scholarly journals In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4040-4048 ◽  
Author(s):  
M Rosenzweig ◽  
DF Marks ◽  
H Zhu ◽  
D Hempel ◽  
KG Mansfield ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Lymphocytes ◽  
Progenitor Cells ◽  
T Cell Differentiation ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow-derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG- 2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome.

Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1431-1439 ◽  
Author(s):  
Ross N. La Motte-Mohs ◽  
Elaine Herer ◽  
Juan Carlos Zúñiga-Pflücker
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cord Blood ◽  
T Cell Development ◽  
T Cell Differentiation ◽  
Human Cord Blood ◽  
Hematopoietic Stem

AbstractThe Notch signaling pathway plays a key role at several stages of T-lymphocyte differentiation. However, it remained unclear whether signals induced by the Notch ligand Delta-like 1 could support full T-cell differentiation from a defined source of human hematopoietic stem cells (HSCs) in vitro. Here, we show that human cord blood–derived HSCs cultured on Delta-like 1–expressing OP9 stromal cells undergo efficient T-cell lineage commitment and sustained T-cell differentiation. A normal stage-specific program of T-cell development was observed, including the generation of CD4 and CD8 αβ–T-cell receptor (TCR)–bearing cells. Induction of T-cell differentiation was dependent on the expression of Delta-like 1 by the OP9 cells. Stimulation of the in vitro–differentiated T cells by TCR engagement induced the expression of T-cell activation markers and costimulatory receptors. These results establish an efficient in vitro coculture system for the generation of T cells from human HSCs, providing a new avenue for the study of early T-cell differentiation and function.

158 Inhibition of PI3Kδ improves tumor specific T cell immunity and metabolic fitness

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A172-A172
Author(s):  
Guillermo Rangel Rivera ◽  
Guillermo Rangel RIvera ◽  
Connor Dwyer ◽  
Dimitrios Arhontoulis ◽  
Hannah Knochelmann ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cell Therapy ◽  
Tumor Immunity ◽  
Mitochondrial Function ◽  
T Cell Differentiation ◽  
Tumor Control ◽  
T Cell Therapy

BackgroundDurable responses have been observed with adoptive T cell therapy (ACT) in some patients. However, current protocols used to expand T cells often exhibit suboptimal tumor control. Failure in these therapies has been attributed to premature differentiation and impaired metabolism of the infused T cells. Previous work done in our lab showed that reduced PI3Kδ signaling improved ACT. Because PI3Kγ and PI3Kδ have critical regulatory roles in T cell differentiation and function, we tested whether inhibiting PI3Kγ could recapitulate or synergize PI3Kδ blockade.MethodsTo test this, we primed melanoma specific CD8+ pmel-1 T cells, which are specific to the glycoprotein 100 epitope, in the presence of PI3Kγ (IPI-459), PI3Kδ (CAL101 or TGR-1202) or PI3Kγ/δ (IPI-145) inhibitors following antigen stimulation with hgp100, and then infused them into 5Gy total body irradiated B16F10 tumor bearing mice. We characterized the phenotype of the transferred product by flow cytometry and then assessed their tumor control by measuring the tumor area every other day with clippers. For metabolic assays we utilized the 2-NBDG glucose uptake dye and the real time energy flux analysis by seahorse.ResultsSole inhibition of PI3Kδ or PI3Kγ in vitro promoted greater tumor immunity and survival compared to dual inhibition. To understand how PI3Kδ or PI3Kγ blockade improved T cell therapy, we assessed their phenotype. CAL101 treatment produced more CD62LhiCD44lo T cells compared to IPI-459, while TGR-1202 enriched mostly CD62LhiCD44hi T cells. Because decreased T cell differentiation is associated with mitochondrial metabolism, we focused on CAL101 treated T cells to study their metabolism. We found that CAL101 decreased glucose uptake and increased mitochondrial respiration in vitro, indicating augmented mitochondrial function.ConclusionsThese findings indicate that blocking PI3Kδ is sufficient to mediate lasting tumor immunity of adoptively transferred T cells by preventing premature differentiation and improving mitochondrial fitness. Our data suggest that addition of CAL101 to ACT expansion protocols could greatly improve T cell therapies for solid tumors by preventing T cell differentiation and improving mitochondrial function.

