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Flowcytometric analysis of pooled IL-3 blast cells and IL3B1 line derived thereof. IL-3-induced blast colonies were developed from 5-FU-treated mice as described in Materials and Methods. On day 6, ^-10' of blast colonies, each consisting of 100-200 cells, were individually lifted up from 90 dishes in total. They were pooled and resuspended for the analysis . 11,3111 was a continuous LGL line, which originated from a single IL-3 blast colony and was propagated in vitro in the presence of both rIL-2 and irradiated PEC. Cells were incubated with various antibodies and analyzed with FACS402 as described in Materials and Methods. Dotted line indicates the negative control, in which cells were treated with the corresponding second antibody alone.
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Blast colonies were developed by rIL-3 from the spleen cells of mice pretreated with 5-fluorouracil (5-FU) in the methylcellulose cultures. When such IL-3-induced blast colonies were individually lifted up and recultured in the presence of rIL-3 and recombinant erythropoietin (rEpo), a variety of hematopoietic colonies developed from every single c...
Citations
... However, several laboratories have provided evidence that T cell precursors in the bone marrow and spleen can differentiate into T cells in vitro in the absence of the thymus. Differentiation required 1-3 wk of culture in media enriched with growth factors such as IL-3 and IL-2 (13)(14)(15)(16)(17). The precursor populations used for in vitro studies were obtained from T cell-depleted bone marrow of athymic or euthymic mice (13,14,17), neonatal spleen (16), and from multilineage hematopoietic colonies from the spleen (15). ...
... Differentiation required 1-3 wk of culture in media enriched with growth factors such as IL-3 and IL-2 (13)(14)(15)(16)(17). The precursor populations used for in vitro studies were obtained from T cell-depleted bone marrow of athymic or euthymic mice (13,14,17), neonatal spleen (16), and from multilineage hematopoietic colonies from the spleen (15). ...
... The rapid renewal of bone marrow T cells in athymic as well as in euthymic mice suggested that the bone marrow is the source of these T cells. Previous studies have indicated that precursor ceils in the bone marrow and spleen can give rise to cells during in vitro culture in the absence of thymic stroma (13)(14)(15)(16)(17). To determine whether C57BL/Ka marrow precursors can generate (x/B T cells in vitro, marrow cells were fractionated on a discontinuous Percoll density gradient, and a low density fraction was obtained to enrich for hematopoietic progenitors (24). ...
The rate of renewal of T lymphocytes in the bone marrow of euthymic C57BL/Ka and athymic nu/nu BALB/c mice was estimated by in vivo labeling with bromodeoxyuridine. T lymphocytes accounted for 16-18% of marrow cells in euthymic mice as judged by immunofluorescent staining with monoclonal antibodies for Thy-1, CD3, and alpha/beta T cell antigen receptor markers. About 70% of marrow cells expressed receptors (Mac-1, Gr-1, B220) for myeloid, macrophage, and B lineage cells. Approximately 13% of cells in the athymic bone marrow expressed alpha/beta T cell receptors. Sorted marrow T cells proliferated in response to stimulation with anti-alpha/beta antibodies in vitro and showed functional rearrangements of V beta and J beta genes. Sorted non-T cells did not respond to stimulation in vitro, and all V beta and J beta gene rearrangements identified were nonfunctional. In vivo labeling studies indicated that approximately 17 x 10(6) bone marrow T cells are renewed daily in euthymic mice and approximately 14 x 10(6) are renewed in athymic mice. Approximately 11 x 10(6) mature B cells (immunoglobulin M+) are renewed daily in the bone marrow of the latter mice. To determine whether marrow precursors can give rise to T cells directly, marrow cells from euthymic and athymic mice were depleted of T cells by cell sorting and incubated in vitro for 48 h in the absence of exogenous growth factors or thymic stromal cells. Examination of the cells after culture showed that 10-12% stained brightly for alpha/beta T cell receptors. Although functional rearrangements of V beta and J beta genes were not detected before culture, the majority of rearrangements were functional after culture. The emergence of the bright alpha/beta T cells in culture was dependent on depletion T cells from the marrow cells before culture. The results suggest that most marrow T cells are generated in the marrow itself.
... We have used micromanipulation of single cells in a two-step culture system to demonstrate the development of both myeloid and lymphoid cells from single lymphohemopoietic progenitors in vitro. Other investigators have reported (16)(17)(18)(19) the development of similar pluripotent progenitors in culture either through identification of T or B lymphocytes in multilineage myeloid colonies or through replating of myeloid Fifty enriched marrow cells were cultured in the presence of Ep (2 units/ml), IL-7 (500 units/ml), and the designated cytokine combinations. Single factors failed to support primary colony formation except for IL-3 on day 11 of incubation. ...
