Hematopoiesis In Immunodeficient Mice Research Articles

Identification of T Cell Receptors Specific to Antigens Presented By HLA-B5401 on IPS Cell-Derived Hematopoietic Stem Cells in a Patient with Acquired Aplastic Anemia Carrying B5401-Lacking Leukocytes

[Background] Cytotoxic T lymphocytes (CTLs) specific to hematopoietic stem cells (HSCs) are thought to play an important role in the development of acquired aplastic anemia (AA). The presence of HLA class I allele-lacking leukocytes as a result of 6pLOH or allelic mutations in about 25% of AA patients strongly suggests that CTLs specific to antigens presented by the specific HLA alleles are involved in the destruction of normal HSCs as well as in the escape of HLA-lacking HSCs. Establishing CTLs from these patients by culturing the patients' CD8+ cells with targeted HSCs may be useful for identifying autoantigens of AA. However, it is generally impossible to obtain sufficient numbers of HSCs from the patients for functional assays and in vitro stimulation of patient's T cells with HSCs may induce clonal expansion of CTLs irrelevant to pathogenesis of AA. To solve these problems, we generated iPS cells (iPSCs) from an untreated AA patient who possessed HLA-B5401-lacking leukocytes for inducing HSCs and attempted to identify cDNAs of T-cell receptor (TCR) α and β chains of predominant T cells in the patient's bone marrow (BM) using a single T-cell analysis, which may specifically recognize autologous HSCs. [Methods] iPSCs were generated from the peripheral blood mononuclear cells of an AA patient (56-year-old man), whose moderate AA later responded to cyclosporine therapy, using Yamanaka's method. The patient's monocytes accounted for 20% wild-type, 20% 6pLOH(+) and 60% B5401-lacking cells at the time of sampling. CD34+ cells were induced from these iPSCs and injected into the BM of C57BL/6.Rag2 mice harboring NOD-Sirpa (BRGS). CD8+ T cells in the BM of the patient were subjected to a single cell sorting followed by cDNA amplification of TCRVβ chains. TCRα chains of five T cells that showed the five most recurrent TCRVβs were determined, and the TCRβ cDNAs were retrovirally transfected to Jurkat cells that lacked TCRβ chain and were transfected with IFN-γ cDNA. Specific responses of each TCR transfectant to iPSC-derived CD34+ cells were determined using ELISA for detecting IFN-γ. [Results] Five iPSC clones, including wild type (n=2), 6pLOH(+) (n=1), and B5401-lacking (B5401[-]) (n=2) clones, that had different mutations in B*54:01 were generated. Three-week culture of the iPSCs in the presence of various growth factors produced hematopoietic cells consisting of 50% CD34+ cells of each phenotype, which were able to reconstitute of BRGS mice with 3.5% to 8.9% human CD45+ cells in the peripheral blood at 9-12 weeks after injection. When the four TCR transfectants were tested for their response to wild-type and B5401-lacking CD34+ cells, all four transfectants secreted higher amounts of IFN-γ in response to wild-type cells than to B5401(-) CD34+ cells. [Conclusions] HSCs with original genotypes capable of reconstituting hematopoiesis in immunodeficient mice were successfully induced from iPSCs that had been generated from an AA patient's monocytes. The TCR transfectants showing a specific response to B5401(+) wild-type CD34+ cells will be useful for identifying the ligand peptides presented by B5401. DisclosuresNo relevant conflicts of interest to declare.

Escape Hematopoiesis By HLA-B5401-Lacking Hematopoietic Stem Progenitor Cells in Male Patients with Acquired Aplastic Anemia

