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Biomedical subjects

I Clark-Lewis

Publications and source records attributed to I Clark-Lewis.

At least 163 records · Page 9Linked to original sources
PubMed1984

In vitro approaches to lymphopoiesis, hemopoiesis, and oncogenesis.

Lymphopoiesis still remains a black box. Much remains to be learned about the identification of the cellular stages and the factors that regulate the rate of production of lymphocytes. Modifications of the Dexter system should assist with answers to these questions. Our work on PSF and that of other groups who have studied what is almost certainly the same factor under names such as hemopoietic growth factor, IL-3, BPA or multi-CSF suggests that this factor may provide an alternative means of generating cells for transplantation and replacement therapy. At present the only established sources of PSF are the activated T cell or tumors in which we think the gene has been anomalously activated, such as WEHI-3B; the long-term bone marrow culture system, however, seems to be defining a factor or influence that may be identical with, or is able to replace, the T cell factor. Finally, our experiments with the heterogeneous P cell lines and the initiation of oncogenesis by activation of the PSF gene raise some caveats about the use of cultured cells for human therapy. In the mouse system the production of immortalized factor-dependent lines appears to be more frequent in cells taken from long-term bone marrow cultures rather than normal bone marrow. Obviously, further information on the mechanism of immortalization and on the frequency and mechanism of activation of PSF genes in such lines will be of great importance in guiding the practical use of in vitro-derived cells and in the understanding of leukemogenesis.

Animals↗
PubMed1984

The in vivo functions and properties of persisting cell-stimulating factor.

We present evidence that persisting (P) cell-stimulating factor (PSF), a T cell lymphokine, is produced and active in vivo. Mice injected in one footpad with keyhole limpet haemocyanin or intravenously with sheep erythrocytes had substantial increases in numbers of splenic P cell precursors; the increase following the sheep erythrocytes did not occur in athymic mice implying a dependence on T lymphocytes. The increase in P cell precursors correlated with the local release of PSF; thus cells from the ipsilateral draining lymph node of mice injected in one footpad with KLH, but not cells from the contralateral node, showed both increased numbers of P cell precursors and the production of PSF. PSF could, in other situations, enter the circulation and exert effects distal to its release. Mice bearing a localized tumour that produced PSF (WEHI-3B), but not those bearing a non-producing subline, showed both a significant increase in P cell precursors in the spleen and bone marrow, and a marked increase in the numbers of mast cells, megakaryocytes, metamyelocytes and polymorphs in the spleen. PSF was detected in the serum of the mice bearing the PSF-producing tumour. Following intravenous injection of PSF into normal mice there was a rapid initial clearance (t 1/2 4 min), followed after 10 mins by a phase of slower clearance (t 1/2 40 min). This was due to removal of PSF rather than inhibition or destruction by serum factors, as when PSF was mixed in vitro with mouse serum for 24 hr at 37 degrees, no activity was lost.

Animals↗
PubMed1983

P-cell stimulating factor: characterization, action on multiple lineages of bone-marrow-derived cells and role in oncogenesis.

T-cell hybridomas have allowed us to define unequivocally a group of 3 distinct molecules, TCGF, T-cell GM-CSF, and PSF, as the products of the activated T-cell. It is becoming increasingly evident that these T-cell-derived molecules, together with a fourth, interferon-gamma, (Wong et al. 1982, 1983), affect a wide range of cell-types. The molecule which we have studied in greatest detail, PSF, probably effects every lineage of non-lymphoid bone-marrow-derived cells. We have evidence that PSF acts in vivo as an important mediator in a pleotropic defence and repair response to antigens that involves all the non-lymphoid elements of the blood. Finally, there is evidence that PSF-dependent cells can become immortal, and that activation and functional expression of the PSF gene can occur in such cells and result in autonomy and tumorigenesis. Clonal sources of T-cell lymphokines and clonal targets for lymphokine assays, formed the basis of recent progress in defining the number and nature of non-antigen-specific T cell products; cloning of the genes coding for these lymphokines should result in a similar impetus to the investigation of the physiology and possible therapeutic role of T-cell lymphokines, and lead to new insights into the control of gene expression and the role of these factors in oncogenesis.

Animals↗
PubMed1983

The size of functional T-lymphocyte pools within thymic medullary and cortical cell subsets.