Opposing Effects of Toll-Like Receptor Ligands and Ascorbic Acid On T and Natural Killer Cell Development From Lymphoid Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1491-1491 ◽  
Author(s):  
Birgitta Mitchell ◽  
Maritza Gonzalez ◽  
Jared Manning ◽  
Gerald J Spangrude
Keyword(s):  
Ascorbic Acid ◽  
Cell Differentiation ◽  
T Cell ◽  
Progenitor Cells ◽  
Nk Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
T Cell Differentiation ◽  
Nk Cell Differentiation

Abstract Abstract 1491 Poster Board I-514 Introduction: A complete understanding of lymphocyte development, particularly factors driving T and natural killer (NK) cell differentiation from progenitor cells, remains an elusive goal in medicine. T and NK cells are key regulators in the defense against infections and malignancies and play a direct causative role in autoimmune diseases and graft-versus-host disease. The OP9-DL1 stromal line is an important tool in the in vitro study of lymphocyte development. Lymphocyte progenitors (KLS,Thy1.1-) harvested from adult murine bone marrow and seeded on this stromal line can be followed through stages of maturation by immunophenotyping. We observed that addition of stem cell factor (SCF), contaminated with lipopolysaccharide (LPS) through its production in E. coli, was particularly effective at promoting NK cell development in the OP9-DL1 culture system. Toll-like receptors, an important component of anti-microbial defense by the innate immune response, recognize LPS and other microbial products. Toll-like receptor ligands (TLR-L) have been shown to enhance NK cell proliferation, however an effect on NK cell differentiation from progenitor cells has not been established. A separate set of experiments led us to hypothesize that ascorbic acid (vitamin C) promotes T cell differentiation. We therefore designed experiments to evaluate the differential effects of TLR-L and ascorbic acid on NK and T cell development from lymphoid progenitors co-cultured with OP9-DL1 stromal cells. Methods: Lymphocyte progenitor cells (KLS,Thy1.1-) were sorted from adult mouse bone marrow and 1000-2000 progenitor cells were seeded per well in a 24 well plate coated with OP9-DL1 stroma. Cultures were supplemented with IL-7 (5 ng/ml), Flt3 ligand (5 ng/ml), and SCF (100 ng/ml) plus one of 5 different TLR-L (TLR1/2, TLR3, TLR4, TLR5, and a crude LPS preparation that likely contains a number of TLR-L), with or without addition of a stabilized form of ascorbic acid. Cells were passaged, counted and re-seeded with fresh media and supplements twice a week over a 30-day period. Immunophenotype and viability were evaluated by flow cytometry. Markers for T cell development included CD44, CD25, CD3, CD4, CD8, T cell receptor beta chain and T cell receptor gamma-delta chains. NK cells were evaluated for the presence of NKp46, NK1.1, and DX5. Results: We observed robust cell expansion, inhibited somewhat by addition of ascorbic acid. The inhibitory effect of ascorbate on expansion was most pronounced in the culture condition lacking TLR-L. T cell differentiation was markedly advanced by the addition of ascorbic acid in the absence of TLR-L, with the majority of cells co-expressing CD4/CD8 and TCRB/CD3. The addition of different TLR-Ls inhibited T cell differentiation, and this inhibition was partially rescued by addition of ascorbic acid. NK cell differentiation, defined as co-expression of NKp46 and NK1.1, was two to three-fold greater with the addition of TLR1/2, TLR4, TLR5, and crude LPS compared to cultures lacking TLR-L addition. In each of these conditions, NK cell differentiation was markedly inhibited by addition of ascorbic acid. Conclusions: Our data supports the hypothesis that both T and NK cell progenitors require Notch signaling for differentiation. In our in vitro model, differentiation of one lineage at the expense of the other can be manipulated with addition of TLR-L or ascorbic acid. Addition of bacterial TLR-L promotes NK cell differentiation at the expense of T cell differentiation; an effect that is partially overcome with the addition of ascorbic acid. The addition of ascorbic acid promotes robust T cell differentiation, and inhibits significant NK cell differentiation in all conditions. The ability of ascorbic acid to promote T cell differentiation appears to dominate over TLR-L promotion of NK lineage differentiation. Further work will include microarray to evaluate these effects at a genetic level. These findings will contribute to our understanding of the immune response under normal and pathologic conditions, and further a model both for study and ex vivo expansion of immune cells for therapeutic use. Disclosures: No relevant conflicts of interest to declare.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

Clinical stem-cell sources contain CD8+CD3+ T-cell receptor–negative cells that facilitate bone marrow repopulation with hematopoietic stem cells

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1735-1738 ◽  
Author(s):  
Stephanie Bridenbaugh ◽  
Linda Kenins ◽  
Emilie Bouliong-Pillai ◽  
Christian P. Kalberer ◽  
Elena Shklovskaya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Hematopoietic Stem ◽  
Cd8 Cells ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8+ cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8+ cells, but the analogous cells in humans have not been identified. Here, we report that clinical stem-cell grafts contain a population of CD8α+CD3ϵ+ T-cell receptor– negative cells with an engraftment facilitating function, named candidate facilitating cells (cFCs). Purified cFC augmented human hematopoiesis in NOD/SCID mice receiving suboptimal doses of human CD34+ cells. In vitro, cFCs cocultured with CD34+ cells increased hematopoietic colony formation, suggesting a direct effect on clonogenic precursors. These results provide evidence for the existence of rare human CD8+CD3+TCR− cells with engraftment facilitating properties, the adoptive transfer of which could improve the therapeutic outcome of stem-cell transplantation.

