© 2000 by Oxford University Press
Journal of the National Cancer Institute Monographs, No. 28, 24-29,
2000
© 2000 Oxford University Press
Hematopoietic Stem Cells in HIV Disease
Presented at the International Symposium on HIV, Leukemia, and Opportunistic Cancers.
Affiliation of authors: D. T. Scadden, H. Shen, T. Cheng, AIDS Research Center and Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston.
Correspondence to: David T. Scadden, M.D., AIDS Research Center and Cancer Center, Massachusetts General Hospital, 149 13th St., Rm. 5212D, Boston, MA 02129 (e-mail: scadden.david{at}mgh.harvard.edu).
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ABSTRACT |
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 TopNotes Abstract IntroductionIn Vivo Evaluation of...Cell Kinetics in Immune...Thymic Function in HIV...Stem Cell Susceptibility to...HIV Receptor/Co-receptor...HIV-1 Co-receptors Function on...In Vivo Corroboration That...Stem-Cell Resistance Mediated at...Stem Cells as Therapeutic...References |
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The hematopoietic stem cell has long been hypothesized to be a target of human immunodeficiency virus type-1 (HIV) infection that limits the potential for compensatory immune cell production. Data have recently emerged documenting stem cell dysfunction in HIV disease and indicating that immune recovery from potent antiretroviral therapy is partly driven by new T-cell generation. Effects of HIV on stem cell physiology, however, appear to be indirect, as stem cells are highly resistant to HIV infection. Despite the presence of surface receptors for HIV, the hematopoietic stem cell is not infectible with HIV. However, stem transduction can be achieved with HIV constructs in which the envelope glycoproteins have been replaced by vesicular stomatitis virus G protein. Therefore, hematopoietic stem cells are likely participants in HIV-related cytopenias, but they are spared direct infection and can serve as a resource for cellular therapies for AIDS.
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INTRODUCTION |
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TopNotesAbstractIntroductionIn Vivo Evaluation of...Cell Kinetics in Immune...Thymic Function in HIV...Stem Cell Susceptibility to...HIV Receptor/Co-receptor...HIV-1 Co-receptors Function on...In Vivo Corroboration That...Stem-Cell Resistance Mediated at...Stem Cells as Therapeutic...References |
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Human immunodeficiency virus (HIV) induces a multitude of alterations in the innate and adaptive immune system that are broad if not equally distributed among virtually every arm of host defense. Because T cells are but one of the cell types affected, the potential for HIV being a disease of stem cells as well as mature effector cells has long been hypothesized. If stem cell dysfunction or destruction plays an important role in HIV disease, the implications are multiple and include limiting immune reconstitution and affecting the potential for stem cell-based gene therapy strategies.
Possible mechanisms include direct virus infection or indirect effects by altering the cellular or cytokine milieu of the bone marrow microenvironment. The issue of direct infection will be addressed in a later section of this paper in which the marked resistance of stem cells to HIV-1 infection is discussed. The lack of infectibility of stem cells, however, is not synonymous with the lack of adverse effects of HIV because of direct interactions with the virus. The virus envelope glycoprotein, gp120, is capable of interacting with the CXCR-4 and of inducing intracellular signaling events as manifest by calcium flux, kinase activation, and even functional changes, such as chemotaxis of some cell types (1,2). Whether it may directly induce altered function of stem cells that express CXCR-4 is not clear, but data suggest that apoptosis may be induced (3).
Alteration in the cellular and cytokine milieu of the bone marrow has been reported by a number of investigators who have demonstrated infection of bone marrow stromal elements and perturbation of either cytokine production or ability to sustain hematopoiesis (4–6).
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IN VIVO EVALUATION OF STEM CELLS IN HIV DISEASE |
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TopNotesAbstractIntroductionIn Vivo Evaluation of...Cell Kinetics in Immune...Thymic Function in HIV...Stem Cell Susceptibility to...HIV Receptor/Co-receptor...HIV-1 Co-receptors Function on...In Vivo Corroboration That...Stem-Cell Resistance Mediated at...Stem Cells as Therapeutic...References |
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Stem cell functional defects in vivo have been demonstrated both in human studies or in animal models. In animal models in which human hematopoietic tissue is engrafted in immunodeficient mice, reduced CD34+ cells and/or decreased colony-forming capacity have been shown to occur after HIV infection (7,8). Efforts to define the stem cell pool in HIV-infected patients have been less uniform in their conclusions, but an important study (9) quantitating circulating CD34+ cells after granulocyte colony-stimulating factor (G-CSF) mobilization has recently been concluded. These studies have shown that a decline of CD34+ numbers is seen with more advanced HIV disease. Patients with lower CD4 cells have lower concentrations of CD34+ cells after G-CSF mobilization. The stem cell numbers that can be harvested from patients with CD4 counts below 200 cells/mm3 may still be adequate for purposes of transplantation, but their concentration per milliliter of blood is demonstrably lower than those patients with more preserved immune function (9).