The frequency of all precursors of T cells capable of proliferation (PTL-p) and of all cytotoxic T-cell clones (CTL-p) was determined for different thymocyte subpopulations using a high cloning-efficiency, Con A and growth factor driven, limit-dilution assay and a lectin-mediated, non-specific cytotoxic readout. As shown previously, more than 99% of precursors were confined to the medullary-type fraction, isolated by fluorescence activated cell sorting as the 14-15% of thymocytes showing low binding of peanut agglutinin (PNA). However, 20-50% of medullary-type cells appeared incapable of responding in a culture system allowing all peripheral T cells to grow, suggesting that the absolute size of the functional pool was 7-12% of all thymocytes. The 3-4% cortisone-resistant fraction of thymus gave a high precursor frequency (PTL-p 1 in 1.3; CTL-p 1 in 6) and a high cloning efficiency per Thy 1 positive cell (80%) which was nevertheless below that of peripheral T cells. However, only 20-25% of the total thymic PTL-p and CTL-p could be recovered in this fraction. Functional precursors were therefore within both the cortisone-sensitive and the cortisone-resistant subgroups of medullary-type thymocytes. Attempts to induce function in PNA+ cortical-type thymocytes by increasing the level of T-cell growth factors in the cultures gave only a marginal increase, the bulk of small cortical cells remained functionally inert. However, the low frequency of precursors found in the PNA+ fraction (around 1% of that in PNA- thymocytes) was not entirely due to PNA- contaminants since a PNA+, high H-2 blast fraction, representing about 4% of all thymocytes, showed a significant, although still low, PTL-p and CTL-p frequency amounting to less than 1% of the total thymus precursor pool. The relevance of this minor subset is discussed.

Animals↗
PubMed1983

Interferon-gamma induces enhanced expression of Ia and H-2 antigens on B lymphoid, macrophage, and myeloid cell lines.

The levels of class II major histocompatibility complex (MHC) antigens (la antigens) on cells of a cultured B lymphoma line (WEHI-279) were significantly increased after 24 hr incubation with medium conditioned by concanavalin A-stimulated mouse or rat spleen cells, or by an azobenzenearsonate- (ABA) specific T cell clone that had been stimulated with ABA-coupled spleen cells or concanavalin A. The levels and properties of the la-inducing activity correlated with those of interferon-gamma (IFN-gamma) measured by inhibition of virus plaque formation. Both the la-inducing activity and the IFN-gamma from the T cell clone had an apparent m.w. of 40,000 determined by gel filtration, were sensitive to treatment with trypsin or exposure to pH 2, but were stable to heat (56 degrees C, 1 hr). The induction of la antigens on WEHI-279 cells was dose-dependent, and the maximum response occurred at a concentration corresponding to 1 to 2 U/ml of antiviral activity. This T cell-derived IFN-gamma-like molecule also increased the expression of cell surface la antigens on another B cell line (WEHI-231), and cell lines of macrophage (J774) and myeloid (WEHI-3B and WEHI-265) origin. Furthermore, in all cases the levels of class I MHC (H-2K or H-2D) antigens were also increased. Similar patterns of induction of Ia and H-2 antigens were obtained with supernatants containing IFN-gamma produced by a monkey cell line (COS) that had been transfected with a plasmid bearing the cloned murine IFN-gamma gene. This activity was sensitive to pH 2 and was not present in the supernatant from COS cells that were not transfected with the murine IFN-gamma gene. These results established that IFN-gamma is the T cell-derived molecule that induces the enhanced expression of Ia and H-2 antigens on B cells and macrophages. A major physiologic role of IFN-gamma may be to regulate immune function through the enhanced expression of MHC antigens.

Animals↗
PubMed1982

Preparation of T cell growth factor free from interferon and factors stimulating hemopoietic cells and mast cells.

A simple two-step method involving ammonium sulfate precipitation followed by hydrophobic chromatography is described for the separation of T cell growth factor (TCGF) from a number of other factors contained in medium conditioned by concanavalin A-stimulated spleen cells. Thus, granulocyte-macrophage colony-stimulating factor, P cell-stimulating activity, pluripotential stem cell-supporting activity and interferon activity were not detected in TCGF partially purified by these steps. T cell-replacing factor co-purified with TCGF. Macrophage activity factor (MAF) co-purified with TCGF, but the ratio of MAF to TCGF activities was reduced more than 20-fold relative to that in crude conditioned medium. All of the factors were present in the 50-80% saturated ammonium sulfate fraction, however, levels of concanavalin A were reduced by 98% in this step. TCGF, separated in this way from these other regulatory factors will be useful in experiments analyzing the actions of TCGF on mixed populations of cells.

Animals↗
PubMed1982

Proliferation and differentiation of single hapten-specific B lymphocytes is promoted by T-cell factor(s) distinct from T-cell growth factor.

