC-mismatched B6×DBA F1 recipients. In contrast to control B6 T cells which led to lethal bone marrow failure in virtually all recipients, DNMAML-expressing Notch-deprived T cells were profoundly deficient at inducing HSC loss in both disease models, leading to markedly improved long-term survival (>90%). Notch-deficient T cells showed a modest decrease in overall expansion within secondary lymphoid organs, but their accumulation in the target bone marrow was preserved. Upon restimulation with anti-CD3 and anti-CD28 antibodies, DNMAML T cells had decreased production of IL-2 and interferon gamma. Activated CD4+ and CD8+ DNMAML T cells had reduced interferon gamma, granzyme B, and perforin transcripts despite preserved induction of the master transcription factors Tb×21 (encoding T-bet) and Eomes. In vivo infusion of CFSE-labeled host-type target cells revealed a decreased cytotoxicity in DNMAML as compared to control B6 T cell recipients. These observations point to a novel spectrum and mechanism of Notch action in mature T cells. Since we have shown recently that canonical Notch signaling is dispensable for the maintenance of adult HSCs (Maillard et al., Cell Stem Cell 2008, 2:356), our findings suggest that Notch inhibition could represent a novel therapeutic modality to target the T cell response and reverse immune-mediated HSC destruction in aplastic anemia. Disclosures: Shan: American Society of Hematology: Research Funding. Zhang:University of Michigan Comprehensive Cancer Center: Research Funding; Damon Runyon Cancer Research Foundation: Research Funding. Maillard:Damon Runyon Cancer Research Foundation: Research Funding; American Society of Hematology: Research Funding; University of Michigan Comprehensive Cancer Center: Research Funding.

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Genetic Associations in Acquired Immune-Mediated Bone Marrow Failure Syndromes: Insights in Aplastic Anemia and Chronic Idiopathic Neutropenia

Clinical and Developmental Immunology ◽

10.1155/2012/123789 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 7

Author(s):

Irene Mavroudi ◽

Helen A. Papadaki

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Transforming Growth Factor ◽

Bone Marrow Failure ◽

Genetic Associations ◽

Hematopoietic Stem ◽

Bone Marrow Failure Syndromes ◽

Immune Mediated ◽

Chronic Idiopathic Neutropenia ◽

Activated T Lymphocytes

Increasing interest on the field of autoimmune diseases has unveiled a plethora of genetic factors that predispose to these diseases. However, in immune-mediated bone marrow failure syndromes, such as acquired aplastic anemia and chronic idiopathic neutropenia, in which the pathophysiology results from a myelosuppressive bone marrow microenvironment mainly due to the presence of activated T lymphocytes, leading to the accelerated apoptotic death of the hematopoietic stem and progenitor cells, such genetic associations have been very limited. Various alleles and haplotypes of human leucocyte antigen (HLA) molecules have been implicated in the predisposition of developing the above diseases, as well as polymorphisms of inhibitory cytokines such as interferon-γ, tumor necrosis factor-α, and transforming growth factor-β1 along with polymorphisms on molecules of the immune system including the T-bet transcription factor and signal transducers and activators of transcription. In some cases, specific polymorphisms have been implicated in the outcome of treatment on those patients.

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Perforin gene mutations in patients with acquired aplastic anemia

Blood ◽

10.1182/blood-2006-12-063495 ◽

2007 ◽

Vol 109 (12) ◽

pp. 5234-5237 ◽

Cited By ~ 34

Author(s):

Elena E. Solomou ◽

Federica Gibellini ◽

Brian Stewart ◽

Daniela Malide ◽

Maria Berg ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Aplastic Anemia ◽

Natural Killer ◽

Nk Cell ◽

Bone Marrow Failure ◽

Genetic Alterations ◽

Cytotoxic Lymphocytes ◽

Activated T Cells ◽

Hematopoietic Stem

Abstract Perforin is a cytolytic protein expressed mainly in activated cytotoxic lymphocytes and natural killer cells. Inherited perforin mutations account for 20% to 40% of familial hemophagocytic lymphohistiocytosis, a fatal disease of early childhood characterized by the absence of functional perforin. Aplastic anemia, the paradigm of immune-mediated bone marrow failure syndromes, is characterized by hematopoietic stem cell destruction by activated T cells and Th1 cytokines. We examined whether mutations in the perforin gene occurred in acquired aplastic anemia. Three nonsynonymous PRF1 mutations among 5 unrelated patients were observed. Four of 5 patients with the mutations showed some hemophagocytosis in the bone marrow at diagnosis. Perforin protein levels in these patients were very low or absent, and perforin granules were completely absent. Natural killer (NK) cell cytotoxicity from these patients was significantly decreased. Our data suggest that PRF1 genetic alterations help explain the aberrant proliferation and activation of cytotoxic T cells and may represent genetic risk factors for bone marrow failure.

