scholarly journals Bystander Proliferation of Piga-Mutated Hematopoietic Progenitor Cells in Acquired Aplastic Anemia Patients Possessing HLA Class I Allele-Lacking Leukocytes

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1308-1308 ◽  
Author(s):  
Takeshi Yoroidaka ◽  
Kohei Hosokawa ◽  
Tatsuya Imi ◽  
Takamasa Katagiri ◽  
Fumihiro Azuma ◽  
...  
Keyword(s):  
T Cells ◽  
Aplastic Anemia ◽  
Progenitor Cells ◽  
Hla Class I ◽  
Survival Advantage ◽  
Class I ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Lineage Diversity

Abstract [Background] Hematopoietic stem progenitor cells (HSPCs) with PIGA mutations are thought to acquire a survival advantage over normal HSPCs under immune attack against HSPCs and produce glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells in patients with acquired aplastic anemia (AA). Various underlying mechanisms of the survival advantage of PIGA-mutated HSPCs have been proposed; however, it remains still unclear how PIGA-mutated HSPCs are immunologically selected in AA. Approximately 15% of AA patients with increased GPI(-) cells possess another aberrant leukocyte subset that lacks the expression of the HLA-class I allele due to a copy number-neutral loss of heterozygosity of the HLA haplotype, which occurs in the short arm of chromosome 6 (6pLOH) as a result of uniparental disomy, or HLA allelic mutations. The presence of HLA-class I allele-lacking leukocytes (HLA-LLs) is considered to be the most compelling evidence to support the involvement of cytotoxic T lymphocytes (CTLs) in the development of bone marrow failure. Charactering GPI(-) leukocytes and platelets in AA patients with HLA-LLs may provide an insight into the mechanism underlying the immune selection of PIGA-mutated HSPCs. [Patients and Methods] We investigated the presence of GPI(-) leukocytes, erythrocytes, and platelets in 63 patients with AA using high-sensitivity flow cytometry (FCM). For the platelet analysis, platelet rich plasma (PRP) was obtained by centrifuging anticoagulated blood at 1000 rpm for 7 minutes with the brake turned off. Thirty microliters of PRP was incubated with monoclonal antibodies specific to CD55-PE, CD59-PE, CD41a-APC and HLA-A2 or A24-FITC for 20 minutes at room temperature in the dark. To prevent doublets, samples were diluted 1 to 100 in PBS and filtered with mesh immediately before the FCM analysis. Thirty of the 63 patients were heterozygous for the HLA-A allele with A24 and A2, and thus the presence of both HLA-LLs and HLA-A allele-lacking platelets could be evaluated by FCM. The lack of the HLA-A allele due to 6pLOH or allelic mutations in all HLA-LL(+) patients was confirmed by a droplet digital PCR or deep sequencing. [Results] Increased GPI(-) granulocytes, which accounted for 0.01-99.8% of the total granulocytes, were detected in 37 (58.7%) patients while HLA-A24 or A2-lacking granulocytes accounted for 0.39-98.3% of the total granulocytes in 20 (66.7%) of the 30 patients. Eight patients possessed both GPI(-) cells and HLA-LLs. In all 8 of these patients, the two aberrant cell populations were mutually exclusive. The analyses of different cell lineages revealed HLA-A allele-lacking cells in all lineages of cells, including granulocytes (Gs), monocytes (Ms), T cells (Ts), B cells (Bs), NK cells (NKs), and platelets (Ps) in 7 of the 8 patients; the remaining one patient had the GMTP pattern. In contrast, the lineage diversity of GPI(-) in the 8 patients was more restricted; GMTBNKP was only detected in 2 patients; the combinations in the other 6 patients were GT (n=1), GMBNKP (n=2), GMTNKP (n= 1) and GMTBP (n= 2). In Case 1, GPI(-) cells were not detected in T cells while HLA-A24(-) cells were detected in all lineages of cells including T cells (Figure 1). The limited lineage diversity of GPI(-) cells was also evident in 6 patients who did not possess HLA-LLs (GMP, GMBP, GMBNKP, GMTNKP, GMTBP) with GPI(-) granulocytes>10% while the GMTBNKP pattern was common in 10 HLA-LL(+) patients who did not possess GPI(-) cells, regardless of their percentage of HLA-A allele-lacking granulocytes. Longitudinal follow-up of 5 patients over a period of 8-27 years showed a decline in the percentage of GPI(-) granulocytes (39.2 to 0.00%, 11.4 to 0.04%, 3.50 to 0.30%, 1.77 to 0.00% and 0.79 to 0.11%) and a reciprocal increase in the percentage of HLA-A allele-lacking granulocytes (80.0 to 95.2%, 92.0 to 99.1%, 24.0 to 24.4%) in 3 patients who had been placed under observation; in two patients (Cases 2 and 3) whose GPI(-) granulocyte percentages had been >10%, the PNH clones were completely replaced by HLA-LL clones during 6 and 8 years, respectively (Figure 2). [Conclusions] The limited diversity of the blood cell lineage and spontaneous decline of GPI(-) cells that coexisted with HLA-LLs suggest that GPI(-) cells are derived from the PIGA-mutated hematopoietic progenitor cells that were allowed to proliferate as a bystander in the environment where the CTL attack against HSPCs is taking place. Disclosures Nakao: Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria.

