Differentiation of CD34(+) haematopoietic stem cells into functional dendritic cells (DC) was investigated using the mAb CMRF-44 and other mAb against DC-associated markers. GM-CSF mobilized peripheral blood stem cells were obtained from healthy donors by leukapheresis. CD34(+) cells were purified using CD34(+)-positive selection,and subsequent immunomagnetic depletion of CD14 and CD2 cells. CD34(+) cells were cultured in medium supplemented with one or more of GM-CSF,TNF-alpha, IL-4 or IL-6. CMRF-44 Ag expression was monitored by flow cytometry, and DC function by allogeneic MLR and tetanus toxoid(TT) presentation assays. CD34(+) cells quickly acquired the CMRF-44 Ag when cultured in the presence of TNF-alpha. By day 3, more than 50% of the cells were double-positive for CD34 and CMRF-44. CD34 expression was gradually lost, so that by day 9, the majority of the cells were CD34(-)/CMRF-44(+).GM-CSF and TNF-alpha also induced CD40 expression, and up-regulation of CD54 and MHC class II on CD34(+) cells; their expression was correlated to the CMRF-44 Ag. Day 3 CD34(+)/CMRF-44(+) cells,but not CD34(+)/CMRF-44(-) cells, become potent APC when cultured further with GM-CSF plus TNF-alpha. These CMRF-44(+) cells were potent inducers of Th1-type immune response in the primary allogeneic MLR and present TT to autologous CD4(+) T cells. TNF-alpha alone is sufficient to induce CMRF-44 expression on CD34(+) cells, but in combination with GM-CSF expands the CMRF-44(+) population. CMRF-44 expression correlates with DC function and may be a useful early marker for commitment of CD34(+) cells to the DC differentiation pathway.

The leukocyte activation marker CD30 is highly expressed on the Reed Sternberg cells of Hodgkin's disease (HD). On normal tissues, CD30 has a restricted expression profile limited to activated T cells, activated B cells, and activated natural killer cells. This expression profile makes CD30 an ideal target for monoclonal antibody (mAb)-based therapies of Hodgkin's disease. CD30 mAbs have been shown to be effective in in vitro and in vivo models of hematologic malignancies such as anaplastic large cell lymphoma, yet these mAb have not been efficacious in HD models. We have found that a mAb against CD30, AC10, was able to inhibit the growth of HD cell lines in vitro. To generate a more clinically relevant molecule, the variable regions from AC10 were cloned into an expression construct containing the human gamma1 heavy chain and kappa light chain constant regions. The resulting chimeric antibody, designated SGN-30, retained the binding and in vitro growth-inhibitory activities of the parental antibody. Treatment of HD cell lines with SGN-30 in vitro resulted in growth arrest in the G(1) phase of the cell cycle and DNA fragmentation consistent with apoptosis in the HD line L540cy. Severe combined immunodeficient mouse xenograft models of disseminated HD treated with SGN-30 produced significant increases in survival. Similarly, xenograft models of localized HD demonstrated dose-dependent reduction in tumor mass in response to SGN-30 therapy. SGN-30 is being developed for the treatment of patients who have HD that is refractory to initial treatment or who have relapsed and have limited therapeutic options.

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)

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.

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.

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 10 20, or 30 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.

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.

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.