scholarly journals Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7

2020 ◽  
Author(s):  
Emilie Coppin ◽  
Bala Sai Sundarasetty ◽  
Susann Rahmig ◽  
Jonas Blume ◽  
Nikita A. Verheyden ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Regulatory Elements ◽  
T Cell Differentiation ◽  
Human Immune System ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Human T Cell ◽  
Human T Cells

AbstractHumanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T cell differentiation remains inefficient. We generated mice expressing human interleukin (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

Hematopoietic Multipotent Progenitor Cells Mobilized by G-CSF Can Inhibit Gvhd

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2969-2969
Author(s):  
Maud D'Aveni ◽  
Julien Rossignol ◽  
Ruddy Montandon ◽  
Marie Bouillie ◽  
Flora Zavala ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Progenitor Cells ◽  
Mechanisms Of Action ◽  
Activated T Cells ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Ifn Γ

Abstract Abstract 2969 Backgound. Acute graft-versus-host-disease (aGVHD) is a frequent life threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Despite the infusion of higher doses of T cells with the use of G-CSF-mobilized HSC grafts, the incidence of aGVHD is not increased. The mechanisms by which G-CSF-mobilized HSC can control GVHD are imperfectly elucidated. We previously described the mobilization of murin hematopoïetic progenitor cells (HPCs) by G-CSF and FLT3 ligand capable of inducing tolerance against autoimmune diabetes in the nude mice (Kared, Immunity 2006). We now show that G-CSF can mobilize murin HPCs with immunoregulatory functions in the allogeneic immune response and describe their mechanisms of action. Methods. Mobilization of HPCs is performed by subcutaneous administration of human recombinant G-CSF at 200μg/kg per day, for 4 consecutive days in the C57BL6 (H-2b) mouse. HPCs are collected in the spleen by FACS sorting according to their phenotype: Lin- Sca1high cKithigh FLT3low CD34+ CD106+ CD127−. In vitro, functions and mechanisms of action were analyzed by co-cultures with i) T cells (from C57BL6) activated by anti-CD28 and -CD3 mAbs or activated by BALB/c (H-2d) allogeneic splenic LPS matured dendritic cells, ii) C57BL6 splenic selected CD4+CD25high T regulatory T cells activated by anti-CD28 and -CD3 mAbs iii) activated antitumor specific CD8 T cells (C57BL6 ovalbumin specific TCR transgenic T cells). These different cultures were performed in the presence or absence of inhibitors of selective cytokines or other regulatory molecules. In vivo, we assessed the effect of donor HPCs on GVHD development by injecting C57BL6 derived HPCs (0.5×106/mouse), splenic T cells (1×106/mouse) and T depleted bone marrow cells (5×106/mouse) into lethally irradiated (8 Gy) Balb/c recipients. Results. In vitro, as compared to controls without HPCs, after 3 days of culture, HPCs: 1) promote the proliferation of natural T regs activated by anti-CD3 and anti-CD28 (>80% at 3 days of culture compared to control <50%), 2) inhibit the proliferation of activated T cells (>80% T cells blocked before 4 divisions as compared to control-T cells alone >80% after 4 divisions- p<0, 001) and 3) induce the apoptosis of activated T cells (30% increased, p=0, 01). The proliferation of T regs was cell contact dependant and required the presence of TGF-b. The inhibition of T cell activation required IFN γ produced by activated T-cells and some contact-dependent stimuli. In such pro-inflammatory conditions, HPCs differentiate after 4 days in myeloid derived suppressor cells (MDSC). These cells could then produce NO in response to IFN γ and suppress the proliferation of activated T cell. However, T cell suppression was not dependant on L-arginine depletion. Induction of apoptosis of T cells was Fas/Fas-L dependant. Although in the presence of HPCs the proliferation of CD8+ T TCR transgenic against the dominant ovalbumin epitope SIINFEKL was reduced, the cytotoxic response against the SIINFEKL-pulsed EL4 cell line was enhanced (cytotoxicity >90% with HPCs versus <90% w/o HPCs, p<0, 001). In addition, HPCs express CCR7 and CD62L, which should allow their migration to the sites of allopriming. In vivo, none of the mice that had received allogeneic HSCT with HPCs developed clinical or histological GVHD signs as compared to 50% of the control allografted mice without HPCs. Conclusion. Hematopoietic progenitor cells acquire an immunosuppressive potential after G-CSF mobilization. These cells can be isolated from mobilized peripheral stem cells and suppress GVHD while possibly preserving the GVL effect. Work is underway in humans to identify and amplify this population ex vivo for potential therapeutic application in allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD69 Association with Jak3/Stat5 Proteins Regulates Th17 Cell Differentiation