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A Murine Model of Bone Marrow Failure Mediated by Disparity in Minor Histocompatibility Antigens.

Blood ◽

10.1182/blood.v106.11.132.132 ◽

2005 ◽

Vol 106 (11) ◽

pp. 132-132

Author(s):

Jichun Chen ◽

Neal S. Young

Keyword(s):

Bone Marrow ◽

T Cells ◽

Lymph Node ◽

Aplastic Anemia ◽

Cd8 T Cells ◽

Bone Marrow Failure ◽

Histocompatibility Antigens ◽

Minor Histocompatibility Antigens ◽

Immune Mediated ◽

Lymph Node Cells

Abstract Destruction of hematopoietic cells in aplastic anemia and other hematologic diseases is mediated in most patients by effector cells of the immune system. We have developed a mouse model of immune-mediated bone marrow failure which employs parental lymph node cell infusion into F1 recipients, resulting in bone marrow hypercellularity, pancytopenia, and death from infection and bleeding, without other organ involvement by graft-versus-host disease (Bloom, ML et al. Exp. Hematol. 32:1163, 2004; Chen, J et al. Blood104:1671, 2004). Because major histocompatibility antigens are not the targets in acquired aplastic anemia, we now have developed a model of murine marrow failure based on disparity of minor histocompatibility antigens. Lymph node cells from C57BL/6 mice were infused into sublethally-irradiated, MHC-matched, C.B10-H2b/LilMce recipients. Animals developed severe pancytopenia and marrow hypoplasia within two-three weeks. CD8+ T lymphocytes were expanded in the blood and infiltrated bone marrow, becoming detectable at day 7 and reaching peak levels at days 10–12. There were no inflammatory responses observed in the skin, intestines, or other visceral organs by gross or microscopic pathological examination. In our experiments we observed a time-dependent expansion followed by contraction of CD8+ T cells specific for a minor histocompatibility antigen H60 as measured by flowcytometry using an H60-specific tetramer. H60 is an antigen peptide derived from a glycoprotein, a known ligand for stimulatory NKG2D receptors, and is immunodominant over other minor antigens in stem cell transplantation. The proportion of H60-specific CD8+ T cells was strongly negatively correlated with peripheral blood white cell, neutrophil, and platelet counts. Isolated H60-specific T-cells from bone marrow of affected animals induced apoptosis in vitro of normal C57BL/6 bone marrow cells in co-culture. The degree of apoptosis was further increased by addition of CD4 T-cells from same affected donors, suggesting a helper lymphocyte effect. The role of H60-specific T cells was demonstrated in further transplant experiments. Infusion of 5 × 106 C57BL/6 lymph node cells that had been depleted of H60-specific T cells was unable to induce marrow failure in C.B10 recipients, while the same number of cells with the addition of 20–90 × 103 H60-specific CD8+ T cells led to thrombocytopenia and leucopenia in recipients. H60-specific T cells thus appear to be key effectors, responsible at least for the initiation of marrow cell destruction. Mice could be treated with cyclosporine at 50 mg/g/day for five days, if treatments were begun at the time of lymph node cell infusion. Immunosuppressive therapy abolished H60-specific CD8+ T cell expansion and attenuated the development of peripheral pancytopenia, effectively rescuing animals. In conclusion, we demonstrate in this model of immune-mediated bone marrow failure that a single clone of peptide-specific T-cells is capable of efficient marrow destruction and the production of aplastic anemia.