Evidence That GPI-AP-Specific CTLs Are Not Involved In The “escape” Of Piga mutant Hematopoietic Stem Cells In Aplastic Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1240-1240
Author(s):  
Takamasa Katagiri ◽  
Hiroyuki Maruyama ◽  
Shigeki Ohtake ◽  
Chizuru Saito ◽  
Kohei Hosokawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Hematopoietic Stem Cells ◽  
Aplastic Anemia ◽  
Survival Advantage ◽  
Lower Sensitivity ◽  
Hematopoietic Stem ◽  
Specific Ctls ◽  
Leukocyte Population ◽  
Lineage Diversity

Abstract Background Hematopoietic stem cells (HSCs) harboring PIGA mutations acquire a survival advantage under immune pressure compared to normal HSCs in patients with acquired aplastic anemia (AA). Cytotoxic T cells (CTLs) specific to glycosylphosphatidylinositol-anchored proteins (GPI-APs) are reportedly involved in this survival advantage, because PIGA mutant HSCs cannot present GPI-AP-derived peptides via class I HLAs. However, there is no convincing evidence that CTLs specific to GPI-AP-derived peptides are involved in the “escape” hematopoiesis by PIGA mutant HSCs. We recently demonstrated that 31.4-99.4% HLA-A allele-lacking leukocytes (HLA-LLs) were detectable in approximately 13% of AA patients as a result of escape hematopoiesis by HSCs with uniparental disomy in the short arm of chromosome 6, and that some patients possessed both GPI-AP-deficient (GPI-AP-) leukocytes and HLA-LLs (Katagiri, et al. Blood 2011). We hypothesized that if GPI-AP-derived peptides serve as a target for CTLs that elicit the development of AA, HLA-LLs may always be detectable only in the GPI-AP+ leukocyte population, because PIGA mutant HSCs do not require the lack of HLA class Is for the escape from the attack by GPI-AP peptide-specific CTLs. Objectives and Methods To examine this hypothesis, the GPI-AP expression was analyzed in various leukocyte lineages in 32 (nine at diagnosis and 23 previously treated) AA patients possessing HLA-LLs by a flow cytometry (FCM) analysis with liquid fluorescent aerolysin. Results A total of 0.01%-50% GPI-AP- granulocytes (GPI-AP- Gs) were detected in 22 (69%) of the 32 HLA-LLs (+) patients. Of the 22 patients possessing both HLA-LLs and increased GPI-AP- Gs, HLA-LLs were detectable in GPI-AP+ cells alone in 19 patients (86%). However, in the remaining three patients, HLA-LLs were shown in both GPI-AP+ and GPI-AP- populations. To determine which mutation occurs first in HSCs with a PIGA mutation and 6pUPD, the lineage diversity of GPI-AP- HLA-LLs was determined in the three patients. In two of the three patients, the lineage diversity of GPI-AP- cells (G/monocytes (M)/T cells (T)/B cells (B)/NK cells (NK)) and G/M/T) was greater than that of the HLA-A-lacking cells (G/M/B/NK and G/M) suggesting that PIGA mutations occurred earlier in the maturation of HSCs than did 6pUPD. The lineage diversity was the same in the GPI-AP- cells and HLA-LLs in one patient Conclusions The presence of HLA-LLs in the GPI-AP- leukocyte population and lower lineage diversity in HLA-LLs than GPI-AP- leukocytes suggest that CTLs specific to GPI-APs are not involved in the escape of PIGA mutant HSCs in AA, and that other mechanisms, such as a lower sensitivity to myelosuppressive cytokines than wild-type HSCs, may contribute to the survival advantage of PIGA mutant HSCs. Disclosures: No relevant conflicts of interest to declare.

