Extensive expansion of primary human gamma delta T cells generates cytotoxic effector memory cells that can be labeled with Feraheme for cellular MRI

Gamma delta T cells (GDTc) comprise a small subset of cytolytic T cells shown to kill malignant cells in vitro and in vivo. We have developed a novel protocol to expand GDTc from human blood whereby GDTc were initially expanded in the presence of alpha beta T cells (ABTc) that were then depleted prior to use. We achieved clinically relevant expansions of up to 18,485-fold total GDTc, with 18,849-fold expansion of the Vδ1 GDTc subset over 21 days. ABTc depletion yielded 88.1 ± 4.2 % GDTc purity, and GDTc continued to expand after separation. Immunophenotyping revealed that expanded GDTc were mostly CD27-CD45RA- and CD27-CD45RA+ effector memory cells. GDTc cytotoxicity against PC-3M prostate cancer, U87 glioblastoma and EM-2 leukemia cells was confirmed. Both expanded Vδ1 and Vδ2 GDTc were cytotoxic to PC-3M in a T cell antigen receptor- and CD18-dependent manner. We are the first to label GDTc with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles for cellular MRI. Using protamine sulfate and magnetofection, we achieved up to 40 % labeling with clinically approved Feraheme (Ferumoxytol), as determined by enumeration of Perls’ Prussian blue-stained cytospins. Electron microscopy at 2,800× magnification verified the presence of internalized clusters of iron oxide; however, high iron uptake correlated negatively with cell viability. We found improved USPIO uptake later in culture. MRI of GDTc in agarose phantoms was performed at 3 Tesla. The signal-to-noise ratios for unlabeled and labeled cells were 56 and 21, respectively. Thus, Feraheme-labeled GDTc could be readily detected in vitro via MRI.     

Age-related changes in human hematopoietic stem/progenitor cells

Adult stem cells are critical for maintaining cellular homeostasis throughout life, yet the effects of age on their regenerative capacity are poorly understood. All lymphoid and myeloid blood cell lineages are continuously generated from hematopoietic stem cells present in human bone marrow. With age, significant changes in the function and composition of mature blood cells are observed. In this study, we report that age-related changes also occur in the human hematopoietic stem cell compartment. We find that the proportion of multipotent CD34(+) CD38(-) cells increases in the bone marrow of elderly (>70 years) individuals. CD34(+) CD38(+) CD90(-) CD45RA(+/-) CD10(-) and CD34(+) CD33(+) myeloid progenitors persist at the same level in the bone marrow, while the frequency of early CD34(+) CD38(+) CD90(-) CD45RA(+) CD10(+) and committed CD34(+) CD19(+) B-lymphoid progenitors decreases with age. In contrast to mice models of aging, transplantation experiments with immunodeficient NOD/SCID/IL-2Rγ null (NSG) mice showed that the frequency of NSG repopulating cells does not change significantly with age, and there is a decrease in myeloid lineage reconstitution. An age-related decrease in the capacity of CD34(+) cells to generate myeloid cells was also seen in colony-forming assays in vitro. Thus, with increasing age, human hematopoietic stem/progenitor cells undergo quantitative changes as well as functional modifications.     

Dynamics of the Splenic Innate-like CD19+CD45Rlo Cell Population from Adult Mice in Homeostatic and Activated Conditions

In the adult spleen, CD19(+)CD45R(-/lo) (19(+)45R(lo)) lymphocytes of embryonic origin exist as a distinct population to that of the conventional B cell lineage. These cells display a plasmablast phenotype, and they spontaneously secrete IgG1 and IgA, whereas the bone marrow population of 19(+)45R(lo) cells contains B1 progenitors. In this study, we show that 19(+)45R(lo) cells are also present in Peyer's patches and in the spleen throughout the life span of wild-type mice, beginning at postnatal day 7. Although this population is heterogeneous, the surface phenotype of most of these cells distinguishes them from follicular, transitional, marginal zone, and B1 cells. In CBA/CaHN mice, few 19(+)45R(lo) cells were detected at postnatal day 7, and none was observed in the adult spleen. Splenic 19(+)45R(lo) cells exhibited homeostatic BrdU uptake in vivo and actively transcribed cell cycle genes. When transferred to immunodeficient RAG2(-/-)γchain(-/-) recipient mice, 19(+)45R(lo) cells survived and differentiated into IgG1- and IgA-plasma cells. Moreover, in vitro stimulation of splenic 19(+)45R(lo) cells with LPS, CpG, BAFF/IL4, and CD40/IL4 induced cell proliferation, IgG1/IgA secretion and the release of IL-10, suggesting a potential immunoregulatory role for this subset of innate-like B cells.     

