Recombinant human c-Mpl ligand (thrombopoietin) not only acts on megakaryocyte progenitors, but also on erythroid and multipotential progenitors in vitro.

To determine the hematopoietic actions of recombinant human c-Mpl ligand (thrombopoietin [TPO]), we studied its effects on the proliferation and differentiation of highly purified CD34+ blood progenitors in plasma-containing and serum-free culture. TPO alone promoted the growth of small megakaryocyte colonies (CFU-Meg) in numbers two to three times greater than those produced by interleukin (IL)-3. The combination of TPO and stem cell factor (SCF) exerted a significant synergistic effect on CFU-Meg formation. In the presence of TPO and IL-3 or granulocyte/macrophage-colony stimulating factor (GM-CSF), a significant number of mixed colonies (CFU-Mix) were observed. The combination of TPO and Epo did not increase the number of CFU-Meg, but did support erythroid-burst (BFU-E) and CFU-Mix colony formation. Interestingly, the combination of TPO with cytokines known to have burst-promoting activity (BPA), including IL-3, GM-CSF, IL-9, and SCF, increased the number of BFU-E and CFU-Mix in the presence of Epo. The BPA of TPO was further investigated by delayed addition of Epo on day 6 after incubation with TPO from day 0. None of the BFU-E or CFU-Mix survived, indicating that TPO acted as a costimulant exclusively for Epo. Moreover, a neutralizing anti-human Mpl receptor polyclonal antibody completely abrogated the BPA of TPO, demonstrating that this effect was mediated through the Mpl receptor. Finally, experiments in single-cell clone sorting and serum-free culture clearly demonstrated that a combination of TPO and Epo directly supported BFU-E and CFU-Mix. These results suggest that TPO acts not only in megakaryocytopoiesis but also in the early stage of hematopoiesis.

Human FLT3 ligand acts on myeloid as well as multipotential progenitors derived from purified CD34+ blood progenitors expressing different levels of c-kit protein.

We studied the effect of human flt3/flk2 ligand (FL) on the proliferation and differentiation of purified CD34+ blood progenitors which express different levels of c-kit protein in clonal cell culture in comparison with that of stem cell factor (SCF). FL alone did not support significant colony formation. However, FL significantly enhanced neutrophil colony (CFU-G) formation in the presence of granulocyte-colony stimulating factor (G-CSF) by peripheral blood (PB)-derived CD34+c-kit- cells which contained a large number of CFU-G. In addition, FL could synergistically increase the number of CFU-G supported by a combination of interleukin (IL)-3 and G-CSF, as did SCF. As we reported previously, SCF showed a significant burst-promoting activity (BPA). In contrast, FL did not exhibit any BPA on PB-derived CD34+c-kithigh cells in which erythroid-burst (BFU-E) was highly enriched. However, FL could synergize with IL-3 or GM-CSF in support of erythrocyte-containing mixed (E-Mix) colony by PB-derived CD34+c-kithigh or low cells in the presence of Epo. Replating of E-Mix colonies derived from CD34+c-kithigh cells supported by IL-3+Epo+SCF yielded more secondary colonies than those supported by IL-3+Epo or IL-3+Epo+FL. When PB-derived CD34+c-kitlow cells which represent a more immature population than CD34+c-kithigh cells were used as the target, number of secondary colonies supported by IL-3+Epo, IL-3+Epo+SCF or IL-3+Epo+FL was comparable. However, the number of lineages expressed in the secondary culture was significantly larger in the primary culture containing IL-3+Epo+FL than in that containing IL-3+Epo. These results suggest that FL not only acts on neutrophilic progenitors, but also on more immature multipotential progenitors.

Thrombopoietin augments ex vivo expansion of human cord blood-derived hematopoietic progenitors in combination with stem cell factor and flt3 ligand.

