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PubMed1982

Clonal analysis of progenitor cell commitment of granulocyte or macrophage production.

Granulocyte-macrophage colony formation by C57BL bone marrow cells was initiated in agar cultures either by the granulocyte-macrophage stimulus, GM-CSF, or by the predominantly macrophage stimulus, M-CSF. After 24 hours, paired daughter cells of granulocyte-macrophage colony-forming cells (GM-CFC) were separated by micromanipulation and one cultured in GM-CSF, the other in M-CSF. From the differentiation pattern of the resulting colonies, irreversible commitment of some cells occurred during the first 24 hours and completion of the first cell division. A similar result was obtained using granddaughter cells present after 24 hours of incubation. However, when intact developing day 2 and day 3 clones were cross-transferred to GM-CSF or M-CSF recipient cultures, irreversible commitment was more obvious. Most M-CSF-initiated clones exhibited irreversible commitment to macrophage formation in GM-CSF cultures and a high proportion of GM-CSF-initiated clones continued to produce granulocyte progeny after transfer to M-CSF. The results indicated that GM-CSF and M-CSF can irreversibly commit the progeny of GM-CFC respectively to granulocyte or macrophage production. While for women GM-CFC this occurs within 24 hours and one cell division, for many cells, the process is slower and requires an incubation period of up to 48 hours and/or several cell divisions. Calculations from the data indicated that two-thirds of GM-CFC in adult C57BL marrow are biresponsive and respond to stimulation both by GM-CSF and M-CSF.

Animals↗
PubMed1982

A clonal analysis of the differentiation of 3T3-L1 preadipose cells: role of insulin.

Cells of the established preadipose line, 3T3-L1, appear to be undifferentiated fibroblasts during exponential growth. When cells become quiescent, a small percentage of them accumulate triglyceride and become morphologically indistinguishable from mature adipocytes. When insulin is added to quiescent cultures, up to 50% of the cells differentiate into adipocytes. The distribution of lipid-containing cells which appear in clusters of varying sizes was analyzed to determine whether commitment to differentiation occurred after quiescence or during exponential growth and whether insulin was required as an inducer of commitment. The spatial arrangement of 3T3-L1 cells at quiescence on some culture dishes was destroyed by replating. This resulted in random distribution of these cells. The distribution of adipocytes among replated and nonreplated cells in these experiments was compared to a computer generated random distribution of differentiated among undifferentiated cells. Dispersal of cells at confluence resulted in a distribution of fat among nonfat cells not significantly different from the computer generated random distribution. In undisturbed cultures, the distribution of fat cells is not random and is consistent with a commitment event in single cells at any cell division during exponential growth followed by divisions of both committed and uncommitted cells. Since insulin affected the number of mature adipocytes only when added after cessation of exponential growth, insulin is not the inducer of commitment but merely enhances lipid production in previously committed cells.

Adipose Tissue↗
PubMed1984

A kinetic and clonal analysis of heterogeneity in K562 cells.

Expression of markers of differentiation was measured in a clone of the continuous cell line K562, derived originally from the cells of a patient with leukemia. Three of the markers were lineage specific, R18 for erythropoiesis and 80H.5 and My-1 for granulopoiesis. The fourth marker was the self-renewal capacity of clonogenic cells. The markers were measured as a function of time in pooled colonies from day 2 to day 12, and at a point of time in individual colonies. Evidence of an orderly pattern of marker appearance and disappearance was not seen. Rather, their expression appeared to occur at random during growth.

Cell Differentiation↗
PubMed1986

Clonal analysis of the population of chondrocytes from the Swarm rat chondrosarcoma in agarose culture.