Hapten-specific B lymphocytes reactive to fluorescein were prepared from mouse spleen, placed singly in 10-microliters culture wells, and stimulated with fluorescein-polymerized flagellin in the presence of conditioned media (CM) from various concanavalin A-stimulated cloned T-cell tumors or hybridomas. Antigen plus appropriate CM triggered 5-9% of the B cells into both clonal proliferation and differentiation into antibody-forming cells. Antigen alone stimulated 0.5-0.8% of B cells and CM alone stimulated less than 0.1%. This bioactivity was termed B-cell growth and differentiation factor(s) (BGDF). Four CM rich in T-cell growth factor (TCGF)--namely, CM from spleen and the lines EL4, T6, and 123--contained BGDF. The lines T19.1 and WEHI-3 lacked BGDF and TCGF. Four lines of evidence suggested that BGDF and TCGF were distinct molecules. First, the BGDF/TCGF ratios in the various CM varied. Second, on gel filtration, TCGF eluted as a sharp peak corresponding to a Mr of about 35,000, whereas BGDF eluted over a range corresponding to a Mr of 25,000-60,000. Third, the activity of TCGF in EL4-CM was markedly reduced by treatment with guanidine HCl while BGDF activity was not. Fourth, BGDF showed more heterogeneity than TCGF on hydrophobic chromatography. All CM or fractions active in promoting B-cell division also promoted differentiation to antibody-forming cells. These results provide unequivocal evidence that antigen and a T-cell product can synergize to directly activate a single B lymphocyte.

Animals↗
PubMed1982

Interferon-gamma-like molecule induces Ia antigens on cultured mast cell progenitors.

Persisting (P) cells (murine cells that resemble mast cells and grow continuously in vitro for prolonged periods in the presence of a specific growth factor) did not express detectable levels of Ia antigens (murine class II major histocompatibility antigens) when their growth was supported by partially purified P cell-stimulating factor. However, when these Ia-negative P cells were transferred to medium conditioned by concanavalin A-stimulated spleen cells, Ia antigens appeared within 24 hr. The increase in Ia antigens was due to induction of synthesis of Ia antigens by P cells and not to absorption of Ia antigens from the conditioned medium or selective growth of Ia-positive cells from a low number of Ia-positive cells in the original population. The Ia-inducing activity was also found in supernatants from antigen-stimulated cloned T-cell lines, but not from certain T-cell hybridomas or the T lymphoma EL-4. The presence of Ia-inducing activity correlated with the presence of interferon-gamma (IFN-gamma). The gel filtration profiles of IFN-gamma activity and Ia-inducing activity were coincident and corresponded to an apparent molecular weight of 40,000-45,000. Both the IFN-gamma and Ia-inducing activity were destroyed by treatment at pH 2. These results indicate that IFN-gamma or a closely related molecule induces Ia antigens on P cells and suggest that regulation of Ia antigen expression may be an important aspect of the effects of IFN-gamma on the immune and hemopoietic systems.

Animals↗
PubMed1982

Biochemical characterization of regulatory factors derived from T cell hybridomas and spleen cells. I. Separation of T cell growth factor and T cell replacing factor from granulocyte-macrophage colony-stimulating factor.

We describe the molecular characteristics of T cell growth factor (TCGF), T cell replacing factor (TRF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by a T cell hybridoma after stimulation with concanavalin A (Con A). All three activities could be separated from Con A itself by ammonium sulfate precipitation. The TRF and TCGF activities had a m.w. of 35,000 to 40,000 on gel filtration in phosphate-buffered saline (PBS). Their m.w. were about 30,000 under dissociating conditions in guanidine hydrochloride and about 35,000 to 40,000 under disulfide reducing conditions, suggesting the molecule(s) lacked noncovalent or disulfide-linked subunit structure. GM-CSF had a m.w. of 25,000 to 30,000 by gel filtration in PBS and about 23,000 in guanidine hydrochloride. TRF and TCGF on the one hand and GM-CSF on the other could be distinguished by the criteria of m.w., relative heat sensitivity, and hydrophobic chromatography. TCGF could not be separated from TRF by any of these methods. In terms of all the above properties, factor derived from the T cell hybridoma and spleen cells appeared identical.

Animals↗
PubMed1982

Biochemical characterization of regulatory factors derived from T cell hybridomas and spleen cells. II. Evidence for glycosylation of T cell growth factor, T cell replacing factor, and granulocyte-macrophage colony-stimulating factor.