Human Vδ1 γδ T cells expanded from peripheral blood exhibit specific cytotoxicity against B-cell chronic lymphocytic leukemia-derived cells

Background aimsThere is increasing interest in using γδ T cells (GDTC) for cancer immunotherapy. Most studies have been concerned with the Vδ2 subset in blood, for which several expansion protocols exist. We have developed a protocol to expand Vδ1 and Vδ2 preferentially from human blood. We have characterized these subsets and their specificities for leukemic targets.MethodsGDTC were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors via positive magnetic cell sorting; their proliferation in vitro was induced by exposure to the mitogen concanavalin A (Con A). CD107 and cytotoxicity (Cr⁵¹-release and flow cytometric) assays were performed. GDTC clones and target cells were immunophenotyped via flow cytometry.ResultsLonger initial exposure to Con A typically resulted in higher Vδ1 prevalence. Vδ1 were activated by and cytotoxic to B-cell chronic lymphocytic leukemia (B-CLL)-derived MEC1 cells, whereas Vδ2 also responded to MEC1 but more so to the Philadelphia chromosome-positive [Ph+] leukemia cell line EM-enhanced green fluorescent protein (2eGFPluc). Vδ2 clone cytotoxicity against EM-2eGFPluc correlated with Vδ2 T-cell antigen receptor (TCR) and receptor found on Natural Killer cells and many T-cells (NKG2D), whereas Vδ1 clone cytotoxicity versus MEC1 correlated with Vδ1 TCR, CD56 and CD95 expression. Vδ1 also killed Epstein-Barr Virus (EBV)-negative B-CLL-derived TMD2 cells. Immunophenotyping revealed reduced HLA-ABC expression on EM-2eGFPluc, whereas MEC1 and TMD2 exhibited higher Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAILR1).ConclusionsOur ability to expand peripheral Vδ1 cells and show their cytotoxicity to B-CLL-derived cell lines suggests that this novel approach to the cellular treatment of B-CLL may be feasible.     

Experimental infection of NOD/SCID mice reconstituted with human CD34+ cells with Epstein-Barr virus

Epstein-Barr virus (EBV)-induced lymphoproliferative disease is an important complication in the context of immune deficiency. Impaired T-cell immunity allows the outgrowth of transformed cells with the subsequent production of predominantly B-cell lymphomas. Currently there is no in vivo model that can adequately recapitulate EBV infection and its association with B-cell lymphomas. NOD/SCID mice engrafted with human CD34(+) cells and reconstituted mainly with human B lymphocytes may serve as a useful xenograft model to study EBV infection and pathogenesis. We therefore infected reconstituted mice with EBV. High levels of viral DNA were detected in the peripheral blood of all infected mice. All infected mice lost weight and showed decreased activity levels. Infected mice presented large visible tumors in multiple organs, most prominently in the spleen. These tumors stained positive for human CD79a, CD20, CD30, and EBV-encoded RNAs and were light chain restricted. Their characterization is consistent with that of large cell immunoblastic lymphoma. In addition, tumor cells expressed EBNA1, LMP1, and LMP2a mRNAs, which is consistent with a type II latency program. EBV(+) lymphoblastoid cell lines expressing human CD45, CD19, CD21, CD23, CD5, and CD30 were readily established from the bone marrow and spleens of infected animals. Finally, we also demonstrate that infection with an enhanced green fluorescent protein (EGFP)-tagged virus can be monitored by the detection of infected EGFP(+) cells and EGFP(+) tumors. These data demonstrate that NOD/SCID mice that are reconstituted with human CD34(+) cells are susceptible to infection by EBV and accurately recapitulate important aspects of EBV pathogenesis.     