We have used a two-step clonal culture system to unequivocally demonstrate that individual primitive lymphohemopoietic progenitor cells have the capacity for differentiation along either the myeloid or the B-lymphoid lineage. Highly enriched murine marrow cells were plated individually in culture by micromanipulation in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium, erythropoietin, steel factor (SF), and interleukin (IL) 7. Forty-five percent of the single cells formed primary colonies expressing multiple hemopoietic lineages. When aliquots from individual colonies were replated in secondary methyl cellulose culture containing SF and IL-7, 41% of the primary colonies gave rise to lymphocyte colonies. Cells of the lymphocyte colonies were blast-like and B220+, sIg-, Mac-1-, Gr-1-, Ly-1-, L3T4-, Ly-2-, and CD3-. Thirty to 70% of the cells were Thy-1+. mu-chain mRNA was detected in most of the cells by in situ hybridization with an antisense RNA probe. When lymphocyte colonies derived from a single cell were pooled and individually injected into scid mice, donor-type IgM was measurable in the serum of mice and spleens contained donor-type B cells. We then carried out initial screening of growth factors to identify growth factors that might replace pokeweed mitogen-stimulated spleen cell conditioned medium in the primary culture. Combinations of two factors that included SF plus IL-6, IL-11, or granulocyte colony-stimulating factor were all effective in the primary culture in the maintenance of the B-lymphoid potential. Interestingly, IL-3 could neither replace nor act synergistically with SF to support the lymphoid potential of the primary cultures. Our observations demonstrate that many primitive progenitors previously believed to be myeloid-committed also possess B-lymphoid potential. This culture system should prove valuable for elucidation of the mechanisms regulating early stages of lymphohemopoiesis.
The treatment of mice with high doses of 5-fluorouracil (5-FU) results in an enrichment of primitive hematopoietic progenitors. Using this procedure, we obtained a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro and could differentiate into not only myeloid cells but also into lymphoid lineage cells. The phenotypes of interleukin-3 (IL-3) induced blast colony cells were Thy-1-positive and lineage-marker-negative. We examined whether these blast colony cells contained primitive hematopoietic stem cells in vivo and could reconstitute hematopoietic tissues in lethally irradiated mice. Blast colony cells could generate macroscopic visible spleen colonies on days 8 and 12, and 5 x 10(3) blast cells were sufficient to protect them from lethally irradiation. It was shown that 6 or 8 weeks after transplantation of 5 x 10(3) blast cells, donor male cells were detected in the spleen and thymus of the female recipients but not in the bone marrow by Southern blot analysis using Y-encoded DNA probe. After 10 weeks, bone marrow cells were partially repopulated from donor cells. In a congenic mouse system, donor-derived cells (Ly5.2) were detected in the thymus and spleen 6 weeks after transplantation. Fluorescence-activated cell sorter analyses showed that B cells and macrophages developed from donor cells in the spleen. In the thymus, donor-derived cells were found in CD4, CD8 double-positive, single-positive, and double-negative populations. Reconstitution of bone marrow was delayed and myeloid and lymphoid cells were detected 10 weeks after transplantation. These results indicate that IL-3-induced blast cells contain the primitive hematopoietic stem cells capable of reconstituting hematopoietic organs in lethally irradiated mice.
Natural cell-mediated cytotoxicity against NK-resistant target tumor cells was found in the peripheral blood of tumor-bearing patients approximately 1 mo after combined chemotherapy. The recognition specificity of these effector cells was broad and had no restriction. From the experiments of negative selection with mAbs and complements, these newly developed killer cells after chemotherapy were thought to be LAK-like cells. Contribution of these LAK-like cells to the mechanism of action of anticancer drugs remains to be clarified.