Isoelectric focusing demonstrated that T cell growth factor (TCGF), T cell replacing factor (TRF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) derived from concanavalin A-stimulated T cell hybridomas and spleen cells are heterogeneous with respect to charge. The spleen cell-derived TCGF and TRF activities focused with isoelectric points (pI) between 3.5 and 6.5 whereas the range for GM-CSF activity was broader (pI, 3.5 to 8.0). The T cell hybridoma-derived activities were slightly more acidic. Neuraminidase treatment of both hybridoma 123 and spleen cell-derived material resulted in a major peak of each activity (TRF/TCGF pI, 4.9; GM-CSF pI, 4.7). Neuraminidase treatment of hybridoma T6-derived material resulted in peaks of TRF and TCGF around 6.0 as well as one around 5.0, suggesting that this charge heterogeneity was due to causes other than variations in the level of sialic acid on the relevant molecules. Tunicamycin-treated spleen cells or hybridoma 123 cells released biologically active TCGF, TRF, and GM-CSF. Each of these three activities from tunicamycin-treated spleen cells focused with pI around 5.0. A major fraction of TRF, TCGF, and GM-CSF activities bound to wheat-germ agglutinin. GM-CSF also bound to concanavalin A and lentil lectin. These results suggest that the molecules responsible for TCGF, TRF, and GM-SCF activities are glycosylated and that the observed heterogeneity in charge and lectin-binding characteristics is due in part to variable glycosylation. Glycosylation was not critical for any of the three biologic activities. No conclusive separation of TRF and TCGF activities was observed in these experiments although GM-CSF differed from TRF and TCGF in that it bound to Concanavalin A.

Carbohydrate Metabolism↗
PubMed1982

A T cell-derived factor stimulating multipotential hemopoietic stem cells: molecular weight and distinction from T cell growth factor and T cell-derived granulocyte-macrophage colony-stimulating factor.

The production of multipotential hemopoietic stem cells (CFUs) in cultures of murine bone marrow cells is supported by medium conditioned by concanavalin A-stimulated T cell hybridoma 123 or spleen cells. We present physiochemical evidence that this activity of these conditioned media, which we have operationally termed CFUs-stimulating activity (CFUs-SA), is due to a new T cell-derived lymphokine. CFUs-SA was shown by gel filtration to reside in a distinct fraction corresponding to a mean apparent m.w. of 29,000, and was distinguishable from T cell growth factor, not only by its lower apparent m.w. but also by chromatography using Phenyl-Sepharose. After isoelectric focusing of neuraminidase-treated conditioned medium, CFUs-SA was found between isoelectric points 5 and 8. In this respect, CFUs-SA appeared similar to another factor, P cell-stimulating factor, which stimulates the growth of persisting (P) cells (homogeneous populations of cells resembling mast cells) and that occurred together with CFUs-SA in both the spleen and hybridoma-conditioned media. Isoelectric focusing separated CFUs-SA from granulocyte-macrophage colony-stimulating factor, which focused as a single peak with an isoelectric point around 5.0. Thus CFUs-SA is due to a T cell-derived factor that is separable from T cell growth factor and T cell-derived granulocyte-macrophage colony-stimulating factor, but has similar properties to P cell-stimulating factor.

Animals↗
PubMed1981

The persisting (P) cell: histamine content, regulation by a T cell-derived factor, origin from a bone marrow precursor, and relationship to mast cells.

Histamine was detected at levels of 100 ng/10(6) cells in the metachromatic granules of the persisting (P) cell, which appears in cultures of murine lymphoid or bone marrow cells and is capable of long-term growth in vitro in the presence of a T cell-derived growth factor. This factor, which we termed P-cell stimulating factor, was distinct from t-cell growth factor and had an apparent molecular weight of 25,000-30,000. P cells did not originate from Thy.1-positive cells nor was the thymus necessary for the development of their precursors. Moreover, P cells grew directly from colonies generated in agar cultures of bone marrow cells, the nature of the colonies indicating that P cells shared a common precursor with hemopoietic cells. Mutant Wf/Wf mice, although deficient in certain mast cells, possessed P-cell precursors. It is hypothesized that P cells are related to a specialized subset of mast cells, derived from a bone marrow progenitor but regulated by activated T cells.

Animals↗
PubMed1981

T cell hybridoma-derived regulatory factors. I. Production of T cell growth factor following stimulation by concanavalin A.

A cloned T cell hybridoma (123) was shown to produce T cell growth factor (TCGF). Supernatants of cultures of hybridoma-123 that had been stimulated with concanavalin A caused T cell blasts or a cloned T cell line to proliferate, allowed a mitogenic response to concanavalin A by thymocytes and by lymph-node cells depleted of accessory cells by treatment with anti-Ia serum and complement, and permitted the generation of both polyclonal and antigen-stimulated cytotoxic T lymphocytes in cultures of thymus cells. These observations suggest that the hybridoma is producing a factor with an identical spectrum of activities to that associated with TCGF derived from mitogen-stimulated spleen cells and indicate that the T cell is the source of TCGF. Together with other evidence that the same hybridoma produces activity affecting B lymphocytes, myeloid progenitor cells, and pluripotential stem cells, these experiments confirm the role of the activated T cell as a regulator of hemopoietic and lymphoid systems. T cell hybridomas should prove invaluable for biochemical and genetic analysis of these factors.

Animals↗