[Background] Leukocytes that lack HLA class I alleles derived from hematopoietic stem progenitor cells (HSPCs) that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations are often detected in acquired aplastic anemia (AA) patients. The presence of HLA class I allele-lacking leukocytes provides compelling evidence that CTLs are involved in the development of AA, but the precise mechanisms underlying HLA missing and clonal hematopoiesis by such HLA(-) HSPCs are unknown. Our recent study showed that, among several HLA-class I alleles that are likely to be lost as a result of 6pLOH, HLA-B*40:02 is the most frequently lost allele in Japanese AA patients. The study also showed that B*54:01 was one of three HLA-alleles that were most likely to be possessed by 6pLOH(+) patients (29% [5/17]) when only patients not carrying HLA-B*40:02 were analyzed. These results prompted us to study the role of HLA-B*54:01 in the pathogenesis of AA in a larger number of patients. [Method] To identify HLA class I alleles other than HLA-B*40:02 that are closely involved in the auto-antigen presentation in AA, we studied leukocytes of 549 AA patients for the presence of 6pLOH as well as HLA alleles that are lost due to 6pLOH. To gain insight into the mechanism underlying clonal hematopoiesis by HLA-B*54:01-lacking HSPCs, we studied HSPCs derived from induced pluripotent stem cells (iPSCs) that were generated from an AA patient possessing B5401-lacking monocytes. We also investigated the association between male AA patients possessing B*54:01 and CAG microsatellites of androgen receptor (AR) gene which are related to transactivation of the AR gene. [Results] 6pLOH was detected in 91 (16.6%) of the total patients and in 48 (10.4%) of the 462 patients not possessing B*40:02. Among the HLA alleles possessed by the 48 patients, B*54:01 was the most frequent (23%). 6pLOH was detected in 17 (34%) of 50 patients possessing B*54:01, and the incidence was markedly higher in males (15/24, 62.5%) than in female patients (2/26, 7.7%, P<0.001). Notably, the 11 patients who lost HLA-B*54:01-containing haplotypes were all male. A flow cytometry analysis revealed the presence of leukocytes that lacked Bw6 (B5401) alone in one (KANA6) of the five 6pLOH(+) patients heterozygous for HLA-Bw (Bw4/Bw6). Deep sequencing of HLA class I alleles in granulocytes from the 11 6pLOH(+) patients revealed a mutation (stop mutation) only in HLA-B*54:01 of KANA6. Reprograming of KANA6's monocytes generated three different iPSCs, including wild-type (WT), 6pLOH(+), and B*54:01-mutated clones, and CD34+ cells that were induced from the three different iPSC clones reconstituted human hematopoiesis in immunodeficient mice to a similar degree, suggesting that B5401-lacking HSPCs without proliferative advantage support hematopoiesis of KANA6. The CAG repeat lengths in male AA patients possessing B*54:01 tented to be shorter than those in the healthy controls (median value 21 vs 23). [Conclusions] Our results suggest that HLA-B*54:01 plays an important role in the autoantigen presentation by HSPCs to T cells in male patients with AA. The shorter CAG repeat lengths of the AR gene that correlate with higher expression levels of AR may explain the male predominance in 6pLOH(+) patients possessing HLA-B*54:01. DisclosuresYoshida:Nihon-shinyaku: Research Funding. Akashi:Novartis pharma: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; MSD: Research Funding; Eisai: Research Funding; Asahi-kasei: Research Funding; Ono Pharmaceutical: Research Funding; Pfizer: Research Funding; Celgene: Research Funding, Speakers Bureau; sanofi: Research Funding; Chugai Pharma: Research Funding; Otsuka Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Taiho Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Eli Lilly Japan: Research Funding. Nakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

Fludarabine as a cost-effective adjuvant to enhance engraftment of human normal and malignant hematopoiesis in immunodeficient mice

There is still an unmet need for xenotransplantation models that efficiently recapitulate normal and malignant human hematopoiesis. Indeed, there are a number of strategies to generate humanized mice and specific protocols, including techniques to optimize the cytokine environment of recipient mice and drug alternatives or complementary to the standard conditioning regimens, that can be significantly modulated. Unfortunately, the high costs related to the use of sophisticated mouse models may limit the application of these models to studies that require an extensive experimental design. Here, using an affordable and convenient method, we demonstrate that the administration of fludarabine (FludaraTM) promotes the extensive and rapid engraftment of human normal hematopoiesis in immunodeficient mice. Quantification of human CD45+ cells in bone marrow revealed approximately a 102-fold increase in mice conditioned with irradiation plus fludarabine. Engrafted cells in the bone marrow included hematopoietic stem cells, as well as myeloid and lymphoid cells. Moreover, this model proved to be sufficient for robust reconstitution of malignant myeloid hematopoiesis, permitting primary acute myeloid leukemia cells to engraft as early as 8 weeks after the transplant. Overall, these results present a novel and affordable model for engraftment of human normal and malignant hematopoiesis in immunodeficient mice.