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Comparison Of Lymphocytic Immune Profiles Of Aplastic Anemia and Hypocellular Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v122.21.3719.3719 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3719-3719

Author(s):

Jeffrey J. Pu ◽

Guillermo Rangel Rivera ◽

Abigail Sido ◽

Arthur Berg ◽

Cinda Boyer ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Myelodysplastic Syndrome ◽

Aplastic Anemia ◽

Cell Population ◽

Bone Marrow Failure ◽

Expression Patterns ◽

Lymphocyte Population ◽

Cd19 Expression

Abstract Background Aplastic anemia (AA) and hypocellular myelodysplastic syndrome (MDS) are two common acquired bone marrow failure diseases. AA is mostly an acquired bone marrow disease caused by cellular and humoral mediated immune attack of hematopoietic stem cells (HSC) due to dysregulation of lymphocytic system, which leads to hematopoietic progenitor cell apoptosis and bone marrow failure. MDS is a group of heterogeneous acquired clonal HSC disorders with ineffective hematopoiesis. Approximately 10% to 20% of MDS manifests a reduced bone marrow cellularity, which comprises hypocellular MDS. There is increasing experimental and clinical indication that an immune-mediated damage to hematopoietic HSCs and changes in the hematopoiesis-supporting microenvironment contribute to the pathogenesis of hypocellular MDS. Because of the similarity of their bone marrow manifestation, hypocellular MDS and AA are often hard to distinguish. Mounting evidence indicates that abnormal activation of cytotoxic T cells plays a crucial role in the pathophysiology of these diseases. One study showed that AA patients have an abnormally activated subpopulation of CD4+ helper cells and a decreased number and function of T regulatory cells in the bone marrow. GVHD mouse models further demonstrated that self-reactive T cells were capable of recognizing non-polymorphic tissue or commensally-derived antigens. Recent literature suggests that immune dysregulation plays a major role in pathogenesis of acquired bone marrow failure disease. However immune profiles of these two diseases have not been thoroughly studied, specially the role of B lymphocyte population. Our study aims to find lymphocytic surface marker expression patterns of hypocellular MDS and AA in both immature cell and lymphocyte populations. Methods This retrospective study analyzed flow cytometry lymphocytic antigen expression profiles from patients diagnosed as AA and hypocellular MDS as per standard criteria. A total of 31 AA and 26 hypocellular MDS patient cases were recruited. The bone marrow aspirate/biopsy data, bone marrow aspiration flow cytometry reports, and Complete Blood Counts (CBC)s from individual patients were analyzed. Using side scatter (SSC) vs. CD45 gating flow cytometry panels, we identified immature cell population (SSClow/CD45low) and lymphocyte population (SSClow/CD45high). We then quantitatively analyzed the expression patterns of 33 cluster differentiation (CD) molecules on individual sample. Finally, we compared the CD expression patterns between AA and hypocellular MDS in both cell populations respectively. Results CD19 expression was significantly higher in AA than in hypocellular MDS in both SSClow/CD45low cell population (P=0.001) and SSClow/CD45high cell population (P=0.003). Hypocellular MDS contains significantly higher CD34high cells than AA in SSClow/CD45low populations (mean:28.5% vs 8.5%; range; 1% to 94% vs 2% to 27%; P=0.04). However, patients with both diseases similarly contains very few CD34high cells in SSClow/CD45high cell population (mean: 0.6% vs 2.6%; range: 0.0% to 2% vs 0.0% to 32%; P=0.99). Conclusion 1. In AA, B cells are highly proliferative in both immature stage and mature stage. This data indicates that B cells which may play a unique role in AA pathogenesis but not in hypocellular MDS. 2. In both AA and hypocellular MDS, the majority of lymphocyte population are mature cells. This data suggests that the pathogeneses of both diseases caused by a persistently dysregulated immune microenvironment, not by an acute insult. CD19 expression pattern may be a useful marker to distinguish AA and hypocellular MDS. Disclosures: No relevant conflicts of interest to declare.

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Macrophage TNF-α licenses donor T cells in murine bone marrow failure and can be implicated in human aplastic anemia

Blood ◽

10.1182/blood-2018-05-844928 ◽

2018 ◽

Vol 132 (26) ◽

pp. 2730-2743 ◽

Cited By ~ 10

Author(s):

Wanling Sun ◽

Zhijie Wu ◽

Zenghua Lin ◽

Maile Hollinger ◽

Jichun Chen ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Aplastic Anemia ◽

