The mdr1-type P-glycoproteins (P-gps) confer multidrug resistance to cancer cells by active extrusion of a wide range of drugs from the cell. To study their physiological roles, we have generated mice genetically deficient in the mdr1b gene [ mdr1b (−/−) mice] and in both the mdr1a and mdr1b genes [ mdr1a/1b (−/−) mice]. In spite of the host of functions speculatively attributed to the mdr1-type P-gps, we found no physiological abnormalities in either strain. Viability, fertility, and a range of histological, hematological, serum–chemical, and immunological parameters were not abnormal in mdr1a/1b (−/−) mice. The high level of mdr1b P-gp normally present in the pregnant uterus did not protect fetuses from a drug (digoxin) in the bloodstream of the mother, although the protein did reduce drug accumulation in the adrenal gland and ovaries. Pharmacologically, mdr1a/1b (−/−) mice behaved similarly to the previously analyzed mdr1a (−/−) mice, displaying, for instance, increased brain penetration and reduced elimination of digoxin. However, both mdr1a and mdr1b P-gps contributed to the extrusion of rhodamine from hematopoietic progenitor cells, suggesting a potential role for the endogenous mdr1-type P-gps in protection of bone marrow against cytotoxic anticancer drugs. This, and the normal viability of mdr1a/1b (−/−) mice, has implications for the use of P-gp-blocking agents in cancer and other chemotherapy. mdr1a/1b (−/−) mice should provide a useful model system to further test the pharmacological roles of the drug-transporting P-gps and to analyze the specificity and effectivity of P-gp-blocking drugs.

Tumor suppressor p53 plays an important role in regulating cell cycle progression and apoptosis. Here we applied RNA interference to study the role of p53 in human hematopoietic development in vivo. An siRNA construct specifically targeting the human tumor-suppressor gene p53 was introduced into human CD34 ؉ progenitor cells by lentivirus-mediated gene transfer, which resulted in more than 95% knockdown of p53. We adapted the human-SCID mouse model to opti- IntroductionThe tumor suppressor p53 plays an important role in regulating the cell cycle and apoptosis in response to DNA damage caused by irradiation or exposure to genotoxic mediators. In addition, p53 can mediate several cellular responses, including cell cycle arrest, senescence, differentiation, and apoptosis, depending on the cell type and the microenvironment. 1 Although mutations occur in the gene encoding p53 in human cancers, including tumors of hematopoietic origin, its function in normal human hematopoietic development remains largely unexplored.Recently, we obtained evidence that p53 plays a role in regulating the replicative lifespan of mature human T cells in vitro through the suppression of human telomerase reverse transcriptase (hTERT) (R.G., E.W., R. Beijersbergen, and H.S., manuscript in preparation). Telomeres are DNA repeats at the distal ends of the chromosomes that protect against chromosome end-to-end fusion. 2 Telomeres are shortened at each cell division, and cells with critically short telomeres undergo cell cycle arrest and become senescent. [3][4][5] hTERT, which prevents telomere shortening, is transiently up-regulated in T cells on stimulation through the T-cell receptor (TCR), 4,5 and expression of a dominant-negative mutant of hTERT significantly decreased the lifespan of CD4 ϩ and CD8 ϩ T cells, 5 indicating that hTERT plays a regulatory role in the lifespan of human T cells. Recently, we observed that the down-regulation of p53 by RNA interference (RNAi) extends the lifespan of mature human T cells and neutralizes the inhibition by dominant-negative hTERT, indicating that p53 regulates hTERT expression in primary human T cells. Given the function of p53 in mature human T cells, we asked whether the p53 loss would affect the homeostatic proliferation of T cells. Mice deficient in p53 did not show obvious defects in T-cell homeostasis. However, T cells from inbred mouse strains have longer telomeres than T cells from humans, resulting in a delay in the onset of replicative senescence and in an extended lifespan of these murine T cells. It is, therefore, of interest to test the effect of p53 inactivation on human T-cell homeostasis.In addition, we examined the role of p53 in thymic T-cell development because studies in the mouse have revealed that p53 is induced after the initiation of TCR rearrangement 6 and that it plays an important role in early T-cell development, specifically in pre-T-cell receptor signaling. 7 Results obtained in mice cannot always be extrapolated to humans, and the role of p53 in human T-cell developm...