We studied the effects of stem cell factor (SCF) and flt3 ligand (FL) on the ex vivo expansion of human umbilical cord blood (CB)-derived CD34+ cells in combination with various cytokines, including interleukin (IL)-3, IL-6, IL-11, and c-Mpl ligand (thrombopoietin, TPO), in a short-term serum-free liquid suspension culture system. Among the two-factor combinations tested, SCF plus IL-3 most effectively expanded committed progenitor cells, including mixed colony-forming units (CFU-Mix). The expansion efficiency (EE) of FL for each progenitor was inferior to that of SCF in the presence of various cytokines, except TPO. IL-6 significantly increased the EE for granulocyte/macrophage colony-forming units (CFU-GM) obtained with SCF + IL-3 or FL + IL-3. Interestingly, TPO markedly augmented the EE for committed progenitors, including CFU-GM, erythroid burst-forming units (BFU-E), and CFU-Mix, in the presence of SCF + IL-3 or FL + IL-3. The combinations of SCF + IL-3 + TPO + IL-6 or IL-11 maximally stimulated the expansion of committed progenitors. The maximum EE for CFU-GM, BFU-E, and CFU-Mix was respectively 197-fold (day 14), 60-fold (day 7) and 51-fold (day 14). Other combinations of cytokines without IL-3 failed to expand effectively these committed progenitors. Our data demonstrate that it is possible to expand human CB-derived committed progenitors in vitro using SCF or FL with several other cytokines including TPO, and that IL-3 is the key cytokine promoting the expansion of human hematopoietic progenitors in the presence of SCF or FL.

Action of human interleukin-4 and stem cell factor on erythroid and mixed colony formation by peripheral blood-derived CD34+ c-kit(high) or CD34+ c-kit(low) cells.

We studied the interaction of interleukin (IL)-4 and other burst-promoting activity (BPA) factors, such as IL-3, granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-9 and stem cell factor (SCF), on erythroid burst-forming unit (BFU-E) and erythrocyte-containing mixed (CFU-Mix) colony formation in serum-free culture. IL-4 alone did not support mixed colony formation in the presence of erythropoietin (Epo). However, IL-4 showed weak but significant BPA when peripheral blood (PB)-derived CD34+c-kit(low) cells were used as the target population. The BPA of IL-4 was much weaker than that of IL-3, which exerted the most potent activity, as previously reported. When CD34+c-kit(high) cells were used as the target, four factors known to have BPA, IL-3, GM-CSF, IL-9 and SCF, could express BPA. In contrast, IL-4, alone failed to support erythroid burst formation. Interestingly, IL-4 showed a remarkable enhancing effect with SCF in promoting the development of erythroid burst and erythrocyte-containing mixed colonies from CD34+c-kit(low) and CD34+c-kit(high) cells. Delayed addition of SCF+Epo or IL-4+Epo to the cultures initiated with either IL-4 or SCF alone clearly demonstrated that SCF was a survival factor for both BFU-E and CFU-Mix progenitors. In contrast, the survival effect of IL-4 was much weaker than that of SCF, and appeared to be more important for progenitors derived from CD34+c-kit(low) cells than for those derived from CD34+c-kit(high) cells. It was recently reported that CD34+c-kit(low) cells represent a more primitive population than CD34+c-kit(high) cells. Taken together, these results suggest that IL-4 helps to recruit primitive progenitor cells in the presence of SCF.

Functional differences between subpopulations of mobilized peripheral blood-derived CD34+ cells expressing different levels of HLA-DR, CD33, CD38 and c-kit antigens.

We have investigated the functional characteristics of peripheral blood-derived CD34+ cells mobilized by a combination of chemotherapy and G-CSF (mobilized peripheral blood-derived [MPB] CD34+ cells). In this study, subpopulations of MPB CD34+ cells have been directly compared in clonal cultures, long-term cultures with bone marrow (BM) stromal cells, and single-cell cultures. MPB CD34+ cells could be subdivided by expression levels of HLA-DR (DR), CD38, CD33 and c-kit antigens. The majority of MPB CD34+ cells expressed DR and CD38 antigens. In contrast, approximately 60% and 20% of the MPB CD34+ cells expressed CD33 and c-kit antigens, respectively. Interestingly, MPB CD34+ cells can be subdivided into three fractions which express high, low or negative levels of c-kit receptor. All types of committed progenitors were observed in populations of CD34+DR+, CD34+DR-, CD34+CD33-, CD34+CD38+ and CD34+ c-kit(low) cells. Colony forming unit-granulocyte/macrophage was highly enriched in the population of CD34+CD33+ cells, whereas BFU-E was highly enriched in the population of CD34+ c-kit(high) cells. In the population of CD34+CD38- cells, however, a few myeloid progenitors were detected. In addition, limiting dilution analyses clearly showed that the long-term culture-initiating cell (LTC-IC) is enriched in the populations of CD34+DR-, CD34+CD33- and CD34+c-kit-(or low) cells, but very few in CD34+ c-kit(high) cells, and that CD38 antigen is not a useful marker for the enrichment of LTC-IC derived from MPB CD34+ cells. Moreover, single cell clone sorting experiments clearly demonstrated the functional differences between CD34+CD38+ and CD34+CD38- cells as well as CD34+ cells expressing different levels of c-kit receptor. Our results suggest that an immunophenotype of LTC-IC is different between BM-, cord blood- and MPB-derived CD34+ cells and that primitive and committed progenitors existing in these sources may be functionally different.