Interleukin-2 (IL-2)-activated murine killer cell lines with macrophage- and B-lymphoblastic-lytic activity were established, and their target specificity, surface markers, recognition-related structures, and requirements for optimal cell growth were characterized. Sustained growth of IL-2-activated lymphocytes was supported by the combination of IL-2 and IL-4-enriched T cell conditioned medium (CM), but was not supported by IL-2 alone or the combination of IL-2 and IL-3-containing CM in the presence of macrophages (Mφ). The established line required continuous contact with Mφ to maintain anti-Mφ cytolytic activity. Flow cytometric analysis showed that the original line isolated by the first cloning was Thy1+, CD4−, and weakly CD8+, FcR+. The majority of these cells were CD3+ and TCR-Vβ8+. From this line, the CD3+, TCR-Vβ8+ and CD3−, TCR-Vβ8− clones were isolated by subcloning. The former clone showed Thy1+, CD3+, CD4−, CD8−, TCR-Vβ8+, FcR+ phenotype, and the latter clone showed Thy1+, CD3−, CD4−, CD8−, TCR-Vβ8−, FcR− phenotype. The original line and subclones showed a similar target specificity and killed resident or thioglycollate (TG)-induced peritoneal Mφ and B-lymphoblasts, but did not kill T-lymphoblasts. Allogeneic Mφ, Mφ-like cell line P388D1, and B cell hybridoma were sensitive, whereas fresh lymphocytes, T cell lymphoma BW5147, natural killer (NK)-sensitive YAC-1, and NK-resistant P815 tumor cells were resistant to lysis by these cytotoxic lines. The addition of anti-H-2 heteroserum, anti-MHC class I, anti-MHC class II, anti-CD3, or anti-TCR-Vβ8 monoclonal antibody (mAb) to assay cultures did not inhibit the anti-Mφ cytolysis by these killer cells. In addition, the CD3−, TCR-Vβ8− clone killed Mφ and B lymphoblasts better than the CD3+, TCR-Vβ8+ clone. These results suggest that cytotoxic lines established in this study do not use the T cell receptor (TCR) molecules to recognize target cells and the MHC molecules are not involved in recognition. Anti-LFA-1 mAb partially inhibited anti-Mφ-lysis, suggesting that the cell contact between targets and effectors is important in cytolysis. Our present data suggest that the culture condition containing IL-2, IL-4, and Mφ may support the continuous growth of non-MHC-restricted killer cells with relative target specificity against Mφ and B-lymphoblasts.
Analysis of TCR of a series of CD4-8- (double negative; DN) alpha beta T cell lines induced with IL-3 revealed that their V gene usage was biased for V alpha 4 and V beta 2. This has been confirmed in the primary short-term cultures. Thus, IL-3 induced the generation of DN alpha beta T cells with predominant V beta 2 gene expression from the CD4+/CD8+ T cell-depleted spleen or bone marrow (BM) cells of both normal and nude BALB/c mice within 10 days. It was further indicated that the V beta 2+ beta-chain genes contained few junctional N regions in both IL-3-induced primary DN alpha beta T cells and continuous lines. Search for the in vivo counterpart of in vitro IL-3-induced DN alpha beta T cells revealed that BM, but not spleens, of normal BALB/c and B6 mice did contain a significant proportion of DN alpha beta T cells, and that the majority of them expressed V beta 2+ beta-chain genes with few junctional N regions. The presence of V beta 2+ DN alpha beta T cells was similarly observed in the BM of BALB/c nude mice, but their proportion varied markedly among various strains of mice, which was not linked to H-2 haplotypes. The results indicated that V beta 2+ DN alpha beta T cells in the BM represented one of the thymus-independent T cell populations, whose development was under the major histocompatibility Ag complex-unlinked genetic control. TCR of these T cells were shown to be functional as judged by the proliferative response to anti-V beta 2 antibody. Taken together, present results suggested that IL-3 could induce differentiation and/or proliferation of DN alpha beta T cells with uniquely limited repertoire, which existed preferentially in BM in vivo, and implied the possible involvement of extrathymic endogenous ligands as a positive selection force.
Nylon wool-passed bone marrow (NW-BM) cells treated with anti-Thy.1 monoclonal antibody and complement were added to a mixed lymphocyte culture which contained a limiting number of lymph node cells, as responder cells, and a sufficient number of mitomycin-c-treated allogeneic spleen cells as stimulator cells. NW-BM cells of the same MHC haplotype as responder cells enhanced the generation of allo-specific cytotoxic T lymphocytes (CTL) not only at a relatively high dose (3 x 10(3) cells/well) of responder cells, but also at an extremely dilute dose (1 x 10(3) cells/well). NW-BM cells which had a third-party MHC haplotype, a haplotype different from both responder and stimulator cells, also enhanced the generation of CTL at relatively high doses, but not at low doses, of responder cells. NW-BM cells which had MHC haplotypes identical with those of responder cells induced CTL from helper T cell-depleted responder cells, but NW-BM cells which had the third-party haplotype did not. These results showed that the enhancing effects of NW-BM cells of the same MHC haplotype as responder cells might be due to a specific helper effect and the enhancing effect of NW-BM cells of the third-party haplotype might be due to a nonspecific filler effect, which only conditioned the cultured cells. It was also found that, to exhibit the helper effect, NW-BM cells had to possess MHC class II, but not MHC class I, molecules in common with CTL precursors. This study showed that in the induction of CTL, prethymic NW-BM cells had a capability comparable to that of mature helper T cells.
