Cord blood natural killer cells are functionally and phenotypically immature but readily respond to interleukin-2 and interleukin-12.

Human umbilical cord blood (CB) is being increasingly used both as an alternative to bone marrow to transplant children and for experimental insight into the ontogenic and maturational characteristics of blood cells. We studied the functional and phenotypic characteristics of CB natural killer (NK) cells because of the possibly important role such cells may play in a transplant setting and to gain insight into the little known ontogenic differences and maturational pathways of NK cells. It was found that CB NK lytic activity is usually deficient and that this deficiency cannot be fully explained by the presence of insufficient percentages of CD56+ cells. Although CD16+CD56+ and CD16-CD56+ NK cell subsets typical of adult peripheral blood (PB) are present, a significant population of CD16+CD56- cells also exists in CB. CB CD16+CD56- cells have little or no lytic capabilities; CB CD16+CD56+ cells vary in their lytic capabilities. Although a decreased ability to bind target cells may contribute to the deficient lytic activity of these CB NK cell subsets, studies suggest that other factors must also play a role. Short-term incubation with interleukin-2 (IL-2) or interleukin-12 (IL-12) substantially increases the lytic capabilities of CB NK cells, and long-term incubations induce lymphokine-activated killer (LAK) cell generation. Cell depletion experiments show that activated CD56+ NK cells are responsible for the lytic activity of CB LAK cells. Flow cytometric analysis reveals that during LAK cell generation, CB undergoes phenotypic changes similar to those of PB except that CD16+CD56- cells are still present.(ABSTRACT TRUNCATED AT 250 WORDS)

Alloantigen priming induces a state of unresponsiveness in human umbilical cord blood T cells.

Induction of alloreactivity in human adult and umbilical cord blood T cells was evaluated in mixed leukocyte culture by exposure to an allogeneic lymphoblastoid line that expresses known costimulatory molecules. Initial exposure to alloantigen-presenting cells (allo-APC) induced strong proliferative responses in both adult and cord blood T cells. However, in contrast to adult T cells, cord blood T cells exhibited little proliferation after restimulation with donor APC. Primed cord blood T cells could respond to interleukin 2 (IL-2), but unresponsiveness to alloantigen was not overcome by addition of exogenous IL-2. Unresponsiveness was long-lasting and appeared to be maintained by a combination of induction of anergy and activity of CD8+ suppressor cells. This information may contribute to use of human cord blood as an allogeneic source of transplantable stem cells.

Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice.

Unseparated or Ficoll-Hypaque (Pharmacia, Piscataway, NJ)--fractionated human cord blood cells were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice. High levels of multilineage engraftment, including myeloid and lymphoid lineages, were obtained with 80% of the donor samples as assessed by DNA analysis, fluorescence-activated cell sorting (FACS), and morphology. In contrast to previous and concurrent studies with adult human bone marrow (BM), treatment with human cytokines was not required to establish high-level human cell engraftment, suggesting that neonatal cells either respond differently to the murine microenvironment or they provide their own cytokines in a paracrine fashion. Committed and multipotential myelo-erythroid progenitors were detected using in vitro colony assays and FACS analysis of the murine BM showed the presence of immature CD34+ cells. In addition, human hematopoiesis was maintained for at least 14 weeks providing further evidence that immature hematopoietic precursors had engrafted the murine BM. This in vivo model for human cord blood-derived hematopoiesis will be useful to gain new insights into the biology of neonatal hematopoietic cells and to evaluate their role in gene therapy. There is growing evidence that there are ontogeny-related changes in immature human hematopoietic cells, and therefore, the animal models we have developed for adult and neonatal human hematopoiesis provide useful tools to evaluate these changes in vivo.

Proliferative and cytotoxic responses of human cord blood T lymphocytes following allogeneic stimulation.

On the basis of the success of recent cord blood transplants, we undertook a comparison of the proliferative and cytotoxic potential, following allogeneic stimulation, of E-rosetted T cells from cord blood or adult peripheral blood (PB3) or bone marrow (BM). The magnitude of the proliferative response of cord blood (CB) T cells to alloantigen was greater than or equal to that of adult PB or BM T cells. Proliferation following culture with IL-2, IL-4, or IL-7 produced a similar result. In contrast, the cytotoxic activity in 1(0), 2(0), or 3(0) mixed leukocyte culture of CB T cells was minimal, typically less than 20% at an effector:target ratio of 100:1. Cytometric analysis of CB T cell populations before and after culture with allostimulation demonstrated similar ratios of CD4+ and CD8+ cells in cultures of CB and adult T cells. Down-regulation of CD45RA antigen expression, a measure of CD4+ cell activation, was comparable in adult and CB cultures. Activation of CD8+ CTL effectors was assessed by the acquisition of CD28 antigen expression. After culture, a smaller proportion of CD8+CD28+ effector cells was present in CB cultures as compared to adult T cell cultures. Thus, following in vitro priming with alloantigen CB T cells generate a vigorous proliferative response yet produce little antigen-specific cytotoxicity. This diminished cytotoxicity may, in part, be related to the low incidence of graft vs host disease thus far noted in human CB transplants.

Allogeneic responses of human umbilical cord blood.

Human umbilical cord blood (CB) is increasingly used as an alternative to bone marrow as a source of stem and progenitor cells for pediatric patients. We have evaluated the alloreactive potential of cord blood T cells in vitro. Our findings demonstrate that in a primary mixed leukocyte culture CB T cells demonstrate strong proliferative responses to allogeneic stimulation but little to no generation of cytotoxic effector function. Furthermore, restimulation of primary cultures results in a state of proliferative unresponsiveness. Such diminished cytotoxic and proliferative responses may, in part, be related to the low incidence of graft vs. host disease thus far noted in human CB transplants.

