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.

Lymphocyte reconstitution in multiple myeloma patients after allogeneic G-CSF-mobilized hematopoietic progenitor cells transplantation from HLA-identical siblings

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7098-7098
Author(s):  
M. Martino ◽  
R. Fedele ◽  
G. Irrera ◽  
G. Messina ◽  
M. Cuzzola ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Progenitor Cells ◽  
Cd8 T Cells ◽  
Immune Reconstitution ◽  
Median Number ◽  
Hematopoietic Progenitor Cells ◽  
Immune Recovery ◽  
Hematopoietic Progenitor

7098 Background: Allogeneic transplantation of G-CSF-mobilized hematopoietic progenitor cells (HPC) results in rapid and complete engraftment in a large proportion of patients and in relatively fast immune recovery. Methods : We have analyzed by flow cytometry the immune reconstitution in 19 patients (pts) affected by multiple myeloma undergone to allogeneic HPC transplant from HLA-identical related donors after nonmyeloablative conditioning regimen with fludarabine 90 mg/m2 and cyclophosphamide 900 mg/m2. In each patient a comparable number of mononuclear cells, CD3+ T lymphocytes and CD34+ progenitor cells was infused. To evaluate the kinetics of the immune reconstitution, the overall number of total lymphocytes, T, B and NK cells of each patient were assessed before and 1, 2, 3, 6, 12, 18, 24, 30, 36 months after allogeneic HPC transplant. Results: Overall T cell reconstitution was in all the pts at 3 months, since at that time the CD3+ T cell median number was 880 cells/microl (r. 589–1,357). However, in all pts high numbers of CD3+ T cells were achieved at 12 months after transplant (median 1,326 cells/microl, r. 850–2,309). The CD4+ T cell median number was 281 cells/microl (r. 185–433) at 6 months, 391 cells/microl (r. 303–505) at 12 months, 603 cells/microl (r. 433–736) at 18 months. The CD8+ T cell median number was increased from the transplant to 18 months in which it was 1,489 cells/microl (r. 760–1,976). The decrease of CD8+ T cells with the normalization of CD4+/CD8+ ratio was observed at 30 months when CD4+ T cells were 650 cells/microl (r. 370–989) and CD8+ T cells were 690 cells/microl (r. 445–1,743). B cells recovery was observed at 18 months with a median number of 194 cells/microl (r. 40–404). The faster reconstitution was documented for NK cells with a median number of 314 cells/microl (r. 61–647) at 2 months. Conclusions: the complete immune reconstitution in our pts was achieved at 30 months after transplant. Our objective is to evaluate if this slow immune recovery is associated with a high incidence of infectious diseases and a low incidence of chronic GVHD. No significant financial relationships to disclose.

Resistance of mature T cells to oncogene transformation

Blood ◽  
2008 ◽  
Vol 112 (6) ◽  
pp. 2278-2286 ◽  
Author(s):  
Sebastian Newrzela ◽  
Kerstin Cornils ◽  
Zhixiong Li ◽  
Christopher Baum ◽  
Martijn H. Brugman ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Severe Combined Immunodeficiency ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Worst Case ◽  
Cell Transplants

AbstractLeukemia caused by retroviral insertional mutagenesis after stem cell gene transfer has been reported in several experimental animals and in patients treated for X-linked severe combined immunodeficiency. Here, we analyzed whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental “worst case scenario,” we transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of gamma retroviral vectors encoding the potent T-cell oncogenes LMO2, TCL1, or ΔTrkA, a constitutively active mutant of TrkA. After transplantation into RAG-1–deficient recipients (Ly5.2), animals that received stem cell transplants developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated polymerase chain reaction analysis revealed monoclonality or oligoclonality of the malignancies. In striking contrast, none of the mice that received T-cell transplants transduced with the same vectors developed leukemia/lymphoma despite persistence of gene-modified cells. Thus, our data provide direct evidence that mature T cells are less prone to transformation than hematopoietic progenitor cells.

Augmented Expression of WT1-Specific TCR and Inhibition of Mispaired-TCR Formation In TCR-Gene Modified T-Cells Can Concomitantly Be Achieved Using a Novel Retroviral Vector with Silencers for Endogenous TCRs

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1020-1020
Author(s):  
Toshiki Ochi ◽  
Hiroshi Fujiwara ◽  
Sachiko Okamoto ◽  
Jun An ◽  
Kozo Nagai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Progenitor Cells ◽  
Cd8 T Cells ◽  
Retroviral Vector ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Nsg Mice