Chondrocytes from the Swarm chondrosarcoma, a transplantable rat tumor, have been difficult to maintain in tissue culture for extended periods due to a time-dependent alteration of the culture to a more fibroblastic phenotype. This feature precluded the use of these cultures to examine chronic conditions that may affect cell metabolism, and the homogeneity or heterogeneity of the tumor cells within the culture population could not be examined. Use of suspension culture in agarose stabilized the chondrocyte phenotype, permitting long-term culture. Clones of tumor chondrocytes were established in agarose and were examined over 2-3 weeks for evidence that the cells were accumulating a proteoglycan-rich extracellular matrix, as determined by positive staining by Alcian blue, and were undergoing cell division. Nearly 90% of the cloned cells exhibited a prominent extracellular matrix by day 7 of culture and greater than 99% did so by day 14. Cell division did not occur to any great extent until days 6-7 of culture. After this lag, the cells appeared to undergo logarithmic growth, with a cell generation time of about 12 days. By 20 days of culture, between 80 and 90% of the initial clones contained multiple cells, indicating that nearly all the cells were in, or had entered, the cell cycle. These results suggest that the chondrocytes from the rat chondrosarcoma form a homogeneous cell population with respect to their ability to synthesize an extra-cellular matrix and divide.

Alcian Blue↗
PubMed1983

Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture. Clonal analysis of effector cells in cytotoxic and proliferative assays.

Lymphocytes from cancer patients were stimulated in mixed culture with autologous tumour (MLTC) or pooled allogeneic lymphocytes (MLC). Both protocols induced increased uptake of 3H-thymidine at 5 days and the appearance of lymphoblasts. Blasts were isolated on discontinuous Percoll gradients and either expanded as bulk cultures or cloned directly under limiting dilution conditions in the presence of conditioned medium containing IL-2. Results with MLTC-blast-CTC have been reported elsewhere. MLC-activated cultures lysed autologous tumour but not autologous lymphoblasts. Lysis of some allogeneic tumours, lymphoblasts from members of the inducing pool, and K562 was also apparent. MLC activated cultures did not undergo restimulation in response to autologous tumour or lymphocytes but were restimulated by leukocytes from pool members. MLTC clones showed autologous tumour-specific cytotoxic activity or cross-reactive proliferative responses with tumours of the same site and histology. The majority of MLC clones cytotoxic for autologous tumour were also specific and did not lyse allogeneic tumour, K562, or lymphoblasts from the inducing pool. Two clones lysed autologous tumour and pool members. None of the clones tested proliferated in response to autologous tumour following MLC activation but some were responsive to pool members and one clone was restimulated by autologous monocytes. No association was found between clone phenotype and function. The implication of these data is that the effector cells with activity against autologous tumour induced in MLC arose largely by transstimulation of in vivo-activated tumour reactive lymphocytes by IL-2 release rather than expansion of NK-like effectors or sharing of antigenic specificities between tumour and allogeneic lymphocytes. Since MLC activation of cancer patients lymphocytes does not induce proliferative responses to autologous tumour it is unlikely to be a useful procedure in preparing cells for immunotherapy protocols.

Antibodies, Monoclonal↗
PubMed1986

Clonal analysis and in situ characterization of lymphocytes infiltrating human breast carcinomas.

T lymphocytes were isolated from tumor biopsies in 13 patients with breast carcinomas. Immunohistology with monoclonal antibodies confirmed the presence of mononuclear cell infiltrates composed primarily of T lymphocytes in all tumors studied. While the proportion of T lymphocytes expressing the T4 or the T8 surface marker varied from tumor to tumor as determined by morphometric analysis, T8+ cells were more numerous than T4+ cells in 8/12 breast tumors studied. Relatively few T cells (less than 10% in 11/12 tumors) were in an activated state as judged by the surface expression of HLA-DR antigens or the receptor for interleukin-2 (IL-2). In 1 case 20% of the infiltrating mononuclear cells were expressing the IL-2 receptor. The tumor infiltrating lymphocytes (TIL) recovered from 10 tumors were cloned in a microculture system that permits proliferation of nearly 100% of normal peripheral blood T lymphocytes (PBL-T). In contrast to normal and autologous PBL-T, frequencies of proliferating T lymphocyte precursors (PTL-P) were depressed (less than 0.01) in 7/10 TIL preparations indicating a decreased responsiveness of TIL to phytohemagglutinin at the single-cell level. The frequency of PTL-P was noticeably higher in 2 cases (0.03 and 0.09) and close to normal in 1 case (0.39). A total of 170 clones were expanded in vitro and analyzed for different functional capabilities. Most of these clones expressed the T4+/T8-phenotype (73%) and strikingly 53% of these T4+/T8- clones were cytolytic in a lectin-dependent assay, a functional subset which is uncommon among normal PBL-T. Some clones (10%) lysed allogeneic breast tumor cells (MCF7). Only 15% of the clones displayed natural killer activity. Among the cytolytic clones, 17 of 31 tested were also IL-2 producers irrespective of the T4 or T8 phenotype. Our results show that human mammary carcinomas contain many infiltrating T cells with cytolytic potential. Interestingly, among the proliferating cytolytic T cell clones (56% of the microcultures), many expressed the T4+/T8- phenotype. These findings may indicate that the in situ cytolytic reaction (against unknown antigens) is associated preferentially with class II antigens.