Generation of Ips Cell-Derived Hematopoietic Progenitor Cells from Patients with Acquired Aplastic Anemia Harboring Copy Number Neutral Loss of Heterozygosity of the Short Arm of Chromosome 6

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2415-2415
Author(s):  
Luis Espinoza ◽  
Yoshinori Yoshida ◽  
Kazuhisa Chonabayashi ◽  
Takamasa Katagiri ◽  
Yoshitaka Zaimoku ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Progenitor Cells ◽  
Copy Number ◽  
Neutral Loss ◽  
Class I ◽  
Hematopoietic Progenitor Cells ◽  
Chromosome 6 ◽  
Hematopoietic Progenitor

Abstract Aplastic anemia (AA) is a syndrome of bone marrow failure characterized by peripheral pancytopenia and marrow hypoplasia. Because of the low number of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow, it has been intrinsically difficult to study AA. Although autoimmunity to HSPCs is considered a key factor responsible for the pathogenesis of this disorder, little is known about the molecular basis of this autoimmunity. We have previously reported that leukocytes lacking HLA class I alleles are frequently detectable in patients with AA and are derived from HSPCs that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH), and thereby escape the cytotoxic T-cell (CTL) attack against HSPCs (Katagiri et al, Blood 2011). Studying HSPCs derived from AA patients with 6pLOH(+) and their 6pLOH(-) counterparts should therefore be useful for identifying specific antigens that are targeted by CTLs in AA. In this study we generated human induced pluripotent stem cells (iPSCs) from monocytes derived from four AA patients with 6pLOH. Our generated iPSCs had normal karyotypes and were positive for human pluripotent stem cell markers such as Oct4 and SSEA4. The proportion of 6pLOH(+) iPSC clones generated from each patient was similar to the proportion 6pLOH(+) cells in the parental monocytes (50%, 100%, 100% and 100%, respectively). Unlike to their parental monocytes and consistent with previous reports, iPSCs poorly expressed class I and class II HLAs. Using an in vitro differentiation system with various hematopoietic cytokines and growth factors, iPSCs were successfully differentiated into CD34+ hematopoietic progenitor cells (iPSC-HPCs). Phenotypic studies revealed that on day 21 of the differentiation process, HPCs expressed CD34 (44.0%), the erythroid cell marker CD235a (10.5%), and CD45 (73.3%) but did not express the lineage-committed marker CD33or CD38, which is a marker expressed by a variety of mature hematopoietic cell types (Figure 1). As expected, both HLA-A alleles were highly expressed by in vitro differentiated HPCs derived from 6pLOH(-) wild type iPSC clones while the one HLA-A allele that was missing on the parental monocytes (HLA-A24) was undetectable in HPCs derived from 6pLOH(+) iPSC clones. Similarly, iPSC-HPCs did not express MHC class I-related chain (MIC)-A/B, the ligands for the NK cell activating receptor NKG2D. To further determine the hematopoietic potential of the iPSC-HPCs, they were cultured in the methylcellulose medium containing various colony stimulating factors. iPSC-HPCs were able to generate various types of colonies including CFU-GEMM (granulocyte, erythrocyte, macrophage, and megakaryocyte) and BFU-E at the plating efficiency of approximately 50%. Our study demonstrated, for the first time, that functional HPCs that lack particular HLA alleles due to 6pLOH can be successfully generated in vitro. The large number of HSCs generated from patients' derived iPSCs may be an excellent tool for searching antigens targeted by CTLs and investigating the pathogenesis of AA and thus developing novel therapeutic approaches. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Minor histocompatibility antigen-specific cytotoxic T cell lines, capable of lysing human hematopoietic progenitor cells, can be generated in vitro by stimulation with HLA-identical bone marrow cells.