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CELL KINETICS IN IMMUNE DECLINE AND REGENERATION |
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TopNotesAbstractIntroductionIn Vivo Evaluation of...Cell Kinetics in Immune...Thymic Function in HIV...Stem Cell Susceptibility to...HIV Receptor/Co-receptor...HIV-1 Co-receptors Function on...In Vivo Corroboration That...Stem-Cell Resistance Mediated at...Stem Cells as Therapeutic...References |
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A causal relationship between lower CD4+ cells and CD34+ cells has been suggested by two recent lines of evidence. The first is the sequential analysis of stem cell and lymphocyte numbers in the previously mentioned mouse models. These studies have indicated that, after acute HIV infection, there is a decline in primitive cell numbers and function that precedes the decline in thymocytes (8). The second is related to the changes seen as viral replication decreases after antiretroviral therapy. Reduced HIV replication is associated with an improvement in CD34+ cells, myeloid colony-forming capacity, and CD4+ T cells (10). The increase in CD4+ cells is not related to improved T-cell survival but rather appears to be due to increased rates of production (11). The initial increase in CD4+ cells may be due to expansion of existing mature memory cells. However, with the use of immunophenotypic markers for naive cells and a recently developed polymerase chain reaction (PCR) method to quantitate cells recently emigrating from the thymus (12), it is clear that de novo generation of T cells is occurring (12–14). Although it has not been rigorously shown that the T-cell regeneration reflects stem cell changes, it is apparent that hematopoiesis rather than improved cell survival times is the driving force of immune reconstitution after antiretroviral treatment. The improvement in T-cell generation may then either reflect improved primitive cell function or number or the improved health of the tissue microenvironments in which the differentiation of primitive cells occurs.
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THYMIC FUNCTION IN HIV DISEASE |
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TopNotesAbstractIntroductionIn Vivo Evaluation of...Cell Kinetics in Immune...Thymic Function in HIV...Stem Cell Susceptibility to...HIV Receptor/Co-receptor...HIV-1 Co-receptors Function on...In Vivo Corroboration That...Stem-Cell Resistance Mediated at...Stem Cells as Therapeutic...References |
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Disruption of thymic architecture has been well described with HIV infection (15–17), but recent data would suggest that the defects in thymic function are neither complete nor irreversible. A radiographically detectable thymus is present in many HIV-infected patients (18); and even in settings of severe dysfunction, T-cell production is occurring. HIV-infected individuals who have had thymectomies for myasthenia gravis have ongoing T-cell generation (19). Also, in the setting of antiretroviral therapy, thymic dysfunction appears to be at least partially reversible. In vivo models have shown improvement in T-cell generation from precursor populations both endogenous and exogenous to the thymus with accompanying control of viremia (13,20). The potential of thymic function is greater than previously thought, and abnormalities of function are likely to be at least partially reversible. Thus, the thymus is considered to be less likely to represent a significant limiting factor in determining the extent to which T-cell numbers will improve after antiretroviral therapy. Rather, the primitive cell pool or other differentiation regulators may provide the difference between those patients who improve to normal or near normal levels and those who do not.
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STEM CELL SUSCEPTIBILITY TO HIV-1 INFECTION |
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TopNotesAbstractIntroductionIn Vivo Evaluation of...Cell Kinetics in Immune...Thymic Function in HIV...Stem Cell Susceptibility to...HIV Receptor/Co-receptor...HIV-1 Co-receptors Function on...In Vivo Corroboration That...Stem-Cell Resistance Mediated at...Stem Cells as Therapeutic...References |
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The emphasis on the stem cell in the context of HIV disease is then shifting in several important ways. The first is recognition that stem cells may indeed play a role in restricting immune restoration. The second is that their longevity presents stem cells as a potentially long-lived reservoir for HIV if infected. And the third is the greater potential for stem cells as a therapeutic tool in the context of gene therapy. If the thymus is not limiting, genetically protected stem cells may indeed be capable of providing the substrate for T-cell generation. Crucial to each of these issues is the infectibility of the stem cell by either HIV-1 or lentivirus vectors derived from HIV-1.
