Human Hemopoietic Cells Research Articles

Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells.

Many cytokines exert their biological effect through members of the hemopoietin receptor family. Using degenerate oligonucleotides to the common WSXWS motif, we have cloned from human hemopoietic cell cDNA libraries various forms of the receptor that was recently shown to bind the obesity hormone, leptin. mRNAs encoding long and short forms of the human leptin receptor were found to be coexpressed in a range of human and murine hemopoietic organs, and a subset of cells from these tissues bound leptin at the cell surface. Ectopic expression in murine Ba/F3 and M1 cell lines revealed that the long, but not the short, form of the leptin receptor can signal proliferation and differentiation, respectively. In cultures of murine or human marrow cells, human leptin exhibited no capacity to stimulate cell survival or proliferation, but it enhanced cytokine production and phagocytosis of Leishmania parasites by murine peritoneal macrophages. Our data provide evidence that, in addition to its role in fat regulation, leptin may also be able to regulate aspects of hemopoiesis and macrophage function.

Establishment of human granulocyte‐macrophage colony stimulating factor producing transgenic SCID mice

Previous work has shown the usefulness of severe combined immunodeficient (SCID) mice as in vivo models for the growth of normal human haemopoietic cells and leukaemic cells. Many approaches have been made to improve the engraftment of human haemopoietic cells in SCID mice. We established transgenic mice producing human granulocyte-macrophage colony stimulating factor (hGM-CSF) with the homozygote of the scid gene. Endogenous serum hGM-CSF levels were detected by ELISA [mean 9585 pg/ml (line A, n = 4); mean 1610 pg/ml (line B, n = 4)]. Expression of hGM-CSF was observed in all organs tested including the heart, lung, liver, kidney, spleen, thymus, bone marrow and brain of hGM-CSF transgenic (hGMTg) mice. Morphological analysis of organs and peripheral blood cell counts showed no differences between hGMTg mice and their littermates. Murine Ba/F3 cells expressing functional hGM-CSF alpha beta receptor (BAF/alpha beta cells) could be successfully engrafted in hGMTg SCID mice. The cells invaded multiple organs and caused death within a few weeks of transplantation, although they infiltrated only the spleen of their littermates. These results showed that these hGM-CSF-producing SCID mice are useful as an in vivo assay system for investigating leukaemogenesis.

Cryopreserved human bone marrow stroma is fully functional in vitro.

Human-marrow long-term culture (LTC) enables maintenance of both stromal and haemopoietic elements of normal bone marrow (NBM) in vitro for 4-6 months. Stroma-based cultures are critical for quantitation of long-term culture initiating cells (LTC-IC), the most primitive human haemopoietic cells measurable in vitro. Supply of NBM can be sporadic, and up to 3-4 weeks in culture is required for stromal maturity. Stroma availability for experimental purposes can therefore be limited. Efforts to produce transformed human and transfected murine stromal cell lines comparable to NBM stroma have had some success. As an alternative, we investigated cryopreserved NBM and cryopreserved performed stroma. Function of cryopreserved and control fresh NBM stroma was similar when evaluated for up to 12 weeks in LTC. We have also demonstrated that stroma derived from cryopreserved NBM or performed cryopreserved NBM stroma can sustain third-party haemopoiesis as efficiently as fresh NBM stroma in LTC. Batched cryopreserved stroma is a convenient, rapidly available, source of functional stroma which avoids the logistic difficulties and lack of standardization associated with stroma from fresh NBM. This important advance will enhance the use of stroma-based LTC in studies of human haemopoiesis.

The role of sheep stroma in human haemopoiesis in the human/sheep chimaeras.

We have previously reported on the successful engraftment and long-term expression of human HSC (haemopoietic stem cells) in sheep following transplantation into pre-immune fetuses. The mechanism(s) underlying the support/maintenance of human haemopoiesis in sheep is not understood. In these studies we examined the ability of sheep stroma to support human haemopoiesis in vitro and compared this with support provided by human stroma. We found that, overall, human stroma was significantly more effective than sheep stroma in supporting human haemopoiesis in vitro. Nevertheless, stroma established from bone marrow of normal sheep was capable of lower-level support of the proliferation and multilineage differentiation of highly purified (CD34+, CD38-) preparations of human HSC in vitro. At lower concentrations, sheep stroma conditioned media (CM) induced human bone marrow mononuclear cells enriched for the CD34-positive fraction to enter the cell cycle at levels comparable to that of human stroma CM. However, unlike human CM, at higher concentrations, sheep CM inhibited human cells from initiating the cell cycle. These results provide a possible explanation for the long-term maintenance of low levels of human haemopoietic cell activity in human/sheep chimaeras.

