Extent Of Engraftment Research Articles

Engraftment of low numbers of pediatric acute lymphoid and myeloid leukemias into NOD/SCID/IL2Rcγnull mice reflects individual leukemogenecity and highly correlates with clinical outcome

Although immortalized cell lines have been extensively used to optimize treatment strategies in cancer, the usefulness of such in vitro systems to recapitulate primary disease is limited. Therefore, the design of in vivo models ideally utilizing patient-derived material is of critical importance. In this regard, NOD.Cg-Prkdc(scid) IL2rg(tmWjl) /Sz (NSG) mice have been reported to provide superior engraftment rates. However, limited data exist on the validity of such a model to constitute a surrogate marker for clinical parameters. We studied primary and serial engraftment on more than 200 NSG mice with 54 primary pediatric B cell precursor acute lymphatic leukemia (B-ALL), myeloid leukemia (AML) and T cell leukemia (T-ALL) samples, characterized the leukemogenic profile and correlated engraftment kinetics with clinical outcome. Median time to engraftment was 7-10 weeks and 90% of the mice engrafted. Male recipients conferred significantly higher engraftment levels than female recipients (p ≤ 0.004). PCR-based minimal residual disease marker expression and fluorescence in situ hybridization confirmed the presence of patient-specific genetic aberrations in mice. Transcriptome cluster analysis of genes known to be important in the leukemogenesis of all three diseases revealed that well-known tumor-regulating genes were expressed to a comparable extent in mice and men. The extent of engraftment and overall survival of NSG mice highly correlated with the individual prognosis of B-ALL, AML and T-ALL patients. Thus, we propose an in vivo model that provides a valuable preclinical tool to explore the heterogeneity of leukemic disease and exploit patient-tailored leukemia-targeting strategies within multivariate analyses.

HIV infection

At a satellite meeting prior to the International AIDS Meeting in Washington DC in July, a great deal of discussion was focused on finding a “cure” for HIV infection [1]. While this objective has been discussed at various times over the past decade, the real incentive for this goal comes from the encouraging findings with Timothy Brown, also known as the “Berlin patient.” This HIV-infected man received hematopoietic stem cells from an HLA-matched donor whose genome naturally lacked CCR5, the major receptor for HIV. Five years after two transplants, Mr. Brown appears to be cured of HIV as well as leukemia [2]. Although potentially infected with both a virus that requires CCR5 for entry and a small population of viruses that can infect cells by another receptor, no infectious virus can be recovered from his blood and other tissues. This finding includes biopsies from the brain, bowel, and lymph nodes. Certainly his “cure” encourages approaches that could mimic this achievement in all HIV-infected individuals.

The Rate of Marrow Recovery and Extent of Donor Engraftment Following Transplantation of Ex Vivo–Expanded Bone Marrow Cells Are Independently Influenced by the Cytokines Used for Expansion

Successful stem cell transplantation depends on cell dose, and this is particularly true for placental/cord blood transplantation in which it has been clearly shown that both the success of engraftment as well as the speed of white cell and platelet recovery are dependent on the nucleated cell dose in the graft. Thus, if stem cell numbers could be increased, the speed as well as the likelihood of engraftment might be improved. We studied the effect of two different cytokine combinations--kit ligand (KL), interleukin-3 (IL-3), and Flt-3 ligand supplemented with thrombopoietin and IL-11 (combination 1) or granulocyte/macrophage colony-stimulating factor (GM-CSF) and G-CSF (combination 2)--for their ability to affect speed and extent of engraftment using limited numbers (5 x 10(4)) of murine bone marrow (BM) light-density (LD) cells or their progeny expanded ex vivo in the presence one or the other cytokine combination for 6 days. With combination 1, we found that speed of platelet recovery was enhanced, but at the expense of white blood cell (WBC) recovery and percent donor engraftment. Furthermore, the cytokine combination that best maintained donor engraftment, combination 2, did so at the expense of platelet recovery. In no case was percent donor engraftment improved over 5 x 10(4) unmanipulated LD BM cells. These results are consistent with the interpretation that immediate recovery of blood cells of different lineages and longterm donor engraftment are separate functions that can be influenced by the choice of cytokines used during the ex vivo expansion process.