Antibodies, Monoclonal↗
PubMed1986

Clonal analysis of cytotoxic T lymphocytes (CTL) against autologous melanoma. Classification based on phenotype, specificity and inhibition by monoclonal antibodies to T cell structures.

This study investigates the nature and specificity of cytotoxic T lymphocytes (CTL) in patients with melanoma which are able to kill autologous melanoma cells. Interleukin 2 (IL2)-dependent T cell clones from two melanoma patients and a normal subject were generated in mixed lymphocyte cultures (MLC) or mixed lymphocyte tumor cell cultures (MLTC) and propagated for prolonged periods in tissue culture. Analysis of their phenotype by a wide range of monoclonal antibodies (M.Abs) revealed two main phenotypes which depended on whether they expressed Fc receptors detected by Leu 11 M.Abs or not. Leu 11- T cells (referred to as Type 1) were inhibited by M.Abs to T3, T8, and a common HLA, ABC antigen. Conversely Leu 11+ T cells (referred to as Type 2) were inhibited by M.Ab to Leu 11 but not by M.Ab to T3, T8 and the HLA, ABC antigen. Subtypes among Type 1 cells were recognized which depended on their specificity. The most restricted were CTL [Type 1(a)] clones generated only in MLTC which recognized the autologous melanoma cell plus 1 of 11 other melanoma target cells. Type 1(b) CTL clones recognized a larger proportion (approximately 50%) of the melanoma cells. A third category [Type 1(c)] recognized antigens on melanoma cells shared with that on the EBV-transformed B cells used as stimulators in the MLC. Type 2 CTL clones had broad specificity to melanoma and nonmelanoma cells, characteristic of that described for lymphokine activated killer (LAK) cells. The latter were MHC unrestricted but further studies are required to clarify whether the Type 1 CTL clones are MHC restricted or not. The CTL activity of all clones was inhibited by M.Ab to the sheep red blood cell receptor and to the T10 antigens. It is suggested that recognition of these different types of CTL clones may assist future studies on the immune response against melanoma and the nature of antigens recognized by CTL.

Antibodies, Monoclonal↗
PubMed1986

The myoblast defect identified in Duchenne muscular dystrophy is not a primary expression of the DMD mutation. Clonal analysis of myoblasts from five double heterozygotes for two X-linked loci: DMD and G6PD.

We previously proposed the hypothesis that the primary expression of the defect in X-linked Duchenne muscular dystrophy (DMD) occurred in the myoblast, or muscle precursor cell. This was based on the observation that the number of viable myoblasts obtained per gram DMD muscle tissue was greatly reduced and those that grew in culture had decreased proliferative capacity and an aberrant distended flat morphology. Here we test that hypothesis by determining whether the expression of the myoblast defect is X-linked. Muscle cells were obtained from five doubly heterozygous carriers of two X-linked loci, DMD and glucose-6-phosphate dehydrogenase (G6PD), and compared with those from five sex- and age-matched controls heterozygous for G6PD only. A total of 1,355 individual clones were determined to be muscle and evaluated at the single cell level for proliferative capacity, morphology, and G6PD isozyme expression. The results demonstrate that the proportion of defective myoblast clones is significantly increased in DMD carriers. However, since this cellular defect does not consistently segregate with a single G6PD phenotype in the myoblast clones derived from any of the carriers, it is unlikely to be the primary expression of the DMD mutant allele.

