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Biomedical subjects

G Pawelec

Publications and source records attributed to G Pawelec.

198 records · Page 11Linked to original sources
PubMed1982

Analysis of the HLA-linked SB gene system with cloned and uncloned alloreactive T-cell lines.

Further enhancement of cellular typing for antigens of the new HLA-linked "SB" gene was undertaken by T-cell expansion and cloning. The PLT reagents which define these antigens could be expanded over 100-fold with interleukin 2 (IL-2), without loss of specificity. Cloning efficiencies in limiting dilution of over 50 percent could be achieved, although only 5 percent of these could be expanded extensively. Detailed analysis was performed with clones from a highly restricted PLT reagent raised between an HLA recombinant donor and his sibling whose only known HLA difference was SB4. The antigens recognized by the 11 independent clones analyzed appeared to segregate in this family with HLA. Although the two SB4-positive haplotypes in the family were essentially indistinguishable by the clones, they detected heterogeneity among unrelated donors matched for SB4 (as defined by bulk PLT reagents). Such heterogeneity did not appear to be due to differences between clones in the kinetics of their responses, but could be explainable by complexity of the SB molecule or by new HLA antigens.

B-Lymphocytes↗
PubMed1981

Restimulation properties of cloned alloactivated lymphocytes: detection of a novel type of HLA-D/DR region determinant and allelic subtypes for HLA-Dw3 using cloned reagents.

Lymphocytes alloactivated by Dw3 homozygous typing cells were cloned by the method of limiting dilution and cultured for prolonged periods using T-cell growth factor and irradiated pooled leukocytes (as feeder cells). Restimulation specificity of two clones functioning as primed lymphocyte typing reagents was investigated in panel and family studies. Cells from one of the clones (12-2) were always specifically stimulated by HLA-Dw3 antigens shared between the original priming cells and the stimulating panel cells. In an informative family KOH, however, cells from this clone seemed to detect a split in the Dw3 cluster. Cells from the other clone (12-8) failed to respond to Dw3 antigens as expressed by the original priming cells or by panel stimulating cells; rather, specificity of restimulation seemed to be associated with the expression of Dw4 antigens. Family segregation analysis did not support this conclusion however, since stimulating products segregated with one of the three Dw3-bearing haplotypes and with none of three Dw4-bearing haplotypes. This suggested that 12-8 cells may be responsive to antigens different from those detected by homozygous typing cells.

Alleles↗
PubMed1981

Cloning of functional human T lymphocytes by limiting dilution: impact of filler cells and interleukin 2 sources on cloning efficiencies.

Conditions for the cloning of alloactivated human lymphocytes by limiting dilution in the presence of interleukin 2 (IL 2) and filler cells have been investigated. Cloning efficiencies were extremely high (at least 40%) when IL 2 derived from pooled peripheral blood mononuclear cells (PBM), stimulated with phytohaemagglutinin in the presence of irradiated Epstein-Barr virus-transformed lymphoid cell lines (LCL), was used in combination with irradiated pooled PBM as filler cells. Cloning efficiencies were reduced almost twofold using autologous filler cells and were further reduced dramatically using autologous IL2, although some clones could still be obtained. In addition, cloning efficiencies were acceptable (over 30%) when IL 2 produced spontaneously from the leukaemic cell Jurkat (M-N) was used. In contrast, sheep erythrocytes and LCL as filler cells failed to allow successful cloning, although LCL were beneficial in the expansion of cloned populations. Other factors being equal, the use of pooled PBM as filler cells and highly active preparations of IL 2 will be the method of choice for cloning functional human T lymphocytes.

Cell Division↗
PubMed1981

Human T lymphocytes grown in T-cell growth factor: functional attributes in MLC, CML, PLT and allogeneic suppression.