Простой способ клонирования эмбриональных гемопоэтических клеток человека

Простой способ клонирования эмбриональных гемопоэтических клеток человека

The expression of the endothelial cell antigen CD34 in demyelinating disease

In this study, the antigenic expression of CD34, a 110 kDa glycoprotein which is expressed on human haemopoietic progenitor cells and vascular endothelium, has been assessed in a variety of neuropathological conditions, including infectious and demyelinating disease. Using immunoperoxidase staining on paraffin sections, the immunohistochemical results show that CD34 antigen is expressed widely on human CNS endothelium in grey and white matter, in the eye including retina, and in the anterior and posterior lobes of the pituitary. In demyelinating disease CD34 antigen expression was not detected in acute lesions, whereas strong expression was observed in old lesions. CD34 endothelial positivity was observed in areas of gliosis, vasogenic oedema, vascular disease and in Alzheimer's and Parkinson's disease pathology. A general pattern emerged, with CD34 antigen reactivity predominantly negative in areas of inflammation with demyelination but positive in adjacent non-inflamed tissue, irrespective of myelin pathology. We conclude that perivascular inflammation is a key factor in the absence of immunoreactivity of CD34 in the CNS in demyelinating disease.

K+ channel antisense oligodeoxynucleotides inhibit cytokine-induced expansion of human hemopoietic progenitors

Primitive human hemopoietic progenitor cells identified by surface membrane markers CD33-CD34+ are capable of expansion into lineage-restricted precursors following in vitro stimulation by hemopoietic regulators such as stem cell factor (SCF) and interleukin-3 (IL-3). In search of ionic currents involved in cytokine-induced progenitor cell growth and differentiation, human umbilical cord blood CD33-CD34+ cells were subjected to perforated patch-clamp recordings following overnight incubation with SCF and/or IL-3. An inward rectifying potassium channel (Kir) was found in 33% of control unstimulated cells, in 34% of cells incubated with IL-3, in 31% of cells incubated with SCF and in 75% of cells incubated with IL-3 plus SCF. Kir activity increased with elevation of extracellular potassium and was blocked by extracellular Cs+ or Ba2+ Antisense oligodeoxynucleotides directed against Kir blocked both mRNA and functional expression of Kir channels. Kir antisense also inhibited the in vitro expansion of cytokine-stimulated CD33-CD34+ cells into erythroid (BFU-E) and myeloid (GM-CFU) progenitors in 7-day suspension cultures. Extracellular Cs+ or Ba2+ induced a similar degree of inhibition (40-60%) of progenitor cell generation. These findings strongly suggest an essential role for Kir in the process of cytokine-induced primitive progenitor cell growth and differentiation.

Induction of Cytochrome P4501 A1 in Haemopoietic Stem Cells by Hydroxylated Metabolites of Benzene

The ability of various metabolites of benzene to regulate the expression of cytochrome P-450 (CYP)1A1 mRNA in human haemopoietic cells was investigated. CYP1A1 mRNA was quantified using a Northern blot technique and high stringency hybridization with a 32P-labelled cDNA probe. Benz[ a]anthracene (BA, 1 or 10 μ m), used as a positive control, induced CYP1A1 mRNA in two out of three human leukaemic haemopoietic stem cell lines (positive: KG-1, U937; negative: HL-60), as well as in long-term bone marrow cultures established from healthy volunteers. In KG-1 and U937 cells, CYP1 Al mRNA induction was studied in the presence of the benzene metabolites, hydroquinone (HQ), p-benzoquinone (BQ), phenol (PHE) and catechol (CAT). HQ and BQ induced CYP1A1 mRNA when added at concentrations of 100 nm or more; CAT was active at a concentration of 1 μ m, whereas PHE had almost no effect, even at the highest concentration used (1 μ m). Maximum mRNA levels induced by 1 μ m HQ were seen at 6 and 12 hr after addition of inducers, and induction was detectable for at least 48 hr. Little, if any, cellular toxicity was seen in clonogenic assays of KG-1 cells at concentrations of maximum induction. In conclusion, CYP1A1 mRNA induction was demonstrated in haemopoietic cells; inducers for CYP1A1 were not only a polycyclic aromatic hydrocarbon (BA), but also, unexpectedly, hydroxylated metabolites of benzene.

