The dendritic cell (DC) compartment comprises subsets of cells with distinct phenotypes. Previously this lab reported methodology for hematopoiesis of dendritic-like cells in vitro dependent on a murine splenic stromal cell line (5G3). Co-cultures of lineage-depleted bone marrow (Lin− BM) over 5G3 continuously produced a distinct population of dendritic-like “L-DC” for up to 35 days. Here the progenitor of L-DC is investigated in relation to known BM-derived hematopoietic progenitors. It is shown here that L-DC-like cells also derive from the CD150+Flt3− long-term reconstituting-hematopoietic stem cells (HSC), and also from the Flt3+ multipotential progenitor subset in BM. Lin− BM co-cultures also produce a transient population of cells resembling conventional (c) DC. Production of cDC-like cells is shown here to be transient and M-CSF dependent, and also appears following co-culture of described common dendritic progenitors or monocyte dendritic progenitors over 5G3. BM cells from C57BL/6-flt3Ltm1lmx and C57BL/6-Csf2tm1Ard mice which lack cDC precursors and monocytes, are shown here to contain L-DC progenitors which can seed 5G3 co-cultures. L-DC are functionally distinct cells, in that they arise independently of M-CSF, and by direct differentiation from HSC.

Murine splenic stroma has been found to provide an in vitro niche for hematopoiesis of dendritic-like APC. Two distinct cell types have been characterized. The novel “L-DC” subset has cross-presenting capacity, leading to activation of CD8+ T cells, but not activating CD4+ T cells, which is consistent with their CD11cloCD11bhiMHC-II− phenotype. For L-DC, an equivalent tissue-specific APC has been found only in spleen. A second population of CD11chiCD11bloMHC-II+ cells resembling conventional dendritic cells (cDC) can activate both CD4 and CD8 T cells. Production of L-DC but not cDC-like cells is now shown to be dependent on contact between the L-DC progenitor and stroma such that the presence of a Transwell membrane can prevent L-DC development. Since L-DC can be produced continuously in vitro in stromal co-cultures overlaid with bone marrow (BM) progenitors, it was hypothesized that L-DC progenitors are self-renewing. The L-DC progenitor is shown here to be defined by the Flt3−c-kit+Lin−Sca-1+ (F−KLS) subset of adult BM which contains primitive HSC. Since the less primitive F+KLS HSC subset also contains L-DC progenitors, Flt3 does not appear to be a defining marker for this progenitor. Precursors of the cDC-like subset are found only within the F+KLS subset and seed production of a transient population of APC. All data identify differentiation of L-DC from HSC, and of cDC-like cells from DC precursors, which occurs independently of inflammatory signals and is dependent on a splenic stromal microenvironment.

Heterogeneity amongst dendritic cell (DC) subsets leads to a spectrum of immune response capacity against pathogens. Several DC subsets in spleen have been described which differ in terms of phenotype and function. We have previously reported a distinct population of CD11cloCD11bhiMHC-II−CD8− dendritic-like “L-DC” in murine spleen, which can also be generated in splenic stromal longterm cultures. Here, the ontogeny of L-DC development in perinatal mice has been compared with other known splenic DC subsets. Flow cytometric analysis has revealed the presence of L-DC at embryonic age (E)18.5 spleen, while plasmacytoid (p)DC and conventional (c)DC appear at 2 and 4 days following birth. Co-cultures of E18.5 spleen above splenic stroma also showed production of only L-DC, while spleen cells from D0 through D5 neonates showed production of both L-DC and cDC-like cells. Addition of an M-CSFR inhibitor to co-cultures revealed that while the development of cDC-like cells depended on M-CSF, many L-DC developed independently of M-CSF. Furthermore, purified hematopoietic stem cells (HSC) and multipotential progenitors (MPP) isolated from neonatal D1 spleen are capable of developing into L-DC in co-cultures. These studies reveal a lineage of dendritic-like cells developing in the spleen microenvironment, and which appear to arise from endogenous progenitors laid down in spleen during embryogenesis.

While spleen and other secondary tissue sites contribute to hematopoiesis, the nature of cells produced and the environment under which this happens are not fully defined. Evidence is reviewed here for hematopoiesis occurring in the spleen microenvironment leading to the production of tissue-specific antigen presenting cells. The novel dendritic-like cell identified in spleen is phenotypically and functionally distinct from other described antigen presenting cells. In order to identify these cells as distinct, it has been necessary to show that their lineage origin and progenitors differ from that of other known dendritic and myeloid cell types. The spleen therefore represents a distinct microenvironment for hematopoiesis of a novel myeloid cell arising from self-renewing hematopoietic stem cells (HSC) or progenitors endogenous to spleen.

A certain number of pediatric cancer patients still succumb to relapse following conventional treatment of their malignancies. One of the mechanisms of relapse is escape from immunity. Adoptive cellular immunotherapy with effector cells has the potential to overcome this escape. In adults, the CD3+ CD56+ cell, a cytokine-induced killer (CIK) cell, appears to be a promising effector cell type with the greatest cytotoxicity. This effector cell type may work in children as well. No similar studies with children have been published. We speculated that expanded CD3+ CD56+ cells obtained from pediatric cancer patients during remission would act similarly against various pediatric tumor cell lines; therefore, we undertook the present study to find support for our speculation. This study was undertaken to generate and expand CD3+ CD56+ CIK cells from normal peripheral blood mononuclear cells (PBL) obtained from 6 children with cancer (2 with acute lymphoblastic leukemia, 2 with large cell lymphoma, and 2 with osteosarcoma) in remission after intensive chemotherapy and to study the cytotoxic activities of these cells against chronic myeloid leukemia cell line K562 t(9;22), 4 pediatric tumor cell lines [infant acute lymphoblastic leukemia RS4 t(4;11), TEL/AML acute lymphoblastic leukemia REH t(12;21), alveolar rhabdomyosarcoma Rh-Cr t(2;13), and Ewing sarcoma EW-Le t(11;22)], and 2 pediatric glioblastoma multiforme cultured cell lines (G74 and G77). CIK cells were generated and expanded in culture medium to which interferon gamma, monoclonal antibody against CD3, and interleukin 2 were added at appropriate times. Cells were counted by flow cytometry. Net lactate dehydrogenase release from target cells incubated with CIK cells was used as an index of CIK cell cytotoxicity against various pediatric tumor cell lines. The results show that after 21 days in culture CD3+ CD56+ CIK cells derived from the 6 pediatric patients accounted for a median of 28.3% of the entire culture (range, 10.7%-36.4%). Before expansion no such cells were found in any of the 6 children. Median lytic activity rates of CIK cells were 45.5% to 64.5%, rates that contrasted drastically to the lytic activity rates of PBL, which were only 8% to 12%. The findings of the present study are encouraging. They provide information for developing adoptive immunotherapy for future clinical trials with pediatric cancer patients, particularly those patients with minimal residual disease after intensive chemotherapy or stem cell transplantation (especially nonmyeloablative transplantation procedures).