Bone Marrow Failure ◽

Murine Bone Marrow ◽

Immune Mediated ◽

Tnf Α ◽

Donor T Cells ◽

Ifn Γ

Abstract Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) have been implicated historically in the immune pathophysiology of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. We recently defined the essential roles of IFN-γ produced by donor T cells and the IFN-γ receptor in the host in murine immune-mediated BM failure models. TNF-α has been assumed to function similarly to IFN-γ. We used our murine models and mice genetically deficient in TNF-α or TNF-α receptors (TNF-αRs) to establish an analogous mechanism. Unexpectedly, infusion of TNF-α−/− donor lymph node (LN) cells into CByB6F1 recipients or injection of FVB LN cells into TNF-αR−/− recipients both induced BM failure, with concurrent marked increases in plasma IFN-γ and TNF-α levels. Surprisingly, in TNF-α−/− recipients, BM damage was attenuated, suggesting that TNF-α of host origin was essential for immune destruction of hematopoiesis. Depletion of host macrophages before LN injection reduced T-cell IFN-γ levels and reduced BM damage, whereas injection of recombinant TNF-α into FVB-LN cell-infused TNF-α−/− recipients increased T-cell IFN-γ expression and accelerated BM damage. Furthermore, infusion of TNF-αR−/− donor LN cells into CByB6F1 recipients reduced BM T-cell infiltration, suppressed T-cell IFN-γ production, and alleviated BM destruction. Thus, TNF-α from host macrophages and TNF-αR expressed on donor effector T cells were critical in the pathogenesis of murine immune-mediated BM failure, acting by modulation of IFN-γ secretion. In AA patients, TNF-α–producing macrophages in the BM were more frequent than in healthy controls, suggesting the involvement of this cytokine and these cells in human disease.

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Effects of Rabbit Anti-Mouse Thymocyte Globulin Treatment in a Splenocyte-Induced Model of Aplastic Anemia

Blood ◽

10.1182/blood.v116.21.1172.1172 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1172-1172

Author(s):

Melanie C Ruzek ◽

Kathleen Phillips ◽

Susan Richards ◽

Khalid Mamlouk ◽

John Williams ◽

...

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Bone Marrow Failure ◽

Complete Response ◽

High Dose ◽

Hematopoietic Stem ◽

Mouse Thymocyte ◽

Immune Mediated ◽

Severe Pancytopenia ◽

High Doses

Abstract Abstract 1172 Acquired aplastic anemia is an immune-mediated disease where destruction of hematopoietic stem cells (HSCs) in the bone marrow results in severe and life-threatening pancytopenia. Thymoglobulin® is often used as immunosuppressive therapy in this disease with up to 80 percent of patients responding to a combination of Thymoglobulin and cyclosporine. In an effort to better understand the activities and mechanism of action of Thymoglobulin we developed a mouse model of immune-mediated aplastic anemia and evaluated a murine surrogate of Thymoglobulin®, rabbit anti-mouse thymocyte globulin (mATG) in this model. We modified a graft-versus-host (GVH)-induced model described in the literature (Bloom, et al., 2004) utilizing HSC-depleted spleen cells transferred from C57BL/6 into CByB6F1 mice instead of lymph node cell transfer. Our modified model shows a cell dose-dependent increase in pancytopenia and lethality. Mice receiving a high dose (100×106) of HSC-depleted splenocytes experienced severe pancytopenia and rapid death occurring around day 21 whereas mice receiving lower doses (70×106, 35×106 and 17×106) of cells showed progressively less pancytopenia and lethality as the dose of cells decreased. Histopathology also showed marked loss of hematopoietic progenitor cells in the bone marrow with little evidence of GVHD in other tissues. Prophylactic administration of mATG (25mg/kg, 2x, day 0 and 3) to mice given high doses of HSC-depleted splenocytes (100×106) resulted in a significant improvement in pancytopenia and survival (70%) in this model. Interestingly, therapeutic administration of mATG was more effective when given later relative to disease induction. Delivery of mATG (25mg/kg, 2x, three days apart) starting on day 3 showed some delay in disease progression (day 30 vs day 21) and mATG started on day 6 slightly increased survival (40%). However, mice receiving mATG starting on days 10 or 14 showed a much greater overall survival of 100% and 60%, respectively, with full rebound of hematopoietic cells in the blood to normal levels. The complete response observed with later mATG administration (day 10 or day 14) mimics the treatment and response of patients given Thymoglobulin®. In summary, we have established a novel model of HSC-depleted splenocyte induction of bone marrow failure in mice that is responsive to therapeutic ATG administration. Studies in this model will aid in further understanding the mechanism of ATG in aplastic anemia and may contribute to the development of potential new therapies. Disclosures: Ruzek: Genzyme: Employment. Phillips:Genzyme: Employment. Richards:Genzyme: Employment. Mamlouk:Genzyme: Employment. Williams:Genzyme: Employment. Garman:Genzyme: Employment.