Differential effects of recombinant human interleukin-13 on the in vitro growth of human haemopoietic progenitor cells.

Effects of recombinant human interleukin (IL)-13 on in vitro haemopoiesis from non-adherent mononuclear cells (NAMC) or highly enriched CD34+ cells of human cord blood (CB) were studied. IL-13 significantly increased megakaryocyte (MK) colony formation from either NAMC or CD34+ cells cultured in a plasma clot system supplemented with aplastic anaemia serum (AAS) and phytohaemagglutinin-stimulated human peripheral blood leucocyte-conditioned medium (PHA-LCM) in a dose-dependent manner. Experiments using a modified plasma clot culture, in which normal AB serum and various cytokines were added to replace AAS and PHA-LCM, demonstrated an increased MK colony number in the presence of IL-13, especially in combination with IL-3. However, IL-13 had no stimulatory effect, but rather a slight inhibitory effect in some cases on granulocyte-macrophage (GM) colony formation in both plasma clot cultures. Furthermore, the growth of GM progenitor cells in a methylcellulose culture system in the presence of IL-3, GM-CSF, Epo, G-CSF or in combination was significantly inhibited by the addition of IL-13. On the other hand, high concentrations (100 ng/ml) of IL-13 were needed to cause a slight inhibition on the growth of BFU-E-derived colonies under the same methylcellulose culture. These results indicate that IL-13, alone and synergistically with the effect of IL-3, promotes MK colony formation, but it inhibits the growth of GM and erythroid progenitor cells in vitro.

Regulatory action of prolactin on the in vitro growth of CD34+ve human hemopoietic progenitor cells.

The pituitary hormone prolactin (Prl) is known to act as a local regulator of immune cell function, and Prl-binding receptors (Prl-R) have been described to share distinctive features with the members of the newly described cytokine/hemopoietin receptor superfamily. Here we show that the hormone can functionally interact with lineage-specific hemopoietic factors. When highly purified progenitor cells (CD34+ve) were seeded in semisolid methylcellulose cultures in the presence of interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), and erythropoietin (Epo), a selective enhancing effect of Prl on the formation of colony forming unit-granulocyte (CFU-G) and burst forming unit-erythroid (BFU-E) colonies was observed. The effect of the hormone was plotted as a bell shaped curve, with the optimal response at the supraphysiological concentration of 50 ng/ml. Limiting dilution analysis showed that Prl acted directly on hemopoietic progenitors. This was confirmed by the observation on the CD34+ve cells of Prl-binding sites reacting with the specific monoclonal antibodies (mAbs), U5 and PrR-7A. Immunoprecipitation of the metabolically labeled CD34+ve cells with the PrR-7A mAb revealed a structure of 43 kD under reducing conditions. Analysis of the early events associated with the Prl/Prl-R interaction showed an increased number of cells engaged in DNA and hemoglobin synthesis. Enhanced erythroid differentiation of CD34+ve cells in the presence of Prl was secondary to upmodulation of receptors for the lineage-specific factor Epo. Together these data demonstrate the existence of a functional interplay between Prl and hemopoietic factors.

Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins.