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HIV RECEPTOR/CO-RECEPTOR EXPRESSION IN HEMATOPOIETIC CELL SUBSETS |
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TopNotesAbstractIntroductionIn Vivo Evaluation of...Cell Kinetics in Immune...Thymic Function in HIV...Stem Cell Susceptibility to...HIV Receptor/Co-receptor...HIV-1 Co-receptors Function on...In Vivo Corroboration That...Stem-Cell Resistance Mediated at...Stem Cells as Therapeutic...References |
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We recently completed studies in which cells representing particular stages of blood cell development were isolated by flow cytometric or functionally based systems (14,21–23). The stem cell population was isolated with the use of the previously described method of selectively killing more mature cells thereby enriching for a cytokine nonresponsive subset with stem-like characteristics. Cells were assessed for expression of CD4, the chemokine receptors (CCR-5 and CXCR-4), messenger RNA, and protein with the use of the techniques adapted for the small numbers of primary cells available from standard donations. Low levels of message for CD4 were detectable by reverse transcription (RT)–PCR in peripheral blood mononuclear cells (PBMCs), myelomonocytic cells (CD11b+), mature T cells (CD3+CD4+), heterogeneous hematopoietic progenitor cells (CD34+), lineage-committed hematopoietic progenitor cells (CD34+CD38+), primitive hematopoietic progenitor cells (CD34+CD38-), and stem cells (G0), but not in NIH3T3 control cells. Similarly, the message for CXCR-4 and CCR-5 was detectable in each hematopoietic cell type tested, although CCR-5 levels in CD34+CD38- cells appeared to be lower as confirmed on multiple independent samples (n = 4). To more precisely define the presence of the receptor transcripts in stem cells, individual cells were isolated by micromanipulation, and single-cell RT–PCR profiles were generated as described previously (24,25). The cells consistently demonstrated detectable CD4, CXCR-4, and CCR-5 messages compared with control cells.
The presence of protein produced from the receptor transcripts was assessed by specific antibody staining and, independently for chemokine receptors, by calcium flux. Anti-CD4 staining analyzed by flow cytometry indicated CD4 expression in subpopulations of CD34+ cells similar to the findings reported by others (3,26–28). CCR-5- and CXCR-4-specific antibodies stained fractions of relevant CD34+ cells with only minimal staining of CD34+CD38- cells with the use of the anti-CCR-5 antibody, consistent with the low transcript levels observed.
Chemokine signaling, as measured by the generation of a calcium flux in cells bearing cognate receptors, was used as a functional assessment of chemokine receptors. Responsiveness of various subsets of CD34+ cells to SDF-1 (ligand for the CXCR-4 receptor) and regulated-on-activation normal T cells expressed and secreted (RANTES), MIP-1a, and MIP-1b (ligands for the CCR-5 receptor) was measured on Indo-1-loaded cells with the use of the fluorescence-activated cell sorter analysis. NIH3T3 cells were used as a cell control, IP-10 (the ligand for CXCR-3) was used as a chemokine control, and measurements were made over time by using the cells before and after exposure to chemokine to establish a target-cell baseline control. Response to SDF-1 was substantial in all populations of CD34+ cells, although increased response was noted in the CD34+CD38- cells, despite no difference in the frequency of CXCR-4 surface protein in that subfraction compared with CD34+CD38+ cells. Similarly, the relationship between detectable surface protein for CCR-5 and response to ligand was not direct. Despite low levels of message and surface CCR-5 in the CD34+CD38- subset, calcium flux was approximately equivalent to other cell fractions when cognate ligands were applied. NIH3T3 cells did not demonstrate calcium flux, and IP-10 did not induce calcium flux except in a huCXCR-3-expressing cell line.
The rarity of stem cells (G0) precluded the routine use of flow cytometry, and, thus, we developed an immunomagnetic bead rosette assay (Fig. 1
). This assay uses the binding of specific monoclonal antibodies to target epitopes on cells similar to immunofluorescence assays. However, instead of fluorescein conjugation, immunomagnetic bead conjugation was used as a means of enhancing the ability to detect antibody binding with the use of microscopy; the size of Dynal beads permitted ready enumeration of rosetted cells and had a low frequency of nonspecific binding (0.7%–6%) when second-step alone or when irrelevant antibody-conjugated beads were used. Estimated frequency of CD4-, CXCR-4-, and CCR-5-expressing cells compared with bead alone or with irrelevant antibody-conjugated bead controls (specific–nonspecific binding) was 12.2%, 23.2%, and 23.6%, respectively. Large-scale stem cell preparation generated by pooling multiple independent marrow preparations permitted flow cytometry analysis of CD34+ and CD4+ cells and demonstrated that a high fraction of these cells co-expressed CXCR-4 and CCR-5, compared with isotype control (Fig. 2).