1991 ◽  
Vol 173 (1) ◽  
pp. 101-109 ◽  
Author(s):  
W A Marijt ◽  
W F Veenhof ◽  
A Brand ◽  
E Goulmy ◽  
W E Fibbe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Hla Class I ◽  
Class I ◽  
Hematopoietic Progenitor Cells ◽  
Cytotoxic T Cell ◽  
Hematopoietic Progenitor ◽  
Marrow Graft

Recipient-antidonor alloreactivity before HLA genotypically identical bone marrow transplantation (BMT) between donor-recipient pairs that are negative in the mixed lymphocyte reaction (MLR), the cell-mediated lympholysis (CML) assay, and the lymphocyte crossmatch was not detectable in the majority of cases, using recipient peripheral blood lymphocytes (PBL) collected before BMT as responder cells and donor PBL as stimulator cells. However, when donor bone marrow mononuclear cells (BMMNC) instead of PBL were used as stimulator cells, we could detect donor-specific alloreactivity in 7 of 10 HLA genotypically identical donor-recipient pairs. To demonstrate that this alloreactivity was minor histocompatibility (mH) antigen specific and not directed against HLA class I splits or variants, two cytotoxic T lymphocyte (CTL) lines were tested in further detail against phytohemagglutinin (PHA) blasts from pairs of HLA genotypically identical siblings positive for the HLA class I restriction molecule. Both CTL lines recognized mH antigens, as illustrated by the differential recognition of PHA blasts of one of the two siblings from several pairs. The potential role of these mH antigen-specific CTLs in bone marrow graft rejection was demonstrated by the mH antigen-specific growth inhibition of hematopoietic progenitor cells from the original bone marrow donor and from HLA class I restriction molecule-positive individuals who expressed the mH antigens on their PBL and BMMNC. Our assay can be used in HLA genotypically identical BMT to detect a recipient-antidonor response, directed against cellularly defined mH antigens expressed on donor HPC, BMMNC, and PBL, before transplantation.

scholarly journals Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 801-807 ◽  
Author(s):  
T Leemhuis ◽  
D Leibowitz ◽  
G Cox ◽  
R Silver ◽  
EF Srour ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Polymerase Chain Reaction Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.

scholarly journals Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 801-807 ◽  
Author(s):  
T Leemhuis ◽  
D Leibowitz ◽  
G Cox ◽  
R Silver ◽  
EF Srour ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Polymerase Chain Reaction Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.

scholarly journals Intracellular Immunization of Rhesus CD34+ Hematopoietic Progenitor Cells With a Hairpin Ribozyme Protects T Cells and Macrophages From Simian Immunodeficiency Virus Infection

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4822-4831 ◽  
Author(s):  
Michael Rosenzweig ◽  
Douglas F. Marks ◽  
Donna Hempel ◽  
Marina Heusch ◽  
Günter Kraus ◽  
...  
Keyword(s):  
T Cells ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hairpin Ribozyme ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Immunodeficiency Virus ◽  
Cell Gene ◽  
Stem Cell Gene Therapy

Abstract Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.

scholarly journals Interferon gamma selectively inhibits very primitive CD342+CD38- and not more mature CD34+CD38+ human hematopoietic progenitor cells.

1994 ◽  
Vol 180 (3) ◽  
pp. 1177-1182 ◽  
Author(s):  
H W Snoeck ◽  
D R Van Bockstaele ◽  
G Nys ◽  
M Lenjou ◽  
F Lardon ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Interferon Gamma ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Ifn Gamma ◽  
Hematopoietic Stem ◽  
First Time ◽  
Dose Dependent