SCID mice as an in vivo model of human cord blood hematopoiesis.

Cord blood is increasingly used as an alternative stem cell source for autologous and allogeneic transplantation, particularly in pediatric patients. We therefore adopted our protocol for transplanting human adult bone marrow cells into severe combined immunodeficient (SCID) mice [1] to develop an in vivo model for cord blood hematopoiesis. Intravenous injection of unfractionated or Ficoll-separated cord blood cells into sublethally irradiated SCID mice led to high levels of human hematopoiesis in the majority of the recipients [2]. Multilineage human hematopoiesis including committed and multipotential myeloerythroid progenitors as well as CD19+ B-lymphoid cells were observed in the murine bone marrow for at least 18 weeks. Together, these data indicate that the SCID mice were engrafted with an immature cell that was able to maintain multiple progenitor lineages in vivo. In contrast to our experiences with adult bone marrow, high levels of human cell engraftment in the mouse could be achieved without exogenous cytokine treatment, suggesting that the cord blood cells respond differently to the murine microenvironment. Alternatively, the cord blood cells might have been able to provide themselves with the necessary growth factors in a paracrine fashion. This model will be useful in gaining new insights into the biology of immature human cord blood progenitors and cord blood transplantation.

High-level multilineage engraftment of human cord blood cells in SCID mice.

Cord blood is increasingly used as an alternative to bone marrow transplantation in pediatric patients. Therefore, to study cord blood-derived hematopoiesis in vivo we are developing a xenogeneic transplantation model using immunodeficient SCID mice.

Inhibition of murine natural killer cell differentiation by dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA) is a naturally occurring steroid. We have previously shown that dietary DHEA (0.45% wt/wt) inhibits murine lymphopoiesis but not myelopoiesis. To assess the effect of DHEA on stages of natural killer (NK) cell differentiation, lethally irradiated mice fed DHEA or not were infused with 10(6) or 20 x 10(6) syngeneic bone marrow cells (BMC) as a source of transplantable NK cell progenitors. The differentiation of progenitor cells to lytic NK cells was assessed by the ability to clear radiolabeled YAC-1 tumor cells from the lungs. DHEA-fed recipients of 10(6) or 20 x 10(6) BMC failed to generate NK activity. Because NK progenitor cells are believed to differentiate into interleukin-2 (IL-2)-responsive precursor cells before maturation, BMC from recipient mice were cultured with IL-2 and the generation of NK cells was assessed. DHEA feeding prevented the generation of IL-2-responsive precursor cells in recipients of 10(6) BMC, but this inhibition was overcome in recipients of 20 x 10(6) BMC. To evaluate the capacity of stem cells to generate NK progenitor cells in DHEA-fed mice, the ability of marrow cells from primary recipients to generate NK activity in irradiated secondary recipients was determined. The production of NK progenitors was inhibited 20-fold. Thus, DHEA appears to inhibit the generation of NK progenitors from more primitive stem cells, the differentiation of progenitors into IL-2-responsive precursors cells and the maturation of IL-2-responsive precursor cells into mature NK cells.

Differential effects of dehydroepiandrosterone (DHEA) on murine lymphopoiesis and myelopoiesis.

Dehydroepiandrosterone (DHEA) inhibits murine lymphopoiesis, especially after sublethal irradiation, without affecting mature lymphocyte function. We demonstrate here that dietary DHEA differentially affected myelopoiesis and lymphopoiesis in sublethally irradiated mice. Erythropoietic progenitor cell and stem cell function was not affected by DHEA although the magnitude of granulopoiesis was slightly reduced. Regeneration of marrow B220+ B cells, natural killer (NK) function, and thymus repopulation were significantly delayed in DHEA-fed mice. Interleukin 2 (IL-2) failed to restore NK activity of DHEA-fed mice to normal levels. Marrow from DHEA-fed mice contained competent thymocyte progenitors capable of repopulating thymi of irradiated hosts fed a control diet but not those fed the DHEA diet. Thus DHEA inhibits lymphopoiesis while sparing myelopoiesis, affecting the growth and maturation of T, B, and NK cells by a mechanism other than the inhibition of IL-2 production.

Mechanisms of chemoprevention by dietary dehydroisoandrosterone. Inhibition of lymphopoiesis.

The ingestion of dehydroisoandrosterone (DHA), a naturally occurring steroid, inhibits the development of autoimmunity, neoplasias, and other disorders of rodents. Potential mechanisms of action include (1) the induction of peroxisomal proliferation and (2) the conversion of DHA to androgens. We evaluated the immune system of mice fed DHA. Dietary DHA had no significant effect on antibody responses, cutaneous sensitivity reactions, natural killer activity, or graft-versus-host reactions. However, a decrease in lymphoid organ cellularity and an absence of splenic germinal centers were observed. We assessed progenitor cell activity in irradiated mice by evaluating the repopulation of marrow and lymphoid organs. Dehydroisoandrosterone feeding resulted in an inhibition of lymphopoiesis but not myelopoiesis. Clofibrate, another peroxisomal proliferator, failed to inhibit lymphocyte repopulation after irradiation. Androgen-non-responsive Tfm/Y mice were as susceptible as control mice to the inhibitory effects of DHA on lymphopoiesis. Thus DHA itself may act on lymphoid progenitor cells and/or the microenvironment.