Expression of CD28 and CTLA4 on T cells in bone morrow of immune-mediated aplastic anemia mice

Abstract Forty-two patients with aplastic anemia (AA) were studied to determine whether or not transfusion-induced sensitization is responsible for the in vitro inhibition by patient lymphocytes of HLA-identical erythroid burst-forming units (BFU-E). The results indicate that lymphocytes from 12 of 34 transfused patients inhibited normal colony growth. In contrast, lymphocytes from none of the 8 untransfused patients demonstrated inhibition. These data were interpreted to mean that coculture studies would not be useful for identifying immune-mediated AA in transfused patients. Therefore, in order to identify possible immune-related AA, we assayed BFU-E from patient blood before and after T-cell depletion. In all 32 patients studied, BFU-E failed to grow from peripheral blood cells before T-cell depletion, but in 8 cases, normal- appearing BFU-E grew after T cells had been removed. Growth of patient BFU-E colonies was inhibited in 6 cases when patient T cells were added back to the culture, indicating that in these 6 patients, an “autoimmune” mechanism may have been present.

Download Full-text

In vitro tests for distinguishing possible immune-mediated aplastic anemia from transfusion-induced sensitization

Blood ◽

10.1182/blood.v55.2.211.bloodjournal552211 ◽

1980 ◽

Vol 55 (2) ◽

pp. 211-215

Author(s):

BJ Torok-Storb ◽

C Sieff ◽

R Storb ◽

J Adamson ◽

ED Thomas

Keyword(s):

T Cells ◽

T Cell ◽

Aplastic Anemia ◽

Colony Growth ◽

Cell Depletion ◽

In Vitro Tests ◽

T Cell Depletion ◽

Immune Mediated ◽

Before And After

Forty-two patients with aplastic anemia (AA) were studied to determine whether or not transfusion-induced sensitization is responsible for the in vitro inhibition by patient lymphocytes of HLA-identical erythroid burst-forming units (BFU-E). The results indicate that lymphocytes from 12 of 34 transfused patients inhibited normal colony growth. In contrast, lymphocytes from none of the 8 untransfused patients demonstrated inhibition. These data were interpreted to mean that coculture studies would not be useful for identifying immune-mediated AA in transfused patients. Therefore, in order to identify possible immune-related AA, we assayed BFU-E from patient blood before and after T-cell depletion. In all 32 patients studied, BFU-E failed to grow from peripheral blood cells before T-cell depletion, but in 8 cases, normal- appearing BFU-E grew after T cells had been removed. Growth of patient BFU-E colonies was inhibited in 6 cases when patient T cells were added back to the culture, indicating that in these 6 patients, an “autoimmune” mechanism may have been present.

Download Full-text

Reduced Regulatory T Cell to Activated T Cell Ratio in a Mouse Model of Immune-Mediated Bone Marrow Failure and Prevention of Aplastic Anemia by Infusion of T Regulatory Cells.

Blood ◽

10.1182/blood.v108.11.121.121 ◽

2006 ◽

Vol 108 (11) ◽

pp. 121-121

Author(s):

Jichun Chen ◽

Neal S. Young

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Aplastic Anemia ◽

Cell Expansion ◽

Cell Ratio ◽

Immune Mediated ◽

Activated T Cell ◽

T Cell Expansion

Abstract We have produced a murine model of immune-mediated bone marrow (BM) failure based on minor histocompatability antigen mismatch. Infusion of C57BL/6 (B6) lymph node (LN) cells into congenic C.B10 mice results 2–5 weeks later in severe marrow hypoplasia and fatal pancytopenia. Expansion of pathogenic T cells, especially cytotoxic T cells specific for the dominant minor antigen H60, is critical in disease progression: infusion of LN cells from H60-matched congenic B6 donors failed to induce BM failure in C.B10 recipients. We observed that the proportion of CD4+CD25+ T cells in the BM increased significantly in LN-cell-infused animals, coordinate with BM invasion by CD4 and CD8 T cells. However, a very large fraction (60–80%) of CD4+CD25+ T cells from BM failure mice did not express intracellular FoxP3, in contrast to CD4+CD25+ cells from normal BM which were 80–90% FoxP3+. In the mouse model, there were significant declines in the ratios between regulatory T cells and total T cells in the BM: CD4+CD25+FoxP3+ (Treg) to CD4+CD25+FoxP3− (activated CD4 cells) ratio decreased from 1 : 0.29 to 1 : 2.10; CD4+CD25+FoxP3+ to CD4+ ratio decreased from 1 : 7.01 to 1 : 19.87 whereas CD4+CD25+FoxP3+ to CD8+ ratio decreased from 1 : 7.94 to 1 : 46.71 when unaffected animals and BM failure mice were compared. Infusion of as few as 5000 CD4+CD25+ T cells obtained from normal B6 mouse spleen (>80% FoxP3+) with allogeneic B6 LN cells prevented H60-specific T cell expansion in C.B10 recipients and suppressed marrow destruction. A reduced regulatory T cell to activated T cell ratio accompanies pathogenic T cell expansion in this model of immune-mediated marrow destruction. Added regulatory T cells can suppress T cell expansion and prevent murine aplastic anemia. Our model concurs with recent observations in human aplastic anemia, in which numbers and activity of circulating regulatory T cells are much reduced in comparison to healthy controls (Solomou EE et al. ASH abstract submitted). Regulatory T cells might be useful in cellular therapy of autoimmune diseases. Figure Figure