MAGE-1 and MAGE-3 or -6 expression in neuroblastoma-related pediatric solid tumors.

MAGE-1 and MAGE-3 or -6 are genes encoding melanoma-rejection antigens recognized by cytotoxic T lymphocytes in an HLA-A1 restriction manner. MAGE-1 and MAGE-3 or -6 were expressed in 5/14 (36%) and 6/14 (43%) neuroblastoma (NB) cell lines, and in 20/41 (49%) and 24/41 (59%) clinical NB-related tumors, respectively. Additionally, they were also expressed in pediatric tumors of other types such as rhabdomyosarcoma and Wilms' tumor. MAGE-1 expression at a functional level in tumor cells was confirmed by the cytotoxicity assay using MAGE-1-specific tumor-infiltrating lymphocytes (TIL). In clinical NB-related tumors, MAGE-3 or -6 expression demonstrated an inverse correlation to clinical stage. Furthermore, although the sample number was small, the incidence of MAGE-1 and/or MAGE-3 or -6 expression was significantly correlated to the absence of metastasis and a more favorable clinical outcome (p < 0.05). These results may suggest that NB cells silent for the expression of MAGE genes escape from the host anti-tumor immune response and consequently retain a growth advantage. Finally, NB-related tumors could be reliable candidates for immunotherapy targeted towards MAGE gene products.

[Neuroblastoma].

Neuroblastoma is the most common and highly malignant tumor. The 2-year survival rate for NB patients for 1970s was 32% in US and 29% in Japan. But, improvement of prognosis was observed by recent advances in surgery, chemotherapy and numerous other supportive therapies. We introduce the some treatment regimens to patients with neuroblastoma which should be selected by the age and the stage at diagnosis and other prognostic factors such as N-myc amplification, trk overexpression, chromosome anomalies (lp-. double minutes, homogeneous staining region) of neuroblastoma cells and histological pathology. As a general rules, patients under 1 year of age without unfavorable prognostic factors should be treated less intensive regimen, even their tumors are progressive stages. Conversely, patients with progressive stages over 1 year of age without unfavorable factors, it is necessary to treat with intensive protocol. Furthermore, to patients of all age group with unfavorable factors, they are given a very strong intensive treatment through advances in supportive therapies such as the new antiemetics, G-CSF, antibiotics, or IVH etc.. Recent treatment regimens to the patients with neuroblastoma are presented.

Increase in T cells bearing the gamma/delta receptor associated with lymphoproliferative disease of granular lymphocytes in an infant with intractable diarrhea.

A two-year-old infant with intractable diarrhea and lymphoproliferative disease of granular lymphocytes attributed to a persistent cytomegalovirus infection showed an increase in cells bearing the gamma/delta T-cell receptor (TCR), which accounted for approximately 20% of total peripheral blood lymphocytes and 40% of CD3+ T cells. Of the gamma/delta TCR+ cells, two-thirds were double negative (CD4-/CD8-) and the other one-third CD8 positive. The majority of gamma/delta+ cells were delta TCS 1 positive. The predominance of delta TCS 1 positive cells was also confirmed on biopsy of lymphoid tissues from the colon. After improvement of watery diarrhea and malnutrition following three-month hyperalimentation, the number of gamma/delta TCR+ cells decreased. The patient subsequently died of pneumonia at the age of 2 years and 11 months. A possible site-specific role for the gamma/delta TCR+ cells, particularly delta TCS 1+ cells, in the human intestine is discussed.