Adult stem cell plasticity: will engineered tissues be rejected?

The dogma that adult tissue-specific stem cells remain committed to supporting only their own tissue has been challenged; a new hypothesis, that adult stem cells demonstrate plasticity in their repertoires, is being tested. This is important because it seems possible that haematopoietic stem cells, for example, could be exploited to generate and perhaps deliver cell-based therapies deep within existing nonhaematopoietic organs. Much of the evidence for plasticity derives from histological studies of tissues from patients or animals that have received grafts of cells or whole organs, from a donor bearing (or lacking) a definitive marker. Detection in the recipient of appropriately differentiated cells bearing the donor marker is indicative of a switch in phenotype of a stem cell or a member of a transit amplifying population or of a differentiated cell. In this review, we discuss evidence for these changes occurring but do not consider the molecular basis of cell commitment. In general, the extent of engraftment is low but may be increased if tissues are damaged. In model systems of liver regeneration, the repeated application of a selection pressure increases levels of engraftment considerably; how this occurs is unclear. Cell fusion plays a part in regeneration and remodelling of the liver, skeletal muscle and even regions of the brain. Genetic disease may be amenable to some forms of cell therapy, yet immune rejection will present challenges. Graft-vs.-host disease will continue to present problems, although this may be avoided if the cells were derived from the recipient or they were tolerized. Despite great expectations for cellular therapies, there are indications that attempts to replace missing proteins could be confounded simply by the development of specific immunity that rejects the new phenotype.

179. Oncoretroviral Vector-Mediated Gene Therapy for Murine WAS Fails To Correct Most of the Deficient Biological Responses to S. pneumonia or M. bovis

The Wiskott-Aldrich syndrome is an X-linked congenital condition manifesting as variable degrees of immunodeficiency and thrombocytopenia. Bone marrow transplantation is apparently curative, but frequent lack of donor availability, and variable outcomes after transplantation with matched unrelated donors, indicate a need for alternative therapies. A murine model of WAS demonstrates features similar to the clinical condition, including lymphocytopenia, thrombocytopenia, and increased susceptibility to certain infectious agents. WAS- mice show improvement in several cellular and physiological parameters after transplantation with WT bone marrow cells. Gene corrected animals, generated by retroviral-vector mediated transduction of murine bone marrow cells followed by transplantation into lethally irradiated recipients, show improvement in the colitis seen in a subset of these animals, and also demonstrate improvement in their deficient secondary T-cell response to influenza inoculation. Here we demonstrate that WAS- mice show increased susceptibility to, and mortality from, pneumonia following intranasal inoculation with S. pneumonia. Specifically, after intranasal inoculation with 105 cfu of luciferase-labeled S. pneumonia, 0 of 10 WT WAS (C57Bl/6, 5th generation backcross from 129SvEv) mice developed pneumonia or died, while 6 of 10 WAS- mice developed pneumonia and died. However, gene-corrected animals did not show a significant improvement in these outcomes. After inoculation with a lower dose (0.5 × 105 cfu) of S. pneumonia, 4 of 7 gene corrected mice, 5 of 6 negative control mice (WAS- mice transplanted with WAS-, GFP-vector transduced bone marrow cells), and 4 of 8 positive controls (WAS- mice transplanted with WT, GFP-vector transduced bone marrow cells) developed pneumonia and died. Susceptibility did not correlate with the extent of engraftment of gene corrected cells in any hematologic lineage. We conclude that diminished resistance of positive control mice was at least partially responsible for the lack of observable phenotypic correction. We have also observed reduced clearance of M. bovis (bcg) from the organs of intravenously inoculated WAS- animals. Here too, however, gene-corrected animals showed only inconsistent indications of improvement in M. bovis clearance. Specifically, gene corrected WAS- mice showed improved clearance from liver, but no overall improvement in clearance from spleen or lung. Clearance correlated with the extent of engraftment of gene corrected lymphocytes and monocytes in liver and spleen, but not in lung. Our results suggest that technical improvements will be required in order to evaluate the biological efficacy of gene therapy for murine WAS. Specifically, we are investigating whether lethal irradiation alone, lack of a perfect genetic match among donors and recipients, or GFP expression diminishes resistance to these agents in recipients of control vector transduced, WT bone marrow cells, as well as whether improved vector design could enhance efficacy.