Download Full-text

Fas- and Fas Ligand Rather Than Perforin-Mediated Apoptosis Has a Dominant Role in Target Cell Death in Murine Models of Immune- Mediated Bone Marrow Failure

Blood ◽

10.1182/blood.v112.11.1039.1039 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1039-1039

Author(s):

Jichun Chen ◽

Stephanie O. Omokaro ◽

Annahita K. Sarcon ◽

Neal Young

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

Aplastic Anemia ◽

T Cell Activation ◽

Cell Activation ◽

Fas Ligand ◽

Wild Type ◽

Cell Destruction ◽

Immune Mediated

Abstract Fas-Fas ligand and perforin-granzyme are two important cell death pathways associated with cytotoxic T cell induced target cell apoptosis. In patients with immune-mediated aplastic anemia, the development of bone marrow (BM) failure is associated with up-regulation in Fas ligand expression on effector cytotoxic T cells and elevated Fas expression on target BM cells. In some aplastic anemia patients, peripheral blood T lymphocytes also carry polymorphisms in the perforin gene which have been associated with familial hemophagocytosis. These findings suggested that Fas ligand/Fas might be the key signaling molecules mediating cell destruction while perforin might also play a role in the development of BM failure in patients with aplastic anemia. We have modeled immune-mediated BM failure in the mouse by infusing allogeneic lymph node (LN) cells from C57BL/6 (B6) donors into sublethally-irradiated CByB6F1 and C.B10 recipients that are mismatched at either major histocompatibility (MHC) or minor histocompatibility (minor-H) loci. Expansion and activation of allogeneic T cells results in increased production of the inflammatory cytokines gamma interferon and tissue necrosis factor alpha in recipient BM, massive BM cell destruction, severe marrow hypoplasia, and fatal pancytopenia. In the current study, we directly tested the roles of Fas, Fas ligand and perforin in the development of BM failure by using murine models with spontaneous mutations at the lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld) loci, or with germline deletion of the gene perforin (prf−/−). Fas and Fas ligand-deficient lpr and gld mutant mice had no evidence of hematopoietic deficiency despite their autoimmune environment and marked lymphoproliferation. LN cells from lpr and gld mice caused significantly less apoptosis to minor-H mismatched C.B10 BM cells when co-cultured in a cytotoxicity assay in vitro, in comparison to LN cells from wild-type B6 mice. Infusion of lpr, gld, and B6 donor LN cells into sub-lethally irradiated CB10 recipients all caused massive T cell expansion in recipient BM with high level expression of CD11a, indicative of T cell activation, but only B6 LN cells caused severe BM destruction. In contrast, recipients of lpr and gld LN cells had only mild to moderate pancytopenia and marrow hypocellularity. We inferred from these results that disruption of the Fas ligand/Fas signaling pathway effectively abrogated immune mediated marrow destruction. To test the role of perforin in BM failure, we first analyzed prf−/−- mice and found no obvious change in cellular composition in lymphohematopoietic tissues in comparison to wild-type B6 controls. LN cells from prf−/− mice showed reduced ability to induce C.B10 BM cell apoptosis in an in vitro cytotoxicity assay when compared to wild-type B6 LN cells. Infusion of 5–10 million prf−/− LN cells into CByB6F1 and C.B10 recipients produced obvious BM failure in both recipient types with pancytopenia and marrow hypoplasia about 80–90% as severe as in control recipients of 5 million B6 LN cells. In both CByB6F1 and C.B10 recipients, infused prf−/− LN cells resulted in less T cell expansion, a similar level of T cell activation, higher proportions of T cells containing gamma-interferon and tissue necrosis factor-alpha, and a higher proportion of T cells expressing the Fas ligand CD178, in comparison to the infused B6 LN cells. We conclude that Fas-Fas ligand-mediated transmembrane signaling provides the major cell death pathway, while perforin-granzyme-mediated exocytosis plays a minor role, in BM cell destruction in animal models of immune-mediated BM failure.

Download Full-text

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.