Cytokines are known to be important regulators of normal hemopoiesis, acting in concert with components of the bone marrow microenvironment. Interactions with this microenvironment are known to regulate the proliferation, differentiation, and homing of hemopoietic progenitor (CD34+) cells. Adhesive interactions with the extracellular matrix retain CD34+ cells in close proximity to cytokines, but may also provide important costimulatory signals. Thus, the functional states of adhesion receptors are critical properties of CD34+ cells, but the physiological mechanisms responsible for regulating functional properties of cell adhesion receptors on primitive hemopoietic cells are still unknown. We confirm that the integrins very late antigen (VLA)-4 and VLA-5 are expressed on the CD34+ cell lines MO7e, TF1, and on normal bone marrow CD34+ progenitor cells, but in a low affinity state, conferring on them a weak adhesive phenotype on fibronectin (Fn). Herein, we show that the cytokines interleukin (IL)-3, granulocyte-macrophage CSF (GM-CSF), and KIT ligand (KL) are physiological activators of VLA-4 and VLA-5 expressed by MO7e, TF1, and normal bone marrow CD34+ progenitor cells. Cytokine-stimulated adhesion on Fn is dose dependent and transient, reaching a maximum between 15 and 30 min and returning to basal levels after 2 h. This cytokine-dependent activation is specific for VLA-4 and VLA-5, since activation of other beta 1 integrins was not observed. The addition of second messenger antagonists staurosporine and W7 abolished all cytokine-stimulated adhesion to Fn. In contrast, genistein inhibited KL-stimulated adhesion, but failed to inhibit GM-CSF- and IL-3-stimulated adhesion. Our data suggest that cytokines GM-CSF and IL-3 specifically stimulate beta 1 integrin function via an "inside-out" mechanism involving protein kinase activity, while KL stimulates integrin activity through a similar, but initially distinct, pathway via the KIT tyrosine-kinase. Thus, in addition to promoting the survival, proliferation, and development of hemopoietic progenitors, cytokines also regulate adhesive interactions between progenitor cells and the bone marrow microenvironment by modifying the functional states of specific integrins. These data are of importance in understanding the fundamental processes of beta 1 integrin activation and cellular response to mitogenic cytokines as well as on the clinical setting where cytokines induce therapeutic mobilization of hematopoietic progenitors.

Characterization of Three cDNAs Encoding Two Isozymes of an Isoaspartyl Protein Carboxyl Methyltransferase from Human Erythroid Leukemia Cells1

Two different sizes (approximately 1.0 and 1.6 kb) of transcripts of an isoaspartyl protein carboxyl methyltransferase (PIMT) were detected in eight cell lines derived from human hemopoietic cells on Northern blot analysis. We found three different sizes of cDNAs (907, 1,553, and 1,600 bp) in human erythroid leukemia cells (HEL) and a unique cDNA sequence corresponding to the 1.0 kb transcript was identified. These three cDNA sequences encoded two isozymes consisting of 226 (isozyme I) and 227 (isozyme II) amino acids. The 1.6 kb transcript was translated into two isozymes (isozyme I and II), while the 1.0 kb transcript was only translated into isozyme I. These results suggest that the two isozymes deduced from the cDNAs of the human erythroid leukemia cells may exist in normal human erythrocytes.

Gp130 and c-Kit signalings synergize for ex vivo expansion of human primitive hemopoietic progenitor cells.

gp130, a signal-transducing receptor component of interleukin 6 (IL-6), associates with an IL-6 and IL-6 receptor (IL-6) complex and transduces signals. To examine the role of gp130 signaling in the expansion of human hemopoietic progenitor cells, we tested the effects of a recombinant soluble human IL-6 receptor (sIL-6R) and/or IL-6 in combination with other cytokines on purified human umbilical cord blood CD34+ cells, using methylcellulose clonal assay and suspension culture in the presence or absence of serum. A combination of sIL-6R and IL-6 (sIL-6R/IL-6), but not sIL-6R or IL-6 alone, was found to dramatically stimulate expansion of hemopoietic progenitor cells as well as CD34+ cells in the presence of stem cell factor. Significant generation of multipotential hemopoietic progenitors over a period of 3 weeks in suspension culture and efficient formation of colonies, especially multilineage and blast cell colonies, in methylcellulose assay supplemented with a combination of sIL-6R/IL-6 together with stem cell factor were observed in serum-containing and serum-free culture. Addition of anti-gp130 monoclonal antibodies or anti-IL-6R monoclonal antibodies to the above cultures dose-dependently inhibited the expansion of progenitor cells in suspension culture and also completely blocked the formation of multilineage colonies in methylcellulose culture. These findings demonstrated that the significant expansion of human primitive hemopoietic progenitors could be achieved with the gp130 and c-Kit signalings initiated by the sIL-6R/IL-6 complex in the presence of stem cell factor and suggested the possible application of this method for ex vivo expansion of CD34+ cells for bone marrow transplantation.