Abstract Abstract 1020 [Purpose] Adoptive engineered T-cell therapy using WT1-specific T-cell receptor gene-transfer is currently considered as a challenging strategy for cancer treatment. However, the formation of shuffled TCR between endogenous and introduced TCR α/β chains still remains a major issue to be solved, not only for the improvement of introduced TCR expression, but also for the prophylaxis of “GVHD-like syndrome” due to newly generated self-reactive T-cells bearing shuffled TCRs. Taking above, we have newly developed an novel WT1-specific TCR retroviral vector with built-in silencers to inhibit endogenous TCRs. In this study, we evaluated the feasibility of this novel WT1-targeting and silencers built-in vector for the clinical application. [Methods] WT1235-243-specific and HLA-A*2402-restricted TCR α/β genes were cloned into this novel retroviral vector with built-in shRNA for endogenous TCRs. (siWT1-TCR) (1) The inhibitory effect on endogenous TCR in gene-modified T-cells was examined using the pre-established CTL clone. (2) Compared with the conventional WT1-TCR vector (without silencers), the functional avidity of siWT1-TCR-gene introduced T-cell was examined, using 51Cr-release assay, CD107a assay and intracellular IFN-γ assay, in vitro. (3) The in vivo anti-leukemia effect of siWT1-TCR introduced CD8+ T-cells was examined using NSG mice. (4) WT1-specific Th1 helper function of siWT1-TCR introduced CD4+ T-cell was examined. (5) The synergistic effect of WT1 peptide stimulation on siWT1-TCR introduced CD8+ T-cell was examined. (6) The on-target adverse effect of siWT1-TCR introduced CD8+ T-cells against autologous hematopoietic progenitor cells were examined using HLA-A*2402+ human cord blood CD34+ and TCR-gene modified autologous CD8+ T-cells, both in vitro and in vivo. [Results] Compared with conventional WT1-TCR vector, siWT1-TCR vector remarkably increased the expression of functional WT1-specific TCR accompanied with the inhibition of endogenous TCR synthesis on gene-modified T-cells. CD8+ and CD4+ T cells engineered with siWT1-TCR gene transfer exerted WT1-specific and leukemia-specific cytotoxicity, and target-responsive Th1 cytokine production, in an HLA-A*2402-restricted fashion, respectively. Mainly because of remarkably increased WT1-specific TCR expression, the anti-leukemia effect exerted by siWT1-TCR introduced CD8+ T-cells was significantly up-regulated, compared with conventional WT1-TCR vector, which reflected the increased the target-responsive granular exocytosis. SiWT1-TCR introduced CD8+ T-cells also exerted in vivo anti-leukemia effect against inoculated leukemia cell lines in NSG mice, furthermore, such siWT1-TCR introduced CD8+ T-cells could kill autologous patients' leukemia cells, but not autologous hematopoietic progenitor cells, in vitro. Eventually, transplanted human hematopoietic progenitor cells which had been precultured with siWT1-TCR introduced autologous CD8+ T-cells into irradiated NSG mice demonstrated that those pre-treated CD34+ cells preserved abilities of engraftment, proliferation and differentiation. Additionally, it was demonstrated that repetitive WT1 peptide stimulations successfully expanded siWT1-TCR introduced CD8+ T-cells, in vitro, which suggests the synergistic effect of combined peptide vaccination in vivo. [Conclusion] Our novel WT1-targeting vector could provide not only the improved functional avidity of gene-modified T-cells, but also a promising option to solve the raised major concern in safety that is the potentially lethal GVHD-like syndrome due to newly generated T-cells bearing self-antigen reactive shuffled TCRs. Based on these pre-clinical observation, we are planning to conduct clinical trials against human hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Generation and function of progenitor T cells from StemRegenin-1–expanded CD34+ human hematopoietic progenitor cells

2019 ◽  
Vol 3 (20) ◽  
pp. 2934-2948
Author(s):  
Jastaranpreet Singh ◽  
Edward L. Y. Chen ◽  
Yan Xing ◽  
Heather E. Stefanski ◽  
Bruce R. Blazar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Generate Functional ◽  
Key Points ◽  
And Function

Key Points CD34+CD7+ as well as CD34−CD7+ cells from SR1-expanded CD34+ HSPCs are effective thymus-reconstituting cells in vivo. CD7+ cells derived from SR1-expanded CD34+ HSPCs generate functional and polyclonal T-cell repertoires in vivo.

scholarly journals HL-T, a new cell line derived from HL-60 promyelocytic leukemia cell cultures expressing terminal transferase and secreting suppressor activity

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1151-1160 ◽  
Author(s):  
E Paietta ◽  
RJ Stockert ◽  
T Calvelli ◽  
P Papenhausen ◽  
SV Seremetis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Cultures ◽  
Germ Line ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Chromosome 9 ◽  
Terminal Transferase

A cell line with immature blast cell morphology was isolated from HL-60 promyelocytic leukemia cell cultures and designated HL-T. This new cell type is biphenotypic, expressing terminal transferase (TdT) together with myelomonocytoid immunologic features. TdT enzymatic activity, undetectable in HL-60, was determined to be 140 to 180 units/10(8) HL-T cells by the dGTP-assay, approximately 20% of the activity found in lymphoblastoid cell lines. HL-T predominantly synthesize the known 58- kDa TdT-protein plus a minor 54/56-kDa doublet. The 58-kDa steady state form is nonglycosylated and is phosphorylated. Precursor antigens S3.13 and MY-10, absent on HL-60, are expressed by HL-T; however, the cells are negative for HLA-Dr. Southern blot analysis by hybridization with immunoglobulin heavy chain (JH) and T cell-receptor chain gene (T beta) probes shows JH to be in the germ-line configuration in both cell lines and the T beta gene to be in germ-line in HL-60 but to be rearranged in HL-T. Truncation of the gene encoding the granulocyte-macrophage-colony- stimulating factor (GM-CSF), as found in HL-60, is not observed in HL- T. HL-T are resistant to differentiation-induction by retinoic acid and 1,25-dihydroxyvitamin D3. Cytogenetically HL-T share with HL-60 a deletion of the short arm of chromosome 9 at breakpoint p13, an aberration frequently found in patients with T cell leukemia. In addition, HL-T display t(8;9)(p11;p24) and trisomy 20. Tetraploidy is observed in 80% of HL-T metaphases with aberrations identical to those in the diploid karyotype. Like HL-60, the new line shows some surface- antigenic-T cell characteristics. Despite an antigenic pattern most consistent with that of helper-inducer T cells (T4+, D44+/-, 4B4+, 2H4- , TQ1+/-), HL-T cells and their conditioned culture medium suppress antigen, mitogen, and mixed-leukocyte-culture-mediated lymphocyte proliferation.

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.

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