2010 ◽  
Vol 30 (20) ◽  
pp. 4877-4889 ◽  
Author(s):  
Pilar Martín ◽  
Manuel Gómez ◽  
Amalia Lamana ◽  
Arantxa Cruz-Adalia ◽  
Marta Ramírez-Huesca ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Th17 Cells ◽  
T Cell Differentiation ◽  
Orphan Receptor ◽  
Interleukin 17 ◽  
Deficient Mice

ABSTRACT T-cell differentiation involves the early decision to commit to a particular pattern of response to an antigen. Here, we show that the leukocyte activation antigen CD69 limits differentiation into proinflammatory helper T cells (Th17 cells). Upon antigen stimulation in vitro, CD4+ T cells from CD69-deficient mice generate an expansion of Th17 cells and the induction of greater mRNA expression of interleukin 17 (IL-17), IL 23 receptor (IL-23R), and the nuclear receptor retinoic acid-related orphan receptor γt (RORγt). In vivo studies with CD69-deficient mice bearing OTII T-cell receptors (TCRs) specific for OVA peptide showed a high proportion of antigen-specific Th17 subpopulation in the draining lymph nodes, as well as in CD69-deficient mice immunized with type II collagen. Biochemical analysis demonstrated that the CD69 cytoplasmic tail associates with the Jak3/Stat5 signaling pathway, which regulates the transcription of RORγt and, consequently, differentiation toward the Th17 lineage. Functional experiments in Th17 cultures demonstrated that the selective inhibition of Jak3 activation enhanced the transcription of RORγt. Moreover, the addition of exogenous IL-2 restored Stat5 phosphorylation and inhibited the enhanced Th17 differentiation in CD69-deficient cells. These results support the early activation receptor CD69 as an intrinsic modulator of the T-cell differentiation program that conditions immune inflammatory processes.

Human CD34+ Cells Express CXCR4 and Its Ligand Stromal Cell–Derived Factor-1. Implications for Infection by T-Cell Tropic Human Immunodeficiency Virus

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 62-73 ◽  
Author(s):  
Alessandro Aiuti ◽  
Lucia Turchetto ◽  
Manuela Cota ◽  
Arcadi Cipponi ◽  
Andrea Brambilla ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Progenitor Cells ◽  
Stromal Cell ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Immunodeficiency Virus ◽  
Stromal Cell Derived Factor ◽  
Hiv 1

Human CD34+ hematopoietic progenitor cells obtained from bone marrow (BM), umbilical cord blood (UCB), and mobilized peripheral blood (MPB) were purified and investigated for the expression of the chemokine receptor CXCR4 and its ligand, stromal cell–derived factor-1 (SDF-1). CXCR4 was found present on the cell surface of all CD34+ cells, although it was expressed at lower density on MPB with respect to BM CD34+ cells. Freshly isolated and in vitro–cultured CD34+ cells also coexpressed SDF-1 mRNA, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Of interest, CD34+/CD38+ committed progenitor cells, unlike primitive CD34+/CD38− cells, expressed SDF-1 mRNA. Supernatants from in vitro–cultured CD34+ cells contained substantial (3 to 8 ng/mL) amounts of SDF-1 by enzyme-linked immunosorbent assay and induced migration of CD34+ cells. Because CD34+ cells express low levels of CD4, the primary receptor of the human immunodeficiency virus (HIV), and CXCR4 is a coreceptor for T-cell tropic (X4) HIV strains, we investigated the susceptibility of CD34+cells to infection by this subset of viruses. Lack of productive infection was almost invariably observed as determined by a conventional RT activity in culture supernatants and by real-time PCR for HIV DNA in CD34+ cells exposed to both laboratory adapted (LAI) and primary (BON) X4 T-cell tropic HIV-1 strain. Soluble gp120 Env (sgp120) from X4 HIV-1 efficiently blocked binding of the anti-CD4 Leu3a monoclonal antibody (MoAb) to either human CD4+ T cells or CD34+ cells. In contrast, sgp120 interfered with an anti-CXCR4 MoAb binding to human T lymphocytes, but not to CD34+ cells. However, CXCR4 on CD34+ cells was downregulated by SDF-1. These results suggest that CXCR4 and its ligand SDF-1 expressed in CD34+ progenitors may play an important role in regulating the local and systemic trafficking of these cells. Moreover, these findings suggest multiple and potentially synergistic mechanisms at the basis of the resistance of CD34+ cells to X4 HIV infection, including their ability to produce SDF-1, and the lack of CXCR4 internalization following gp120 binding to CD4.

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