IL-7 enhances the responsiveness of human T cells that develop in the bone marrow of athymic mice

The beige/nude/xid/human (bnx/hu) model of human hematopoiesis provides a unique opportunity to study extrathymic human T lymphocyte development in an in vivo system. Purified human hematopoietic stem cells develop into mature T lymphocytes and immature progenitors in the bone marrow of athymic bnx mice. The human T cells are all TCR alpha beta(+) and display a restricted TCRV beta repertoire. In the current studies, we examined the effects of systemic human IL-7 (huIL-7) administration on the phenotype and the activation status of the bnx/hu T cells. In the majority of the mice that did not have huIL-7 administration, a higher frequency of human CD3(+)/CD8(+) than CD3(+)/CD4(+) T cells developed in the bone marrow. This phenomenon is also frequently observed in human bone marrow transplant recipients. Extremely low levels of IL-2 were expressed by human CD3(+) cells isolated from these mice, in response to PMA plus ionomycin and to CD3 and CD28 cross-linking. IL-4 was not expressed by cells exposed to either stimulus, demonstrating a profound inability of the bnx/hu T cells to produce this cytokine. Systemic production of huIL-7 from engineered stromal cells transplanted into the mice increased the human CD4 to CD8 ratios, and increased the ratio of memory to naive CD4(+) and CD8(+) T cells. The human CD3(+) cells recovered from mice that had systemic huIL-7 and equivalent numbers of CD3(+)/CD4(+) and CD3(+)/CD8(+) cells in the marrow were still unable to produce IL-4 in response to any condition tested, but were capable of normal levels of IL-2 production following stimulation.     

Human umbilical cord blood cells improve cardiac function after myocardial infarction

Human umbilical cord blood (UCB) contains an abundance of immature stem/progenitor cells and has been clinically used as an alternative to bone marrow transplantation. In addition, cord blood can be obtained non-invasively, in contrast to invasive bone marrow aspiration. We investigated the potential of human UCB CD34(+) cells to improve cardiac function following myocardial infarction. Myocardial infarction was induced in Wistar rats by ligation of the left coronary artery. Either 2x10(5) human UCB CD34(+) cells or equivalent cell-free medium was injected into the injured myocardium of the rats following induction of myocardial infarction. CD34(+) cell transplantation significantly improved ventricular function as compared to the control group. Immunofluorescence staining for human CD34, CD45, and PECAM-1 revealed surviving cells in the myocardium. Our findings suggest that transplanted human cells survived and improved cardiac function following myocardial infarction. These results may show the usefulness of UCB CD34(+) cells for myocardial infarction.     

Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells

Ethical considerations constrain the in vivo study of human hemopoietic stem cells (HSC). To overcome this limitation, small animal models of human HSC engraftment have been used. We report the development and characterization of a new genetic stock of IL-2R common gamma-chain deficient NOD/LtSz-scid (NOD-scid IL2Rgamma(null)) mice and document their ability to support human mobilized blood HSC engraftment and multilineage differentiation. NOD-scid IL2Rgamma(null) mice are deficient in mature lymphocytes and NK cells, survive beyond 16 mo of age, and even after sublethal irradiation resist lymphoma development. Engraftment of NOD-scid IL2Rgamma(null) mice with human HSC generate 6-fold higher percentages of human CD45(+) cells in host bone marrow than with similarly treated NOD-scid mice. These human cells include B cells, NK cells, myeloid cells, plasmacytoid dendritic cells, and HSC. Spleens from engrafted NOD-scid IL2Rgamma(null) mice contain human Ig(+) B cells and lower numbers of human CD3(+) T cells. Coadministration of human Fc-IL7 fusion protein results in high percentages of human CD4(+)CD8(+) thymocytes as well human CD4(+)CD8(-) and CD4(-)CD8(+) peripheral blood and splenic T cells. De novo human T cell development in NOD-scid IL2Rgamma(null) mice was validated by 1) high levels of TCR excision circles, 2) complex TCRbeta repertoire diversity, and 3) proliferative responses to PHA and streptococcal superantigen, streptococcal pyrogenic exotoxin. Thus, NOD-scid IL2Rgamma(null) mice engrafted with human mobilized blood stem cells provide a new in vivo long-lived model of robust multilineage human HSC engraftment.     