Adult↗
PubMed1985

Intercellular NOR-Ag-variability in man. II. Search for determining factors, clonal analysis.

Intercellular, nonartifactual variability of nucleolar organizer region (NOR)-Ag-staining was studied in cultured human peripheral blood lymphocytes, skin and embryonic fibroblasts. No differences in number and character of variable NORs and intensity of their staining were observed between lymphocytes stimulated to proliferate with phytohemagglutinin and pokeweed mitogen, as well as lymphocytes of first- and second division. The number of NOR associations per cell and the number of associated chromosomes per association were also similar. In a given individual these criteria were similar in lymphocytes and fibroblasts. In all nine clones derived from three independent parental fibroblast cultures the intercellular NOR-Ag-variability was similar to that observed in a given parental cell line. A significant decrease in the number of metaphases containing NOR associations was observed in second-division lymphocytes compared with first-division ones, as well as in skin fibroblasts compared with lymphocytes.

Cell Count↗
PubMed1985

Clonal analysis of two mutations in the large subunit of RNA polymerase II of Drosophila.

Two mutations in the gene, RpII215, were analyzed to determine their effects on cell differentiation and proliferation. The mutations differ in that one, RpII215ts (ts), only displays a conditional recessive lethality, while the other, RpII215Ubl (Ubl), is a recessive lethal mutation that also displays a dominant mutant phenotype similar to that caused by the mutation Ultrabithorax (Ubx). Ubl causes a partial transformation of the haltere into a wing; however, this transformation is more complete in flies carrying both Ubl and Ubx. The present study shows that patches of Ubl/-tissue in gynandromorphs are morphologically normal. cuticle that has lost the wild-type copy of the RpII215 locus fails to show a haltere to wing transformation, nor does it show the synergistic enhancement of Ubx by Ubl. We conclude that an interaction between the two RpII215 alleles, Ubl and RpII215+, is responsible for the mutant phenotype. Gynandromorphs carrying the ts allele, when raised at permissive temperature, display larger patches of ts/-cuticle than expected, possibly indicating that the proliferation of ts/+ cells is reduced. This might result from an antagonistic interaction between different RpII215 alleles. Classical negative complementation does not appear to be the cause of the antagonistic interactions described above, as only one RpII215 subunit is thought to be present in an active multimeric polymerase enzyme. We have therefore coined the term 'negative heterosis' to describe the aforementioned interactions. We also observed that the effects of mutationally altered RNA polymerase II on somatic cells are different from its effects on germ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Clonal analysis of B- and T-cell responses to Ia antigens. I. Topology of epitope regions on I-Ak and I-Ek molecules analyzed with 35 monoclonal alloantibodies.

Thirty-five Iak-specific monoclonal alloantibodies, derived from hybridomas constructed by fusion between mouse myeloma and spleen cells from A.TH alloimmune mice (Is anti-Ik), have been used to estimate the allotypic polyporphism of the Ik-gene products. Cross-blocking studies using 17 mAb specific for the I-A molecule indicated that six determinants, which were associated with the conventional specificities Ia.2 and Ia.19, were organized in at least three distinct polymorphic areas of the I-Ak molecules. Similarly, another group of six determinants, which did not correspond to previously described conventional Ia specificities, were found to be topologically heterogeneous. By contrast, the five epitopes associated with the Ia. 1 specificity were clustered into a single region of this molecule. In addition the potentiation of binding observed between mAb specific for topologically distinct epitope regions of the I-Ak molecule, suggested that the latter may undergo conformational changes after binding of a given mAb. A similar analysis of 17 mAb specific for the I-Ek molecule indicated that specificity Ia. 7 of the E alpha chain (as defined in this series by eight mAb) was composed of three topologically distinct polymorphic areas, one of which is also spatially related to a complex cluster of eight new determinants of the I-Ek molecule. Finally, one mAb identified a so far undescribed shared determinant of the I-Ak and I-Ek molecules. The present results, which provide a new estimate of the allotypic polymorphism of the Iak antigens, are discussed with regard to their functional, biochemical, and evolutionary implications.

Animals↗