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Auto-Reactive Glycosyl-Phosphatidyl-Inositol (GPI) Specific T Cells Are Present in Most Patients with Idiopathic Aplastic Anemia (IAA)

Blood ◽

10.1182/blood.v126.23.2409.2409 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2409-2409 ◽

Cited By ~ 1

Author(s):

Lucia Gargiulo ◽

Yoshitaka Zaimoku ◽

Barbara Scappini ◽

Hiroyuki Maruyama ◽

Rie Ohumi ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Aplastic Anemia ◽

Bone Marrow Failure ◽

Immunosuppressive Treatment ◽

Healthy Controls ◽

Hematopoietic Stem ◽

Immune Attack ◽

Negative Population

Abstract Idiopathic aplastic anemia (IAA) is an acquired bone marrow disease probably caused by an auto-immune attack against hematopoietic stem cells (HSCs), which leads to bone marrow failure. Many abnormalities have been observed in the T cell compartment, but the putative auto-antigen(s) remain elusive. A large body of evidence links paroxysmal nocturnal hemoglobinuria (PNH) to IAA, supporting the notion that autoimmunity is a key pathogenic mechanism in both diseases. In PNH, auto-reactive T cells may be the 'noxious agent' capable of killing GPI positive HSCs while sparing GPI negative HSCs. Recently, CD1d restricted, GPI-specific T-cells have been demonstrated in PNH patients. Here, we investigate whether CD1d restricted, GPI-specific T cells are also present in IAA patients. When peripheral blood mononuclear cells (PBMNCs) from 14 newly diagnosed IAA patients [12 of whom had a small percentage (between 0.003% and 5%) of GPI-negative granulocytes] were co-cultured with antigen presenting cells (APCs) expressing CD1d and competent for the synthesis of GPI, we detected GPI specific T cells (CD8+CD1d/GPI dimer+ T cells) in 10 out of 14 patients (71%) at a significantly higher abundance than in co-culture experiments performed with PBMNCs from healthy controls (Fig. 1). In fact, the frequency of CD8+CD1d/GPI dimer+ T cells was below the cutoff value of 0.35% in all the 15 healthy controls but only in 4 out 14 IAA patients (Fisher test, P <0.00005). The frequency of GPI specific T cells in IAA patients was similar to that previously found in PNH patients (Fig.1). In the same experimental setting, in IAA we found increased frequency of INFγ producing T cells (CD8+INFγ+ T cells) than in healthy controls: interestingly, in the 4 patients with no detectable CD8+CD1d/h-GPI dimer+ T cells, we found INFγ producing T cells regardless of the GPI stimulus. Furthermore, we have observed a trend towards the association of higher frequency of CD8+CD1d/GPI dimer+ T cells and severe IAA versus non-severe IAA. In 5 of the 10 IAA patients with detectable CD8+CD1d/GPI dimer+ T cells we tested sequential samples; we observed that response to immunosuppressive treatment (IST) was associated with significant decrement of both GPI specific and INFγ producing T cells. In keeping with these data, GPI specific T cells were absent in 2 AA patients in remission after engraftment of HSC transplants. We then similarly analyzed 27 IAA patients who were already receiving IST. 8 of these patients did not have any GPI-negative granulocyte population, 10 had a small PNH clone (between 0.003% and 5%) and 9 had more than 5% of GPI-negative granulocytes. GPI specific T cells were detected in 20 out of these 27 (74%) patients, regardless of the presence/absence of a PNH (GPI-negative) population; the frequency of GPI specific T cells was lower than in patients at diagnosis. In these 20 patients, INFγ producing T cells were detected only when co-cultured in presence of GPI on CD1d+ APC; in the remaining 7 patients with no detectable CD8+CD1d/h-GPI dimer+ T cells, INFγ producing T cells were present regardless of any GPI stimulus. In one patient with a congenital form of mild AA due to a TERC mutation, GPI specific T cells were present. This finding might signify that although congenital AA patients are innately predisposed to AA, an auto-immune attack may be still needed to produce full-blown AA. We conclude that GPI specific T cells are present in about 70% of IAA patients regardless of the presence/absence of a PNH (GPI-negative) population; and that the presence and size of a PNH cell population results from the existence of a PIG-A mutant HSC and from its degree of 'stemness'. The presence of GPI specific T cells in the large majority of AA patients suggests that in most of these cases, GPI is the target of the auto-immune attack potentially resulting in the suppression of hematopoiesis. In the remaining IAA patients, different and still unknown auto-immune targets are presumably involved in the T-cell attack. These data strongly support the hypothesis that AA and PNH could be different clinical expression of the same pathogenetic mechanism, namely the auto-immune suppression of the GPI+ (normal) HSCs. Disclosures No relevant conflicts of interest to declare.