Characterization of CD34+ Human Hemopoietic Progenitor Cells from the Peripheral Blood: Enzyme-, Carbohydrate- and Immunocytochemistry, Morphometry, and Ultrastructure

Following an immunomagnetic isolation and enrichment procedure, CD34+ cells were harvested from the peripheral blood of about 50 healthy donors. A battery of cytochemical staining reactions, monoclonal and carbohydrate-specific antibodies, proliferation markers and lectins was applied on smears and sections from paraffin-embedded pellets. Additionally, a morphometric analysis and ultrastructural investigation was carried out. More than 95% of the total yield of progenitor cells expressed CD34 and CD43 (MT1) and of these about 90% CD45 (LCA) and 25% CD45A (MT2). The CD34+/CD45RA-population was thought to represent very primitive, probably not lineage-restricted stem cells. On the other hand, reactivity with ANAE, CD11c, CD15, CD20, Ret40f, KiM1P, and CD61 (ranging between 1 to 20%) was considered to indicate a transition into more differentiated elements of hemopoiesis. The failure to detect any staining with proliferation markers (Ki-67/MIB 1, PCNA, KiS1) was in keeping with a quiescent status. Carbohydrate antigens revealed a pattern which underlines the fact that the CD34 and CD43 antigens belong to the family of heavily O-glycosylated sialomucins. Blood group antigens which are located at the peripheral regions of mucin-oligosaccharides (H type 2, Lewis, Lewis) could be demonstrated, but not A, B, Sialyl-Lewis and Lewis. Morphometric analysis revealed that CD34+ progenitors were larger than small lymphocytes. Electron microscopy showed a relatively primitive cytoplasmic organization and numerous tiny magnetic beads clustered at the plasma membrane.

CD34+ Human Hemopoietic Progenitor Cells of the Bone Marrow Differ from Those of the Peripheral Blood: An Immunocytochemical and Morphometric Study

CD34+ progenitor cells were harvested from bone marrow and peripheral blood from 10 healthy donors by immunomagnetic isolation and enrichment procedures. The CD34+ cell population was investigated using a battery of enzyme reactions and monoclonal antibodies on cytospin preparations. Additionally, morphometric measurements were carried out and also liquid suspension culture studies were performed to ascertain vitality and stem cell character. More than 95% of the total yield of medullary CD34 progenitors expressed CD45 (LCA), CD43 (MT1) and beta-glucuronidase. Reactivity with CD33 (My9), CD15 (LeuM1), CD38 (Leu17), CD20 (L26) and Ret40f (glycophorin C) was assumed to be in keeping with a transition into more differentiated elements of the various hemopoietic lineages. Morphometric analysis revealed conspicuous heterogeneity of the CD34+ cell population considering size measurements. This finding was in line with the diversities of antigen expression, indicating the more committed nature of CD34+ stem cells derived from the bone marrow in comparison with those progenitors isolated from the peripheral blood. Moreover, proliferation marker staining by PCNA disclosed a positivity in a considerable number of progenitors in contrast to the findings in CD34+ cells that are found in the peripheral blood.

Radiation-induced genomic instability: delayed cytogenetic aberrations and apoptosis in primary human bone marrow cells.

Transmissible chromosomal instability, characterized by non-clonal cytogenetic aberrations with a high frequency of chromatid-type aberrations together with a lower frequency of chromosome-type aberrations, has been demonstrated in the clonal descendants of human haemopoietic stem cells after alpha- but not X-irradiation. Comparable cytogenetic abnormalities have also been demonstrated in non-clonal cultures of alpha-irradiated primary human bone marrow, but a different pattern of delayed aberrations, mainly of chromosome-type, was found after X-irradiation in non-clonal cultures. In clonal analyses, delayed apoptotic cell death was evident after both X- and alpha-irradiation. It is suggested that the type of radiation exposure, the type of cell and its genetically determined susceptibility are factors that may influence the expression of delayed effects of radiation.

Gene transfer into human hemopoietic progenitor cells

Considerable progress is being made in the transfer of genetic material to hematopoietic stem cells. In this chapter we describe how gene transfer is being used to: mark marrow and peripheral blood progenitor cells prior to autologous transplantation, to track their fate on reinfusion and to detect contaminating tumorigenic cells; modulate immunocyte function--important in immunologic disorders and perhaps in cancer therapy; generate tumor vaccines from tumor cells isolated from marrow; correct single gene defects--the 'classical' concept of gene therapy; and finally to modify the drug sensitivity of progenitor cells--enabling them to resist the suppressive effects of cytotoxic drugs during cancer therapy and perhaps providing a mechanism for in vivo selection of gene modified cells.