A Study of the Regenerative Potential of Bone Marrow Cells of Donor Mice that Carry the <Emphasis Type="Italic">egfp</Emphasis> Gene in Irradiated Mice

A study of the regenerative potential of bone marrow cells of donor mice that express the enhanced green fluorescent protein was conducted in mice irradiated at a dose of 7 Gy. Expression of this protein allowed us to carry out monitoring of the presence of donor cells in recipient blood over the entire lifespan of the recipient. The lifespan of young recipients increased by 93% after transplantation; for old recipients it increased by 15%. Total acceptance of the bone marrow, spleen, thymus, and blood of the recipient with donor bone marrow cells was demonstrated over the entire life of the recipient. Only the donor colonies were detected with the studied irradiation dose and number of transplanted cells (11.7 ± 0.4) · 10⁶ on the spleen surface. The percentage of bone marrow and spleen cells that expressed the CD117 and CD34 stem cell markers in the recipient mice was above the control level for a long period of time after the irradiation. More than half of the cells with CD117, CD34, CD90.2, and CD45R/B220 phenotypes in the studied organs were donor cells. Further detailed study of the peculiarities of the engraftment of bone marrow cells, both without preliminary treatment of recipients and after the effects of extreme factors, will allow improvement of the methods of cell therapy.     

CXCR2-Dependent Endothelial Progenitor Cell Mobilization in Pancreatic Cancer Growth

Neovascularization is essential for tumor growth. We have previously reported that the chemokine receptor CXCR2 is an important regulator in tumor angiogenesis. Here we report that the mobilization of bone marrow (BM)-derived endothelial progenitor cells (EPCs) is impaired in CXCR2 knockout mice harboring pancreatic cancers. The circulating levels of EPCs (positive for CD34, CD117, CD133, or CD146) are decreased in the bone marrow and/or blood of tumor-bearing CXCR2 knockout mice. CXCR2 gene knockout reduced BM-derived EPC proliferation, differentiation, and vasculogenesis in vitro. EPCs double positive for CD34 and CD133 increased tumor angiogenesis and pancreatic cancer growth in vivo. In addition, CD133(+) and CD146(+) EPCs in human pancreatic cancer are increased compared with normal pancreas tissue. These findings indicate a role of BM-derived EPC in pancreatic cancer growth and provide a cellular mechanism for CXCR2 mediated tumor neovascularization.     

Generation of an HIV-1-resistant immune system with CD34(+) hematopoietic stem cells transduced with a triple-combination anti-HIV lentiviral vector

HIV gene therapy has the potential to offer an alternative to the use of current small-molecule antiretroviral drugs as a treatment strategy for HIV-infected individuals. Therapies designed to administer HIV-resistant stem cells to an infected patient may also provide a functional cure, as observed in a bone marrow transplant performed with hematopoietic stem cells (HSCs) homozygous for the CCR5-Δ32-bp allele. In our current studies, preclinical evaluation of a combination anti-HIV lentiviral vector was performed, in vivo, in humanized NOD-RAG1(-/-) IL2rγ(-/-) knockout mice. This combination vector, which displays strong preintegration inhibition of HIV-1 infection in vitro, contains a human/rhesus macaque TRIM5α isoform, a CCR5 short hairpin RNA (shRNA), and a TAR decoy. Multilineage hematopoiesis from anti-HIV lentiviral vector-transduced human CD34(+) HSCs was observed in the peripheral blood and in various lymphoid organs, including the thymus, spleen, and bone marrow, of engrafted mice. Anti-HIV vector-transduced CD34(+) cells displayed normal development of immune cells, including T cells, B cells, and macrophages. The anti-HIV vector-transduced cells also displayed knockdown of cell surface CCR5 due to the expression of the CCR5 shRNA. After in vivo challenge with either an R5-tropic BaL-1 or X4-tropic NL4-3 strain of HIV-1, maintenance of human CD4(+) cell levels and a selective survival advantage of anti-HIV gene-modified cells were observed in engrafted mice. The data provided from our study confirm the safety and efficacy of this combination anti-HIV lentiviral vector in a hematopoietic stem cell gene therapy setting for HIV and validates its potential application in future clinical trials.     