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Single-cell digital fluorescence imaging was used to document stem cell (G0) calcium flux in response to chemokines. MIP-1a, MIP-1b, and SDF-1 generated evidence of calcium flux (Fig. 2
, B) and thereby confirmed the functional status of surface CCR-5 and CXCR-4 on stem cells. In contrast, IP-10 did not induce flux, whereas inducing calcium flux in huCXCR-3 transduced control cells. |
HIV-1 CO-RECEPTORS FUNCTION ON CD34+ CELLS BUT NOT G0 STEM CELLS |
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TopNotesAbstractIntroductionIn Vivo Evaluation of...Cell Kinetics in Immune...Thymic Function in HIV...Stem Cell Susceptibility to...HIV Receptor/Co-receptor...HIV-1 Co-receptors Function on...In Vivo Corroboration That...Stem-Cell Resistance Mediated at...Stem Cells as Therapeutic...References |
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Definition of the functional characteristics of the co-receptor molecules was further pursued through exposure of cells to stocks of infectious HIV-1. Given the presence of identifiable receptors for M-tropic strains (with the use of CCR-5) and T-tropic strains (with the use of CXCR-4), appropriate virus envelopes were used (HIV-1Ba–L and HIV-1HxB–2, respectively). Following exposure to concentrated stocks of virus, infection was evaluated for 1) the presence of HIV DNA, indicating virus entry and RT, and 2) the production of HIV-1 p24 antigen after addition of highly infectible cell lines, indicating completion of a replicative virus life cycle and passage of the virus. Virus DNA was detectable at the level of a single cell diluted to 10-4 in titration experiments of ACH-2 cells that contained a single proviral copy per cell (data not shown). HIV-1 DNA was evident in all subsets of cells exposed to infectious, but not heat-inactivated virus, with the notable exception of the G0 stem cells (Fig. 3
, A). G0 cells independently isolated from independent normal donors (three are shown) were consistently negative for viral DNA. CD34+-cell fractions other than stem cells had detectable viral DNA that was inhibitable at a level of approximately 50% by preincubation with cognate ligands for CCR-5, MIP-1a, MIP-1b, and RANTES (data not shown). The non-stem cell fractions also demonstrated productive completion of the virus life cycle by passage of HIV-1 to the readily infectible indicator cell lines PM-1 (Fig. 3, B) or H9 (data not shown) when HIV-1Ba–L or HIV-1HxB–2, respectively, were used. Prompt production and passage of the virus to PM-1 was noted from the relatively mature CD34+CD38+ population of cells. Although CD34+CD38- cells had clearly identifiable virus DNA present, passage of infectious virions was very much delayed with p24 antigen detectable on co-cultivation with PM-1 only after prolonged periods (approximately 21 days). In no instance was p24 detectable on co-cultivation of indicator (PM-1 or H9) cells with virus-exposed G0 cells, including experiments extended out to 35 days.
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IN VIVO CORROBORATION THAT STEM CELLS ARE RESISTANT TO HIV-1 INFECTION |
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TopNotesAbstractIntroductionIn Vivo Evaluation of...Cell Kinetics in Immune...Thymic Function in HIV...Stem Cell Susceptibility to...HIV Receptor/Co-receptor...HIV-1 Co-receptors Function on...In Vivo Corroboration That...Stem-Cell Resistance Mediated at...Stem Cells as Therapeutic...References |
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To determine if stem cells were infected in vivo, bone marrow samples were obtained from HIV-1-infected patients with high levels of circulating virus and with low blood cell counts. With the use of multiple independent patient samples, HIV DNA was identified in PBMCs and bone marrow mononuclear cells (BMMC), but there was consistently no detectable HIV DNA in G0 cells (n = 7) (Fig. 4
).