To assess the effects of interferon gamma (IFN-gamma) on very primitive hematopoietic progenitor cells, CD34(2+)CD38- human bone marrow cells were isolated and cultured in a two-stage culture system, consisting of a primary liquid culture phase followed by a secondary semisolid colony assay. CD34(2+)CD38- cells needed at least the presence of interleukin 3 (IL-3) and kit ligand (KL) together with either IL-1, IL-6, or granulocyte-colony-stimulating factor (G-CSF) in the primary liquid phase in order to proliferate and differentiate into secondary colony-forming cells (CFC). Addition of IFN-gamma to the primary liquid cultures inhibited cell proliferation and generation of secondary CFC in a dose-dependent way. This was a direct effect since it was also seen in primary single cell cultures of CD34(2+)CD38- cells. The proliferation of more mature CD34+CD38+ cells, however, was not inhibited by IFN-gamma, demonstrating for the first time that IFN-gamma is a specific and direct hematopoietic stem cell inhibitor. IFN-gamma, moreover, preserves the viability of CD34(2+)CD38- cells in the absence of other cytokines. IFN-gamma could, therefore, play a role in the protection of the stem cell compartment from exhaustion in situations of hematopoietic stress and may be useful as stem cell protecting agent against chemotherapy for cancer.

Inhibition of Hematopoietic Progenitor Cells by CD4+ T Cells Specific for an Endogenous Peroxisomal Protein, DRS-1: A Possible Mechanism for Bone Marrow Failure in Individuals Carrying HLA-DR15.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1345-1345
Author(s):  
Xingmin Feng ◽  
Tatsuya Chuhjo ◽  
Xuzhang Lu ◽  
Hiroyuki Takamatsu ◽  
Chiharu Sugimori ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cd4 T Cells ◽  
Leukemia Cell ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Normal Individuals ◽  
Organ Specific

Abstract A large body of evidence has suggested that acquired aplastic anemia (AA) of patients carrying HLA-DR15 is a kind of organ-specific autoimmune disease where hematopoietic progenitor cells in bone marrow are attacked by CD4+ T cells recognizing endogenous antigens. We recently identified diazepam-binding inhibitor-related protein 1 (DRS-1) as a candidate autoantigen capable of provoking immune system attack against hematopoietic progenitor cells in AA (Blood, 2004). Although in other organ-specific autoimmune diseases such as insulin-dependent diabetes mellitus and primary biliary cirrhosis, cytoplasmic proteins including glutamic acid decarboxylase 65 and pyruvate dehydrogenase complex have been shown to serve as autoantigens and mediate organ damages by CD4+ T cells, it remains unclear whether a peroxisomal protein like DRS-1 can be processed in hematopoietic progenitor cells, presented by HLA-DR15, and eventually serve as a target antigen of specific CD4+ T cells, leading to killing of hematopoietic progenitor cells themselves. To clarify these issues, we established a CD4+ T-cell line specific to a DRS-1 peptide (amino acid residues 191–204) from an AA patient carrying HLA-DR15 who had exhibited a high titer of anti-DRS-1 antibody as well as a high frequency of T-cell precursors specific to DRS-1, and then examined the cytotoxicity of the DRS-1-specific T-cell line against (1) autologous lymphoblastoid cell line (LCL) cells transfected with full length DRS-1 cDNA using a lentiviral vector, (2) myeloid leukemia cell lines carrying HLA-DR15 (KH88 and SAS413) and a leukemia cell line not carrying HLA-DR15 (K562), and (3) CD34+ progenitor cells from normal individuals. When all leukemia cell lines and LCL cells were examined for DRS-1 expression using Western blotting with specific monoclonal antibodies, DRS-1 protein was detected in DRS-1-transfected LCL cells, KH88 and K562, but not in nontransfected LCL cells and SAS413. Overexpression of DRS-1 gene by the CD34+ cells from normal individuals was ascertained by real-time PCR. In the 51Cr release assay, DRS-1-specific T cells showed cytotoxicity against only DRS-1-transfected LCL cells and KH88 in a dose-dependent manner (Figure), indicating that the T cell line requires presence of both DRS-1 and HLA-DR15 on target cells to exert cytotoxicity. When the DRS-1-specific T cells were incubated with CD34+ cells isolated from normal individuals with or without HLA-DR15 at an 10:1 ratio for 4 hours and cultured in a methylcellulose medium supplemented with colony-stimulating factors, the numbers of CFU-GM and BFU-E colonies derived from an HLA-DR15+ individual were 60.0% and 52.9% of a control whereas those derived from an HLA-DR15− individual were 90.1% and 88.2%. These findings indicate that hematopoietic progenitor cells in individuals with HLA-DR15 can present DRS-1 through the DR molecule and a breakdown of immune tolerance to DRS-1 may lead to development of AA.