急性淋巴细胞白血病CD34+CD38-细胞在NOD/SCID小鼠体内的自我更新与增值能力

目的 探索急性淋巴细胞白血病(ALL)患者CD34+ CD38-细胞移植到NOD/SCID小鼠体内建立白血病的可行性、自我更新与增殖潜能.方法 分选并鉴定ALL患者骨髓CD34+ CD38-细胞及对照CD34- CD38+细胞后,经尾静脉分别注射104个细胞于亚致死剂量射线照射的NOD/SCID小鼠体内,连续监测小鼠状态以及外周血血象改变,对濒死或死亡小鼠进行骨髓检查、肝脾病理学检查.结果 接种从ALL患者分选的CD34+ CD38-细胞到NOD/SCID小鼠体内后4周,小鼠外周血白细胞上升,到8周左右达高峰,约15×109～20× 109/L,原始及幼稚淋巴细胞明显增多.骨髓象显示以原始及幼稚淋巴细胞增生为主,约为40％,且肝脾组织也有白细胞浸润,明显高于接种了对照组CD34- CD38+细胞的NOD/SCID小鼠.结论 ALL患者CD34+CD38-细胞可以成功移植NOD/SCID 小鼠,在小鼠体内增殖形成白血病,说明该群细胞具有自我更新和增殖的潜能,可作为探索白血病起始细胞研究的重要载体.     

Induction of graft versus leukemia effects by cell-mediated lymphokine-activated immunotherapy after syngeneic bone marrow transplantation in murine B cell leukemia

 The feasibility of inducing graft versus leukemia (GVL) effects with allogeneic T cells in recipients of autologous bone marrow transplantation (BMT) was studied in a murine model (BCL 1) of human B cell leukemia/lymphoma. Allogeneic cell therapy, induced by infusion with peripheral blood lymphocytes, a mixture of allogeneic spleen and lymph node cells and allogeneic activated cell therapy, induced by in vitro recombinant-interleukin-2(rIL-2)-activated allogeneic bone marrow cells in tumor-bearing mice, prevented disease development in adoptive BALB/c recipients. Concomitant in vivo activation of allogeneic lymphocytes with rIL-2 suppressed even more effectively the development of leukemia in secondary adoptive recipients of spleen cells obtained from treated mice. In contrast, in vivo administration of rIL-2 after syngeneic BMT, with or without equal numbers of syngeneic lymphocytes, led to disease development in secondary recipients. Our data suggest that effective cell therapy can be achieved after SBMT by allogeneic but not syngeneic lymphocytes and that anti-leukemic effects induced by allogeneic lymphocytes can be further enhanced by in vitro or in vivo activation of allogeneic effector cells with rIL-2. Therefore, cell therapy by allogeneic lymphocytes following autologous BMT could become an effective method for inducing GVL-like effects on minimal residual disease provided that graft versus host disease can be prevented or adequately controlled. Received: 14 May 1996 / Accepted: 6 August 1996     

Adoptive transfer of PR1 cytotoxic T lymphocytes associated with reduced leukemia burden in a mouse acute myeloid leukemia xenograft model

Background aimsTumor antigen-specific cytotoxic T lymphocytes (CTL) have been used in the treatment of human cancer, including leukemia. Several studies have established PR1 peptide, an HLA-A2.1-restricted peptide derived from proteinase 3 (P3), as a human leukemia-associated antigen. PR1-specific CTL elicited in vitro from healthy donors have been shown to lyse P3-expressing AML cells from patients. We investigated whether PR1-CTL can be adoptively transferred into NOD/SCID mice to eliminate human leukemia cells.MethodsPR1-CTL were generated in bulk culture from peripheral blood mononuclear cells (PBMC) stimulated with autologous dendritic cells. Human acute myeloid leukemia (AML) patient samples were injected and engrafted in murine bone marrow at 2 weeks post-transfer.ResultsFollowing adoptive transfer, bone marrow aspirate from mice that received AML alone had 72–88% blasts in a hypercellular marrow, whereas mice that received AML plus PR1-CTL co-infusion had normal hematopoietic elements and only 3–18% blasts in a hypocellular marrow. The PR1-CTL persisted in the bone marrow and liver and maintained a CD45RA^? CD28⁺ effector phenotype.ConclusionsWe found that adoptive transfer of PR1-CTL generated in vitro is associated with reduced AML cells in NOD/SCID mice. PR1-CTL can migrate to the sites of disease and maintain their capacity to kill the AML cells. The surface phenotype of PR1-CTL was consistent with their trafficking pattern in both vascular and end-organ tissues.     