The Kinetics and Extent of Engraftment of Chronic Myelogenous Leukemia Cells in Non-Obese Diabetic/Severe Combined Immunodeficiency Mice Reflect the Phase of the Donor’s Disease: An In Vivo Model of Chronic Myelogenous Leukemia Biology

In vitro studies have provided little consensus on the kinetic abnormality underlying the myeloid expansion of chronic myelogenous leukemia (CML). Transplantation of human CML cells into non-obese diabetic mice with severe immunodeficiency disease (NOD/SCID mice) may therefore be a useful model. A CML cell line (BV173) and peripheral blood cells collected from CML patients in chronic phase (CP), accelerated phase (AP), or blastic phase (BP) were injected into preirradiated NOD/SCID mice. Animals were killed at serial intervals; cell suspensions and/or tissue sections from different organs were studied by immunohistochemistry and/or flow cytometry using antihuman CD45 monoclonal antibodies (MoAbs), and by fluorescence in situ hybridization (FISH) for the BCR-ABL fusion gene. One hour after injection, cells were sequestered in the lungs and liver, but 2 weeks later they were no longer detectable in either site. Similar short-term kinetics were observed using51Cr-labeled cells. The first signs of engraftment for BV173, AP, and BP cells were detected in the bone marrow (BM) at 4 weeks. At 8 weeks the median percentages of human cells in murine marrow were 4% (range, 1 to 9) for CP, 11% (range, 5 to 36) for AP, 38.5% (range, 18 to 79) for BP, and 54% (range, 31 to 69) for BV173. CP cells progressively infiltrated BM (21%) and spleen (6%) by 18 to 20 weeks; no animals injected with the cell line or BP cells survived beyond 12 weeks. The rate of increase in human cell numbers was higher for BP (7.3%/week) as compared with CP (0.9%/week) and AP (0.5%/week). FISH analysis with BCR and ABL probes showed that some of the human cells engrafting after injection of CP cells lacked a BCR-ABL gene and were presumably normal. We conclude that CML cells proliferate in NOD/SCID mice with kinetics that recapitulate the phase of the donor’s disease, thus providing an in vivo model of CML biology. © 1998 by The American Society of Hematology.

Recombinant human growth hormone promotes human lymphocyte engraftment in immunodeficient mice and results in an increased incidence of human Epstein Barr virus-induced B-cell lymphoma

Recombinant human growth hormone (rhGH) previously has been demonstrated to promote human or mouse T-cell engraftment in immunodeficient mice. We then wanted to examine long-term effects of rhGH on human cell engraftment in these mice. Mice with severe combined immune deficiency (SCID) were given human peripheral blood lymphocytes or human bone marrow cells and daily injections of rhGH (20 μg ip every other day). Upon later assessment for engraftment by flow cytometric analysis, it was determined that rhGH strongly promoted human T-cell engraftment in the thymus and spleens of these mice. However, there was considerable variability in both the incidence and extent of engraftment which appears to be due to donor-to-donor variation. Additionally, rhGH promoted B lymphomagenesis in these mice since long-term treatment of these xenogeneic chimeras with rhGH resulted in the increased incidence of human Epstein-Barr virus (EBV)-infected B-cell lymphoma. Thus, while rhGH can be used to optimize human T-cell engraftment in SCID mice, it also increases the likelihood of B-cell lymphoma generation when the donor is EBV infected. The results suggest that the activation of human T cells by rhGH results in an increased ability of these cells to traffic to the peripheral lymphoid organs of the SCID mice and results in a lymphoid microenvironment conductive to the outgrowth of EBV-transformed B lymphocytes.