Download Full-text

Intrinsic impairment of CD4+CD25+ regulatory T cells in acquired aplastic anemia

Blood ◽

10.1182/blood-2011-11-390708 ◽

2012 ◽

Vol 120 (8) ◽

pp. 1624-1632 ◽

Cited By ~ 60

Author(s):

Jun Shi ◽

Meili Ge ◽

Shihong Lu ◽

Xingxin Li ◽

Yingqi Shao ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Aplastic Anemia ◽

Critical Role ◽

Interferon Γ ◽

Effector T Cells ◽

Immune Mediated ◽

Main Target ◽

Lower Ratio

Abstract Acquired aplastic anemia (AA) is an immune-mediated bone marrow (BM) failure attacked by autoreactive effector T cells and BM is the main target organ. CD4+CD25+ regulatory T cells (Tregs) were believed to control development and progression of autoimmunity by suppressing autoreactive effector T cells, but little was known regarding the function of Tregs in AA. Our study demonstrated that both peripheral blood (PB) and BM had decreased frequencies of Tregs, accompanied with a reversed lower ratio of Treg frequencies between BM and PB in AA. PB Tregs in AA had impaired migratory ability because of lower CXCR4 (but not for CXCR7) expression. Interestingly, we first showed that impairment of Treg-mediated immunosuppression was intrinsic to Tregs, rather than resistance of effector T cells to suppression in AA by coculture assays and criss-cross experiments in vitro. Furthermore, Tregs in AA were less able to inhibit interferon-γ production by effector T cells. Defective immunosuppression by Tregs could contribute to impaired hematopoiesis conducted by effector T cells in vitro. Our study provided powerful evidence that impairment of Tregs played a critical role in the pathophysiology of AA. Thus, patients with AA might greatly benefit from a Treg-oriented immunosuppressive strategy.

Download Full-text

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.

Download Full-text

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

Blood ◽

10.1182/blood.v110.11.1703.1703 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1703-1703

Author(s):

Bianca Serio ◽

Ziad Peerwani ◽

Ramon Tiu ◽

Jennifer Powers ◽

Erik Hsi ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Aplastic Anemia ◽

Immunohistochemical Staining ◽

Bone Marrow Failure ◽

Peripheral Tolerance ◽

Hematopoietic Stem ◽

Central Tolerance ◽

Immune Mediated

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.

Download Full-text

The Epigenetic Regulator Ezh2 Is An Effective Target For Controlling Th1 Cell-Mediated Acquired Aplastic Anemia In Mice

Blood ◽

10.1182/blood.v122.21.2467.2467 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2467-2467

Author(s):

Qing Tong ◽

Shan He ◽

Fang Xie ◽

Amanda Wong ◽

Kazuhiro Mochizuki ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

Aplastic Anemia ◽

Cd4 T Cells ◽

Regulatory Region ◽

Th1 Cells ◽

Th1 Cell ◽

Immune Mediated ◽

Tbx21 Gene

Abstract Severe acquired aplastic anemia (AA) is a fatal disorder characterized by immune-mediated destruction of hematopoietic stem and progenitor cells. Evidence in most AA patients indicates that IFN-g-producing T helper (Th)1 effector CD4+ T cells are important for mediating bone marrow (BM) failure in AA. However, the efficacy of standard therapies that typically include antithymocyte globulin and cyclosporine A is limited, and novel approaches are urgently needed. Ezh2 is a histone methyltransferase that specifically catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3) and acts primarily as a gene silencer. We investigated whether Ezh2 regulatory control governs Th1 immune-mediated cytopenias in AA. We tested this hypothesis in a mouse model of AA using genetic approaches of Ezh2 inhibition. In naïve CD4+ T cells, high levels of H3K27me3 are correlated with repressed expression of IFNG and TBX21, the gene that encodes T-bet, which is essential for inducing IFN-g expression. Upon Th1 cell differentiation, the regulatory regions of both IFNG and TBX21 gene loci show a marked reduction of H3K27me3. We found that Ezh2 is required to induce Th1 cell differentiation and T cell-mediated AA in mice. Conditionally deleting Ezh2 in mature T cells had the effect of dramatically reducing the production of Th1 cells secreting high levels of IFN-g in vivo, decreasing BM-infiltrating Th1 cells during active disease, and rescuing mice from BM failure. In vitro culture assays confirmed that Ezh2-deficient T cells showed significantly reduced production of IFN-g under Th1-skewing conditions compared to wild-type (WT) T cells. This effect of Ezh2 deficiency on Th1 cell differentiation was accompanied by a marked decrease in the expression of both IFNG and TBX21 genes. These results stand in sharp contrast to the conventional view that Ezh2 and its catalyzed H3K27me3 may repress gene expression, and the corollary that loss of Ezh2 may result in increased production of IFN-g and T-bet. Using chromatin immunoprecipitation assay, we found that upon Th1 cell differentiation in vitro, naïve WT CD4+ T cells showed a significant reduction of H3K27me3 at the regulatory region of both IFNG and TBX21 gene loci, in agreement with previous reports. In contrast, high levels of Ezh2 were detected at the regulatory region of the TBX21 gene in activated WT CD4+ T cells, suggesting that Ezh2 may be required to promote TBX21 transcription during Th1 cell development. To test this possibility, we infected Ezh2-deficient CD4+ T cells with a retrovirus construct encoding T-bet. Ectopic expression of T-bet rescued Th1 cell differentiation of Ezh2-deficient T cells in vitro. Collectively, our findings identify a critical role for Ezh2 in regulating Th1 responses and AA. Given the availability of Ezh2-specific inhibitors newly developed for cancer therapy in clinical trials, we propose that targeting Ezh2 should be investigated as a new strategy for treating AA in patients. Disclosures: No relevant conflicts of interest to declare.

Download Full-text