Unbiased selection of bone marrow derived cells as carriers for cancer gene therapy

BACKGROUND: There is currently great interest in development of cell-based carriers for delivery of viral vectors to metastatic tumors. To date, several cell carriers have been tested based largely upon their predicted tumor-localizing properties. However, cell types may exist which can be mobilized from the circulation by a tumor which have not yet been identified. Here we use an unbiased screen of bone marrow (BM) cells to identify cells which localize to tumors and which might serve as effective candidate cell carriers without any prior prediction or selection. METHODS: Unsorted BM cells from green fluorescent protein (GFP)-transgenic donor mice were adoptively transferred into C57Bl/6 mice bearing pre-established subcutaneous B16 melanoma tumors. Forty-eight hours and eight days later, tumors, organs and blood were analyzed for GFP-expressing cells by flow cytometry. The phenotype of GFP cells in organs was determined by co-staining with specific cell surface markers. RESULTS: CD45(+) hematopoietic cells were readily detected in tumor, spleen, bone marrow, blood and lung at both time points. Within these CD45(+) cell populations, preferential accumulation in the tumor was observed of cells expressing Sca-1, c-kit, NK1.1, Thy1.2, CD14, Mac-3 and/or CD11c. Lymphodepletion increased homing to spleen and bone marrow, but not to tumors. CONCLUSIONS: We have used an in vivo screen to identify populations of BM-derived donor cells which accumulate within tumors. These studies will direct rational selection of specific cell types which can be tested in standardized assays of cell carrier efficiency for the treatment of metastatic tumors.     

Providing a microenvironment for the development of human CD34+ hematopoietic cells in SCID mice

In order to develop a convenient small-animal model that can support the differentiation of human bone-marrow-derived CD34+ cells, we transplanted SCID mice with an immortalized human stromal cell line, Lof(11–10). The Lof(11–10) cell line has been characterized to produce human cytokines capable of supporting primitive human hematopoietic cell proliferation in vitro. Intraperitoneal injection of Lof(11–10) cells into irradiated SCID mice by itself resulted in a dose-dependent survival of the mice from lethal irradiation. The radioprotective survival was reflected by an increase in the growth and number of mouse bone-marrow-derived committed hematopoietic progenitors. The Lof(11–10) cells localized to the spleen, but not to the bone marrow of these animals and resulted in detectable levels of circulating human IL-6 in their plasma. Secondary intravenous injections of either human or simian CD34+ cells into the Lof(11–10)-transplanted SCID mice resulted in engraftment of injected cells within the bone marrow of these mice. The utility of this small-animal model that allows the growth and differentiation of human CD34+ cells and its potential use in clinical gene therapy protocols are discussed.     

Differential role for cyclic AMP response element binding protein-1 in multiple stages of B cell development, differentiation, and survival

CREB-1 is expressed in the bone marrow and in developing B cells. To determine the role of CREB-1 in developing B cells in the bone marrow, several lines of transgenic (Tg) mice overexpressing a dominant-negative Ser(119-ala) phosphomutant CREB-1 in the bone marrow were generated. Analysis of RNA and protein revealed expression of the transgene in the bone marrow. Flow cytometric analysis of bone marrow cells from Tg mice revealed approximately 70% increase in pre-B1 (CD43(+)B220(+)CD24(+(int))) and approximately 60% decreased pre-BII (CD43(+)B220(+)CD24(++(high))) cells, indicating a developmental block in pre-BI to pre-BII transition. Consistent with this, the Tg mice showed approximately 4-fold decrease in immature and mature B cells in the bone marrow. RT-PCR analysis of RNA from Tg mice revealed increased JunB and c-Jun in pre-BII cells associated with decreased S-phase entry. Adoptive transfer of bone marrow cells into RAG-2(-/-) mice resulted in reconstitution of non-Tg but not Tg bone marrow-derived CD43(+)B220(+)CD24(high) population that is normally absent in RAG-2(-/-) mice. In the periphery, the Tg mice exhibited decreased CD21(dim)CD23(high)IgM(+) follicular B cells in the spleen and increased B1a and B1b B cells in the peritoneum. While exhibiting normal Ab responses to T-independent Ags and primary response to the T-dependent Ag DNP-keyhole limpet hemocyanin, the Tg mice exhibited severely impaired secondary Ab responses. These studies provide the first evidence for a differential role for CRE-binding proteins in multiple stages of B cell development, functional maturation, and B1 and B2 B cells.     