The role of IL-4 in proliferation and differentiation of human NK cells was studied using newly established sublines of an IL-4-dependent NK cell clone (IL4d-NK cells) and an IL-2-dependent NK cell clone (IL2d-NK cells) derived from a parental conditioned medium-dependent NK cell clone (CM-NK cells). IL-4 induced the higher proliferation of CM-NK cells, but abolished their NK activity and decreased CD16 and CD56 Ag expression. In contrast, IL-2 induced the higher NK activity and increased CD16 and CD56 Ag expression. Addition of anti-IL-4 antibody to the culture of CM-NK cells with CM inhibited the proliferation, but slightly increased NK activity, and largely increased CD56 Ag expression. Addition of anti-IL-2 antibody to the culture of CM-NK cells with CM inhibited both proliferation and cytotoxicity. Proliferation of IL4d-NK cells, which is totally dependent on rIL-4, is greater than that of IL2d-NK cells, which was greater than parental CM-NK cells. Morphologically, IL4d-NK cells are small and round, whereas IL2d-NK cells are large and elongated. Anti-IL-4 antibody inhibited proliferation of IL4d-NK but not IL2d-NK cells, whereas anti-IL-2 antibody inhibited that of IL2d-NK but not IL4d-NK cells. IL-2 was not detected in the supernatant from IL4d-NK cells, nor was IL-2-mRNA expressed in IL4d-NK cells. In contrast, IFN-gamma production and protein expression in IL4d- and IL2d-NK cells were detected. NK cell activation markers (CD16 and CD56) were expressed on IL2d-NK cells but not IL4d-NK cells. IL4d-NK cells were not cytotoxic to any tumor cells tested, whereas IL2d-NK cells displayed potent NK activity and lymphokine-activated killer activity. IL4d-NK cells failed to bind K562 tumor cells, whereas one-third of the IL2d-NK cells did. IL4d-NK cells responded to rIL-2, proliferated, and differentiated into cytotoxic NK cells, whereas IL2d-NK cells failed to respond to rIL-4 and died. These results raise a possibility that IL4d-NK cells or IL2d-NK cells primarily represent the immunologic properties of immature or activated types of human NK cells, respectively. Our results provide the first evidence of the capability of IL-4 to support continuous proliferation of a lymphocyte clone with immature NK cell characteristics and to stimulate IFN-gamma production in the clone. IL-4 is suggested as a potential growth factor for certain types of human NK cell progenitors.
The treatment of mice with high doses of 5-fluorouracil (5-FU) results in an enrichment of primitive hematopoietic progenitors. Using this procedure, we obtained a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro and could differentiate into not only myeloid cells but also into lymphoid lineage cells. The phenotypes of interleukin-3 (IL-3) induced blast colony cells were Thy-1-positive and lineage-marker-negative. We examined whether these blast colony cells contained primitive hematopoietic stem cells in vivo and could reconstitute hematopoietic tissues in lethally irradiated mice. Blast colony cells could generate macroscopic visible spleen colonies on days 8 and 12, and 5 x 10(3) blast cells were sufficient to protect them from lethally irradiation. It was shown that 6 or 8 weeks after transplantation of 5 x 10(3) blast cells, donor male cells were detected in the spleen and thymus of the female recipients but not in the bone marrow by Southern blot analysis using Y-encoded DNA probe. After 10 weeks, bone marrow cells were partially repopulated from donor cells. In a congenic mouse system, donor-derived cells (Ly5.2) were detected in the thymus and spleen 6 weeks after transplantation. Fluorescence-activated cell sorter analyses showed that B cells and macrophages developed from donor cells in the spleen. In the thymus, donor-derived cells were found in CD4, CD8 double-positive, single-positive, and double-negative populations. Reconstitution of bone marrow was delayed and myeloid and lymphoid cells were detected 10 weeks after transplantation. These results indicate that IL-3-induced blast cells contain the primitive hematopoietic stem cells capable of reconstituting hematopoietic organs in lethally irradiated mice.
Nine patients with a lymphoproliferative disorder characterized by a persistent expansion of large granular lymphocytes (LGL) and an increased proportion of cells labelling with natural killer (NK) and T cell markers were identified. The six patients with an expansion of alpha beta CD3/TcR positive cells were shown to have rearranged T cell receptor (TcR) genes whereas three patients whose LGL lacked CD3/alpha beta TcR on the surface had no beta TcR rearrangement detected. Eight of the nine patients were shown to exhibit non-MHC-restricted cytotoxic activity against K562; this activity was inhibited by CD45 and CD45-associated monoclonal antibodies known to inhibit normal non-MHC-restricted cytotoxicity but not specific MHC-restricted cytotoxic T cell activity. In contrast, the CD3 monoclonal antibody OKT3 did not inhibit but redirected LGL non-MHC-restricted cytotoxicity against K562. Following modulation of the CD3 molecule, the LGL were still capable of cytolysis of K562 targets, but additional OKT3 could no longer redirect cytolysis. The data indicate that the CD3/TcR complex on the LGL clones in patients with large granular lymphoproliferative disease is not the receptor for antigen on K562 cells, although it retains functional capabilities. Thus the CD3/TcR positive subset appears to have bipotential cytotoxic characteristics involving additional unique receptors for non-MHC-restricted cytotoxicity.