Flt3 Ligand Negatively Regulates In Vitro Migration, Intracellular Signaling Activated by SDF-1α/CXCR4 and In Vivo Homing of Hematopoietic Progenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2674-2674
Author(s):  
Seiji Fukuda ◽  
Hal E. Broxmeyer ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Leukemia Cells ◽  
Flt3 Ligand ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The Flt3 receptor tyrosine kinase (Flt3) is expressed on primitive normal and transformed hematopoietic cells and Flt3 ligand (FL) facilitates hematopoietic stem cell mobilization in vivo. The CXC chemokine SDF-1α(CXCL12) attracts primitive hematopoietic cells to the bone marrow microenvironment while disruption of interaction between SDF-1α and its receptor CXCR4 within bone marrow may facilitate their mobilization to the peripheral circulation. We have previously shown that Flt3 ligand has chemokinetic activity and synergistically increases migration of CD34+ cells and Ba/F3-Flt3 cells to SDF-1α in short-term migration assays; this was associated with synergistic phosphorylation of MAPKp42/p44, CREB and Akt. Consistent with these findings, over-expression of constitutively active ITD (internal tandem duplication) Flt3 found in patients with AML dramatically increased migration to SDF-1α in Ba/F3 cells. Since FL can induce mobilization of hematopoietic stem cells, we examined if FL could antagonize SDF-1α/CXCR4 function and evaluated the effect of FL on in vivo homing of normal hematopoietic progenitor cells. FL synergistically increased migration of human RS4;11 acute leukemia cells, which co-express wild-type Flt3 and CXCR4, to SDF-1α in short term migration assay. Exogenous FL had no effect on SDF-1α induced migration of MV4-11 cells that express ITD-Flt3 and CXCR4 however migration to SDF-1α was partially blocked by treatment with the tyrosine kinase inhibitor AG1296, which inhibits Flt3 kinase activity. These results suggest that FL/Flt3 signaling positively regulates SDF-1α mediated chemotaxis of human acute leukemia cells in short-term assays in vitro, similar to that seen with normal CD34+ cells. In contrast to the enhancing effect of FL on SDF-1α, prolonged incubation of RS4;11 and THP-1 acute myeloid leukemia cells, which also express Flt3 and CXCR4, with FL for 48hr, significantly inhibited migration to SDF-1α, coincident with reduction of cell surface CXCR4. Similarly, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulates CXCR4 expression, inhibits SDF-1α-mediated phosphorylation of MAPKp42/p44, CREB and Akt and impairs migration to SDF-1α. Despite reduction of surface CXCR4, CXCR4 mRNA and intracellular CXCR4 in Ba/F3-Flt3 cells were equivalent in cells incubated with or without FL, determined by RT-PCR and flow cytometry after cell permeabilization, suggesting that the reduction of cell surface CXCR4 expression is due to accelerated internalization of CXCR4. Furthermore, incubation of Ba/F3-Flt3 cells with FL for 48hr or over-expression of ITD-Flt3 in Ba/F3 cells significantly reduced adhesion to VCAM1. Consistent with the negative effect of FL on in vitro migration and adhesion to VCAM1, pretreatment of mouse bone marrow cells with 100ng/ml of FL decreased in vivo homing of CFU-GM to recipient marrow by 36±7% (P<0.01), indicating that FL can negatively regulate in vivo homing of hematopoietic progenitor cells. These findings indicate that short term effect of FL can provide stimulatory signals whereas prolonged exposure has negative effects on SDF-1α/CXCR4-mediated signaling and migration and suggest that the FL/Flt3 axis regulates hematopoietic cell trafficking in vivo. Manipulation of SDF-1α/CXCR4 and FL/Flt3 interaction could be clinically useful for hematopoietic cell transplantation and for treatment of hematopoietic malignancies in which both Flt3 and CXCR4 are expressed.

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