Significance of the SCID Mouse Models for the Study of Normal and Leukemic Human Hemopoiesis and Gene Therapy

The hemopoietic system is organized as a hierarchy with all myeloid and lymphoid cells arising from a small number of pluripotent stem cells. As current in vitro methods do not assay stem cells, transplantation-based models for normal and leukemic human hemopoietic cells have been developed using immune-deficient mice as recipients. In vivo models were established for adult, neonatal, and fetal human hemopoiesis, for human leukemias and for genetic disorders and gene therapy. This transplantation system can be used to characterize and purify candidate human stem cell populations. Moreover, recent studies have provided strong support for the concept that there is a hierarchy of stem and progenitor cells for human leukemias. The SCID mouse models enable analysis of the biology of and metabolization of cyto-static drugs by leukemic stem cells. In addition, they are an important tool in developing and improving gene therapy protocols by assessing the efficiency of gene transfer into primitive genetically deficient stem cells following engraftment in immune-deficient mice. Potentials and limitations of the different approaches are discussed.

Retrovirus‐mediated transfer of the multidrug resistance gene into human haemopoietic progenitor cells

We report the utilization of cord blood (CB) or bone marrow (BM) derived low density or purified CD34+ cells as a target for human multidrug resistance (MDR1) gene transfer. Cells were cocultivated for 48 h with an irradiated MDR1 retroviral producer line. Since some degree of MDR1 gene expression has been reported to occur in haemopoietic progenitor cells and in peripheral blood cells, efficiency of MDR1 gene transfer was assessed by: (1) Drug selection and culture in presence of 50 ng/ml doxorubicin, 10 ng/ml colchicine and 0.85 micrograms/ml taxol. In uninfected control, 1-2% of CFU-GM and CFU-GEMM were found to be drug-resistant, while 14-31% of original clonogenic activity was found after 2 weeks of culture of transduced cells. Efficiency of MDR1 transfer was significantly enhanced by prestimulation with cytokines, and found to be significantly superior in CB-derived compared to BM-derived progenitors. (2) Analysis of MDR1 gene expression by evaluating MDR1 mRNA through polymerase chain reaction. MDR1 expression was very low in cultures of uninfected controls, whereas, after drug selection, MDR1 mRNA levels in transduced cells was as high as in the MDR1 retroviral producer line (positive controls). (3) Flow cytometric analysis of the expression of CD34 and P-glycoprotein, the product of the MDR1 gene. After MDR1 transduction and 2 weeks of culture, membrane expression of P-glycoprotein was found on 17-25% of viable CD34+ cells. (4) Cytochemical localization by APAAP staining of P-glycoprotein. No specific localization was found in untransduced controls, whereas transduced and cultured CB-cells expressed P-glycoprotein on plasma and nuclei membrane. In conclusion, MDR1 gene transfer into CB- and BM-derived progenitor cells seems a feasible and attractive approach to generate a drug-resistant haemopoiesis.

Severe combined immunodeficient mice engrafted with human splenocytes have functional human T cells and reject human allografts.

Previous studies have shown that human hemopoietic cells can be adoptively transferred into immunodeficient C.B-17 scid/scid (SCID) mice that lack autologous T and B lymphocytes, to generate chimeric animals. The future development of novel immunomodulatory drugs in transplantation will depend increasingly on experimental animal models to investigate the properties of the agents on human cells before starting clinical trials. However, in previous models of SCID mice engrafted with human PBLs, human T cells have been found either to be in an unresponsive state, unable to respond to mitogenic stimulations in vitro, or to mediate skin graft rejection only when HLA-primed in vivo before their adoptive transfer into SCID mice. In addition, T cells and other leukocyte subsets engraft quite poorly in the lymphoid tissues of the animals. In an attempt to develop a useful model for transplantation research, we have inoculated SCID mice with fresh human splenocytes from cadaveric organ donors (hu-Spl-SCID mice). In this model, various leukocyte subsets engraft effectively in different lymphoid compartments. In addition, human T cells retain their proliferative responses to mitogens and to alloantigens when tested 3 wk after engraftment into SCID mice. Finally, mice engrafted with unprimed human spleen cells acutely reject human foreskin allografts. Treatment of hu-Spl-SCID mice with OKT3, an immunosuppressive mAb directed against the human CD3 complex associated with the TCR, prevents the rejection of most human skin allografts, indicating a major role for human T cells in this phenomenon. Thus, this hu-Spl-SCID model may be useful for the study of immunosuppressive therapies in a preclinical in vivo setting.