Disparate in vivo efficacy of FTY720 in xenograft models of Philadelphia positive and negative B-lineage acute lymphoblastic leukemia

Most patients with acute lymphoblastic leukemia (ALL) respond well to standard chemotherapy-based treatments. However a significant proportion of patients, particularly adult patients, relapse with the majority dying of leukemia. FTY720 is an immunosuppressive drug that was recently approved for the treatment of multiple sclerosis and is currently under pre-clinical investigation as a therapy for a number of hematological malignancies. Using human ALL xenografts in NOD/SCIDγc(-/-) mice, we show for the first time that three Ph(+) human ALL xenografts responded to FTY720 with an 80 ± 12% (p = 0.048) reduction in overall disease when treatment was commenced early. In contrast, treatment of mice with FTY720 did not result in reduced leukemia compared to controls using four separate human Ph(-) ALL xenografts. Although FTY720 reactivated PP2A in vitro, this reactivation was not required for death of Ph(-) ALL cells. The plasma levels of FTY720 achieved in the mice were in the high nanomolar range. However, the response seen in the Ph(+) ALL xenografts when treatment was initiated early implies that in vivo efficacy may be obtained with substantially lower drug concentrations than those required in vitro. Our data suggest that while FTY720 may have potential as a treatment for Ph(+) ALL it will not be a useful agent for the treatment of Ph(-) B-ALL.     

Colony-forming activity of hematopoietic stem cells in tolerant mice as affected by antigens

The effect of sheep red blood cells (SRBC) and human red blood cells (HRBC) on the amount of CFUs in the bone marrow and spleen of (CBA X C57BL/6) FI SRBC-tolerant mice was studied. The increase in the number of bone marrow and spleen CFUs was demonstrated in SRBC-tolerant mice injected with HRBC. Using SRBC test injection the increase in CFUs amount was observed in the spleen, but not the bone marrow, where the amount of CFUs remained unchanged.     

Xenogeneic Graft-versus-Host-Disease in NOD-scid IL-2Rγ(null) Mice Display a T-Effector Memory Phenotype

The occurrence of Graft-versus-Host Disease (GvHD) is a prevalent and potentially lethal complication that develops following hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic-GvHD based upon immunodeficient strains injected with human peripheral blood mononuclear cells (PBMC; "Hu-PBMC mice") are important tools to study human immune function in vivo. The recent introduction of targeted deletions at the interleukin-2 common gamma chain (IL-2Rγ(null)), notably the NOD-scid IL-2Rγ(null) (NSG) and BALB/c-Rag2(null) IL-2Rγ(null) (BRG) mice, has led to improved human cell engraftment. Despite their widespread use, a comprehensive characterisation of engraftment and GvHD development in the Hu-PBMC NSG and BRG models has never been performed in parallel. We compared engrafted human lymphocyte populations in the peripheral blood, spleens, lymph nodes and bone marrow of these mice. Kinetics of engraftment differed between the two strains, in particular a significantly faster expansion of the human CD45(+) compartment and higher engraftment levels of CD3(+) T-cells were observed in NSG mice, which may explain the faster rate of GvHD development in this model. The pathogenesis of human GvHD involves anti-host effector cell reactivity and cutaneous tissue infiltration. Despite this, the presence of T-cell subsets and tissue homing markers has only recently been characterised in the peripheral blood of patients and has never been properly defined in Hu-PBMC models of GvHD. Engrafted human cells in NSG mice shows a prevalence of tissue homing cells with a T-effector memory (T(EM)) phenotype and high levels of cutaneous lymphocyte antigen (CLA) expression. Characterization of Hu-PBMC mice provides a strong preclinical platform for the application of novel immunotherapies targeting T(EM)-cell driven GvHD.