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Relatively Low Sensitivity of CD109(-) Hematopoietic Stem/Progenitor Cells (HSPCs) to TGF-β: A Possible Mechanism Responsible for the Preferential Commitment of Piga Mutant HSPCs in Immune-Mediated Bone Marrow Failure

Blood ◽

10.1182/blood.v128.22.3911.3911 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3911-3911

Author(s):

Noriharu Nakagawa ◽

Kohei Hosokawa ◽

Luis Espinoza ◽

Kana Maruyama ◽

Takamasa Katagiri ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Aplastic Anemia ◽

Progenitor Cells ◽

Research Funding ◽

Lower Sensitivity ◽

Wild Type ◽

Hematopoietic Stem ◽

Immune Mediated ◽

Cd34 Cells

Abstract [Background] An increase in the numbers of glycosylphosphatidylinositol-anchored protein-deficient [GPI(-)] blood cells is often detected in patients with acquired aplastic anemia (AA) and low-risk myelodysplastic syndrome (MDS), and is associated with good response of their bone marrow (BM) failure to immunosuppressive therapy. Although some immune mechanisms are thought to play a role in the preferential commitment of hematopoietic stem/progenitor cells (HSPCs) with PIGA mutations in such BM failure patients, the exact mechanisms are unknown. Our previous studies suggested that GPI(-)T cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) were less susceptible to TGF-ƒÀ-mediated inhibition of proliferation triggered by anti-CD3 and anti-CD28 antibodies than GPI(+)T cells of the same patient. The lower sensitivity of PIGA mutant HSPCs to TGF-ƒÀ, a cytokine capable of inhibiting the cell cycling of dormant HSPCs, than GPI(+) HSPCs may also explain the preferential commitment of GPI(-) HSPCs in immune-mediated BM failure. However, little is known about the GPI-APs that affect the sensitivity of HSPCs to TGF-ƒÀ. [Objectives/Methods] We assessed the roles of GPI-APs in the signal transduction of CD34(+) cells of a PNH patient and the myeloid leukemia cell line TF-1 in response to TGF-ƒÀ. We also assessed the TGF-ƒÀ-mediated inhibition of cell proliferation in TF-1 cells with or without a PIGA mutation. CD109, a GPI-AP that serves as a TGF-ƒÀ co-receptor in human keratinocytes, of TF-1 cells, was knocked out from TF-1 cells using a CRISPR-Cas 9 system, and the sensitivity to TGF-ƒÀ was compared between CD109(+) and CD109(-) TF-1 cells. [Results] The treatment of BM mononuclear cells from a florid PNH patient with TGF-ƒÀ induced SMAD2 phosphorylation in GPI(-) CD34(+) cells to a lesser degree than in GPI(+) CD34(+) cells (fold increase in pSMAD2 MFI, 1.0 vs. 2.6, Figure 1). TGF inhibited PIGA-mutant TF-1 (PNH-TF-1) proliferation to a lesser degree (percentage of inhibition, 19%}13%) than wild-type TF-1 cells (67%}3%) in an MTT-based proliferation assay. Transfection of PIGA into PNH-TF-1 cells restored GPI-AP expression as well as sensitivity to TGF-ƒÀ (53%}10% vs. 19%}13% in PNH-TF-1 cells). CD109 coimmunoprecipitated with TGF-ƒÀ in TF-1 cells, and its expression was confirmed on BM CD34+ cells of healthy individuals, particularly CD34+CD38+CD123-CD45RA- megakaryocyte-erythroid progenitor cells, as well as on TF-1 cells. The pSMAD2 induction in CD109(-) TF-1 cells by TGF-ƒÀ was less pronounced (relative increase in pSMAD2 MFI, 7.65}2.15 vs. 10.74}2.28) than that in CD109(+) TF-1 cells (Figure 2). [Conclusions] CD109 deficiency is involved in the lower sensitivity of GPI(-) HSPCs to TGF-ƒÀ than GPI(+) HSPCs. This deficiency may account for the preferential activation of PIGA mutant HSPCs in immune-mediated BM failure, in which TGF-ƒÀ suppresses activation of wild-type HSPCs. Disclosures Hosokawa: Aplastic Anemia and MDS International Foundation: Research Funding. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

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