Cytokine and integrin stimulation synergize to promote higher levels of GATA-2, c-myb, and CD34 protein in primary human hematopoietic progenitors from bone marrow

AbstractWe have previously shown that engagement of the integrins VLA-4 and VLA-5 to the fibronectin fragment CH-296 in combination with cytokines sustained the capacity of cultured human CD34+ cells to undergo hematopoiesis in immunodeficient mice for 7 to 12 months, whereas this capacity was rapidly lost in cells cultured in suspension with the same cytokines. In the current study, we assessed the molecular pathways that might explain the loss of long-term engraftment capacity in cells cultured in suspension. Although the cell cycle profile was similar between cells cultured in suspension versus on fibronectin, levels of cell death were higher in the suspended cultures. While the CDK inhibitors p27Kip1 and p57Kip2 were present at equal levels in cells from both cultures, low levels of p21Cip1 were detectable only in the cytoplasmic compartment of cells cultured in suspension. Cytoplasmic location of p21Cip1 has been linked to monocytic differentiation. The levels of c-myb and GATA-2, transcription factors associated with stem cell maintenance, were higher in cells cultured on fibronectin as compared with suspension. In contrast, the levels of PU.1, which is induced during myeloid differentiation, were higher in cells cultured in suspension. There were no significant differences in surface expression of CD34 on the cells after culture, but total CD34 protein, assessed by immunoblotting, was significantly higher in cells cultured on fibronectin. Our data suggest that, in the presence of cytokines, the engagement of VLA-4 and VLA-5 integrins to the fibronectin fragment CH-296 preserves the expression of specific transcription factors associated with primitive stem cell maintenance. In contrast, a lack of integrin engagement leads to the induction of cellular markers associated with myeloid differentiation.

Prolonged multilineage clonal hematopoiesis in a rhesus recipient of CD34 positive cells marked with a RD114 pseudotyped oncoretroviral vector

The ability to efficiently transfer a gene into repopulating hematopoietic stem cells would create many therapeutic opportunities. We have evaluated the ability of particles bearing an alternative envelope protein, that of the feline endogenous virus (RD114), to transduce stem cells in a nonhuman primate autologous transplantation model using rhesus macaques. We have previously shown this pseudotyped vector to be superior to the amphotropic vector at transducing cells in umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice. Gene transfer efficiency as reflected by the number of genetically modified cells in hematopoietic tissues varied among the five monkeys studied from low levels (<1%) in three animals to much higher levels in two (20–60%). An animal that exhibited extremely high levels for several weeks was found by vector genome insertion site analysis to have reconstitution predominantly with a single clone of cells. This variability among animals is in keeping with computer simulations of reconstitution with limiting numbers of stem cells genetically modified at about 10% efficiency. Our studies provide insights into the biology of hematopoietic reconstitution and suggest approaches for increasing stem cell targeted gene transfer efficiency.

In vitro and in vivo hematopoietic potential of human stem cells residing in muscle tissue.

We studied the in vitro and in vivo hematopoietic potential of human stem cells residing in muscle tissue collected from adults with head and neck cancer. Adherent muscle cells were cultured in F12 medium with 10% fetal bovine serum and transplanted into immunodeficient mice. On day 12 we obtained a median of 500,000 adherent cells per gram muscle sample. Thy-1, endoglin, HER2/neu, and P1H12 were expressed in the majority of cells. CD34, VEGFR2, c-kit, VCAM-1, and CXCR4 were expressed in 0.5-1.5%, 1-5%, 1-15%, 9-15%, and 30% of cells, respectively. Immunodeficient mice transplanted with fresh muscle cells or less than 500,000 cultured cells showed little or no human engraftment. In mice transplanted with more than 500,000 cultured cells, up to 14% human CD45(+) hematopoietic cells (including myeloid and lymphoid subsets) were detected by flow cytometry. Engraftment was confirmed by polymerase chain reaction, Southern blotting, and DNA sequencing. Liver, muscle, and spleen evaluated for human DNA were positive in the majority of mice showing hematopoietic engraftment in the bone marrow. In vivo hematopoietic engraftment potential was maintained in cultured CD45(-) muscle cells transduced with the green fluorescence protein gene. Human stem cells residing in muscle tissue can generate multilineage hematopoiesis in immunodeficient mice. Surprisingly, this hematopoietic potential increased in cultured versus fresh cells from muscle tissue.