The homeostatic control mechanisms regulating human leukocyte numbers are poorly understood. Here, we assessed the role of phagocytes in this process using human immune system (HIS) BALB/c Rag2 −/− IL-2Rγc −/− mice in which human leukocytes are generated from transplanted hematopoietic progenitor cells. Interactions between signal regulatory protein alpha (SIRPα; expressed on phagocytes) and CD47 (expressed on hematopoietic cells) negatively regulate phagocyte activity of macrophages and other phagocytic cells. We previously showed that B cells develop and survive robustly in HIS mice, whereas T and natural killer (NK) cells survive poorly. Because human CD47 does not interact with BALB/c mouse SIRPα, we introduced functional CD47/SIRPα interactions in HIS mice by transducing mouse CD47 into human progenitor cells. Here, we show that this procedure resulted in a dramatic and selective improvement of progenitor cell engraftment and human T-and NK-cell homeostasis in HIS mouse peripheral lymphoid organs. The amount of engrafted human B cells also increased but much less than that of T and NK cells, and total plasma IgM and IgG concentrations increased 68-and 35-fold, respectively. Whereas T cells exhibit an activated/memory phenotype in the absence of functional CD47/SIRPα interactions, human T cells accumulated as CD4 + or CD8 + single-positive, naive, resting T cells in the presence of functional CD47/SIRPα interactions. Thus, in addition to signals mediated by T cell receptor (TCR)/MHC and/or IL/IL receptor interactions, sensing of cell surface CD47 expression by phagocyte SIRPα is a critical determinant of T-and NK-cell homeostasis under steady-state conditions in vivo.hematopoiesis | humanized mice | leukocyte homeostasis

The only peptide of Sendai virus that is recognized by cytotoxic T lymphocytes (CTL) in B6 mice was found with (g) the use of recombinant vaccinia virus constructs containing separate genes of Sendai virus and (d) a set of overlapping peptides completely spanning the identified nudeoprotein (NP) gene product. This immunodominant NP peptide is recognized by Sendai virus-specific CTL that are known to have therapeutic effects in vivo. By subcutaneous i'mmunization, this peptide induced Sendai virus and NP peptide-specific CTL memory responses in vivo. Most importantly, mice that had been immunizd with this peptide were protected against a lethal virus dose, indicating that viral peptides can be used as antiviral T-cell vaccines. The induction of T-cell memory by free peptide immunization potentially has wide applicability in biology and medicine, including protection against infectious disease.

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The development of plasmacytoid dendritic cells (pDC2) from human CD34 ؉ stem cells in vivo was studied in RAG-2 ؊/؊ interleukin (IL)-2R␥ ؊/؊ mice that lack functional T and B cells and natural killer cells. CD34 ؉ cells isolated from fetal liver or thymus were labeled with 5-and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and were injected into a human thymus grafted subcutaneously in the RAG-2 ؊/؊ IL-2R␥ ؊/؊ mice. One to 4 weeks later the CFSE label was found not only in T cells but also in CD123 ؉/high CD4 ؉ CD45RA ؉ pDC2, indicating that the CD34 ؉ cells can develop into pDC2 within a thymus. In addition to pDC2, CFSElabeled dendritic cells with a mature phenotype, determined by the cell surface markers CD11c, CD83, and CD80, were found in the injected human thymus graft. pDC2 was not found in the periphery of mice carrying a human thymic graft, indicating that the intrathymic pDC2 failed to emigrate from the thymus. We also demonstrate that pDC2 can develop outside the thymus because relatively high percentages of pDC2 were found in the periphery after the intravenous injection of CD34 ؉ CD38 ؊ fetal liver cells in RAG-2 ؊/؊ IL-2R␥ ؊/؊ mice without a human thymus graft. These data indicate that the thymus and the peripheral pDC2 develop independently of each other. (Blood. 2002;99: 2752-2759

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