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STEM-CELL RESISTANCE MEDIATED AT THE LEVEL OF VIRUS FUSION AND ENTRY |
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TopNotesAbstractIntroductionIn Vivo Evaluation of...Cell Kinetics in Immune...Thymic Function in HIV...Stem Cell Susceptibility to...HIV Receptor/Co-receptor...HIV-1 Co-receptors Function on...In Vivo Corroboration That...Stem-Cell Resistance Mediated at...Stem Cells as Therapeutic...References |
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RT in quiescent cells may be incomplete and, in lymphocytes, may result in partial complementary DNA intermediates (29). To evaluate this possibility in stem cells, PCR primer pairs corresponding to 5' of the primer binding site and U3–U5 portions of the HIV-1 genome reverse transcribed even in quiescent lymphocyte populations were used (labeled TC-1 in Fig. 5
). To assess if the level of block to infection was at the receptor level or following receptor interaction, an HIV-1 green fluorescent protein (GFP)-encoding construct pseudotyped with either T-tropic (HXB2), M-tropic (YU-2), or dual tropic (89.6) HIV-1 envelopes were compared with the same virus construct pseudotyped with the vesicular stomatitis virus G (VSV G) protein. VSV G permits virus fusion with the cell membrane via mechanisms that bypass those mediated by CD4 and CCR-5 (30). Only the VSV G-pseudotyped virus was capable of infecting the stem cell population (Fig. 5, A). No HIV DNA was detectable in stem cells when HIV-1 envelopes or heat-inactivated VSV G-envelope pseudotypes were used as assayed by either PCR or for GFP expression by fluorescence microscopy. HIV-1 envelope pseudotypes were capable of infecting Jurkat or primary mononuclear control cells. These data demonstrate that stem cells have a block to HIV-1 infection and that the level of the block is in the steps of viral-cell membrane fusion and entry. Steps downstream of these events are intact as evident from the VSV G-pseudotype infection.
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An independent stem cell purification process with the use of Rhodamine 123 and Hoechst 33342 staining as defined by others (22) was used to exclude the possibility that the selection method induced alterations in the ability of the stem cells to be infected. The exclusion of Rhodamine 123 and low-intensity staining with Hoechst 33342 in CD34+ cells has been shown to associate with a population capable of functioning as stem cells in in vitro and in vivo experiments (31). After exposure to the virus, the cells corresponding to a more mature population (bright/bright) acquired detectable HIV DNA, but stem cells (dim/dim) had neither late nor early RT products detectable (Fig. 5
, B) identical to what was seen with G0 cells. Furthermore, these cells were readily infectible with the virus when the envelope was VSV G. These data confirm the resistance of stem cells to HIV-1 infection and demonstrate that the block can be overcome if an alternative CXCR-4- and CCR-5-independent mechanism of envelope-cell membrane fusion is used. |
STEM CELLS AS THERAPEUTIC TOOLS |
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TopNotesAbstractIntroductionIn Vivo Evaluation of...Cell Kinetics in Immune...Thymic Function in HIV...Stem Cell Susceptibility to...HIV Receptor/Co-receptor...HIV-1 Co-receptors Function on...In Vivo Corroboration That...Stem-Cell Resistance Mediated at...Stem Cells as Therapeutic...References |
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The data presented here indicate that the hematopoietic stem cell is resistant to HIV-1 infection in vitro and in vivo despite receptor and co-receptor expression. The stem cell is, therefore, not a potential long-lived reservoir of virus and is an appropriate cell to consider in autologous gene therapy approaches to acquired immunodeficiency syndrome (AIDS). To the extent that this cell can be recovered from AIDS patients, it may be envisioned to be a virus-free cell type that may be transduced with anti-HIV constructs for possible immune reconstitution. The ability to transduce the stem cell with HIV-based constructs was also documented in this study but was restricted to constructs pseudotyped in VSV G. There appears to be no block to such constructs entering quiescent stem cells and, at least transiently, expressing a transgene. Integration of the transgene or durable expression was not tested in this study, but another report (32) has indicated that HIV-based vectors may indeed result in sustained transgene expression in transplanted hematopoietic cells. Thus, stem cells may ultimately be manipulated to serve as a component of therapies of the future designed to replace the damaged immune system of HIV-1-infected individuals.
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NOTES |
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Supported by Public Health Service grants HL44851 (National Heart, Lung, and Blood Institute) and DK50234 and DK02761 (National Institute of Diabetes and Digestive and Kidney Diseases), National Institutes of Health, Department of Health and Human Services; by the Richard Saltonstall Charitable Foundation; and by the Center for AIDS Research.
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TopNotesAbstractIntroductionIn Vivo Evaluation of...Cell Kinetics in Immune...Thymic Function in HIV...Stem Cell Susceptibility to...HIV Receptor/Co-receptor...HIV-1 Co-receptors Function on...In Vivo Corroboration That...Stem-Cell Resistance Mediated at...Stem Cells as Therapeutic...References |
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