RD114‐Pseudotyped Oncoretroviral Vectors

Limited functional expression of the viral envelope receptor is a recognized barrier to efficient oncoretroviral mediated gene transfer. To circumvent this barrier we evaluated a number of envelope proteins with respect to gene transfer efficiency into primitive human hematopoietic stem cell populations. We observed that oncoretroviral vectors pseudotyped with the envelope protein of feline endogenous virus (RD114) could efficiently transduce human repopulating cells capable of establishing multilineage hematopoiesis in immunodeficient mice after a single exposure to RD114-pseudotyped vector. Comparable rates of gene transfer with amphotropic and GALV-pseudotyped vectors have been reported, but only after multiple exposures to the viral supernatant. Oncoretroviral vectors pseudotyped with the RD114 or the amphotropic envelopes had similar stability in vitro, indicating that the increased efficiency in gene transfer is at the receptor level likely due to increased receptor expression or an increased receptor affinity for the RD114 envelope. We also found that RD114-pseudotype vectors can be efficiently concentrated, thereby removing any adverse effects of the conditioned media to the long-term repopulating potential of the target human hematopoietic stem cell. These studies demonstrate the potential of RD114-pseudotyped vectors for clinical use.

Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein

Limited expression of the amphotropic envelope receptor is a recognized barrier to efficient oncoretroviral vector–mediated gene transfer. Human hematopoietic cell lines and cord blood–derived CD34+ and CD34+, CD38− cell populations and the progenitors contained therein were transduced far more efficiently with oncoretroviral particles pseudotyped with the envelope protein of feline endogenous virus (RD114) than with conventional amphotropic vector particles. Similarly, human repopulating cells from umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice were efficiently transduced with RD114-pseudotyped particles, whereas amphotropic particles were ineffective at introducing the proviral genome. After only a single exposure of CD34+ cord blood cells to RD114-pseudotyped particles, all engrafted nonobese diabetic/severe combined immunodeficiency mice (15 of 15) contained genetically modified human bone marrow cells. Human cells that were positive for enhanced green fluorescent protein represented as much as 90% of the graft. The use of RD114-pseudotyped vectors may be advantageous for therapeutic gene transfer into hematopoietic stem cells.

Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein

AbstractLimited expression of the amphotropic envelope receptor is a recognized barrier to efficient oncoretroviral vector–mediated gene transfer. Human hematopoietic cell lines and cord blood–derived CD34+ and CD34+, CD38− cell populations and the progenitors contained therein were transduced far more efficiently with oncoretroviral particles pseudotyped with the envelope protein of feline endogenous virus (RD114) than with conventional amphotropic vector particles. Similarly, human repopulating cells from umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice were efficiently transduced with RD114-pseudotyped particles, whereas amphotropic particles were ineffective at introducing the proviral genome. After only a single exposure of CD34+ cord blood cells to RD114-pseudotyped particles, all engrafted nonobese diabetic/severe combined immunodeficiency mice (15 of 15) contained genetically modified human bone marrow cells. Human cells that were positive for enhanced green fluorescent protein represented as much as 90% of the graft. The use of RD114-pseudotyped vectors may be advantageous for therapeutic gene transfer into hematopoietic stem cells.

Sustained Human Hematopoiesis in Immunodeficient Mice by Cotransplantation of Marrow Stroma Expressing Human Interleukin-3: Analysis of Gene Transduction of Long-Lived Progenitors

We have developed a novel cotransplantation system in which gene-transduced human CD34+ progenitor cells are transplanted into immunodeficient (bnx) mice together with primary human bone marrow (BM) stromal cells engineered to produce human interleukin-3 (IL-3). The IL-3-secreting stroma produced sustained circulating levels of human IL-3 for at least 4 months in the mice. The IL-3-secreting stroma, but not control stroma, supported human hematopoiesis from the cotransplanted human BM CD34+ progenitors for up to 9 months, such that an average of 6% of the hematopoietic cells removed from the mice were of human origin (human CD45+). Human multilineage progenitors were readily detected as colony-forming units from the mouse marrow over this time period. Retroviral-mediated transfer of the neomycin phosphotransferase gene or a human glucocerebrosidase cDNA into the human CD34+ progenitor cells was performed in vitro before cotransplantation. Human multilineage progenitors were recovered from the marrow of the mice 4 to 9 months later and were shown to contain the transduced genes. Mature human blood cells marked by vector DNA circulated in the murine peripheral blood throughout this time period. This xenograft system will be useful in the study of gene transduction of human hematopoietic stem cells, by tracing the development of individually marked BM stem cells into mature blood cells of different lineages.