Conditions for the production of highly effective human T-cell growth factor (TCGF) and its screening in a thymidine incorporation assay are described. Culture medium supplemented with optimum concentrations of TCGF was used in bulk culture systems for the expansion of alloactivated and non-alloactivated T-cell populations. Surface marker studies of cells grown in TCGF-dependent culture showed that the majority were T cells expressing DR antigens. These cells retained their proliferative responsiveness in mixed leucocyte cultures but were unable to stimulate proliferation of normal allogeneic lymphocytes. Cultured cells which derived from mixed leucocyte cultures retained immunological specificity compared with cells not exposed to TCGF both in terms of proliferative responses caused by allogeneic cells in the absence of TCGF, and in terms of cytotoxic activity against 51Cr-labelled target cells. In addition they were capable of suppressing tritiated thymidine incorporation in mixed leucocyte cultures. These experiments show that continuous culture with TCGF can expand a variety of T-cell subpopulations with retention of distinct immunological function.

Antigens, Surface↗
PubMed1980

Restimulation properties of human alloreactive cloned T-cell lines. Dissection of HLA-D-region alleles in population studies and in family segregation analysis.

Alloactivated human lymphocytes were cloned by limiting dilution. After 1 month in culture with T-cell growth factor several clones incorporated tritiated thymidine when stimulated with the appropriate allogeneic cells. Specificity of restimulation of two primed lymphocyte clones, designated 12-2 and 12-8, was studied in detail after varying periods of culture (up to 50 days). Clone 12-2 cells were stimulated only by cells expressing the HLA-Dw antigens of the original priming cells (Dw3); furthermore, this primed lymphocyte reagent specifically recognized antigens associated with only one of the three distinct Dw3-bearing haplotypes from an informative family (KOH). Clone 12-8 cells, on the other hand, failed to recognize Dw3 antigens in the random panel or on homozygous typing cells (including the original priming cell), but were strongly restimulated by certain cells expressing Dw4 antigens. In addition, within family KOH, these restimulating products segregated with another one of the three Dw3-bearing haplotypes but with none of the three Dw4-bearing haplotypes. These two clones exemplify a hitherto unknown precision in cellular typing of the HLA-D region. Clone 12-2 allows the discrimination of a probably rare and as yet undetected HLA-Dw3 subtypic specificity. Clone 12-8, on the other hand, apparently identifies an allelic system segregating with HLA but distinct from the HLA-D determinants definable by HTC-typing.

Alleles↗
PubMed1980

The induction of suppressor cells in mixed leucocyte cultures and in mixed leucocyte-non-lymphoid cell cultures.

X-ray resistant porcine suppressor T cells expressing Ia-like antigens were obtained from mixed cultures of leucocytes and tissue cells (cultured kidney cells, liver cells, endothelial cells, fibroblasts or X-irradiated leucocytes), and were assayed by their ability to suppress lymphocyte proliferation in a second mixed culture. All tissues tested induced suppressor cells although quantitative differences existed between them. Suppressor cell induction was under genetic control by at least two loci, one of which was within the major histocompatibility (MHC) complex. Suppressor cell function was restricted by the MHC type of the responding cell but not the stimulating cell in the second culture.

Animals↗
PubMed1979

Stimulation of lymphocyte proliferation by non-lymphoid porcine tissue cells.

Cultured porcine non-lymphoid cells, characterized by biochemical and morphological criteria, were derived from different tissues of individuals typed by serological and mixed lymphocyte culture methods for gene products of the major histocompatibility complex. These cultured cells have been used as stimulators in mixed lymphocyte-tissue cell cultures in order to investigate (1) the magnitude, kinetics and dose-dependence of lymphocyte transformation caused by tissue cells compared with that caused by lymphocytes as stimulators; (2) the relationship between the expression of serologically detected Ia-like antigens by tissue cells and their ability to cause lymphocyte transformation; (3) the genetic control of stimulation by tissue cells and by lymphocytes and (4) the expression and genetic control of lymphocyte stimulatory properties restricted to tissue cells and absent from lymphocytes. It has been shown that some but not all kinds of tissue cells can stimulate allogeneic lymphocytes strongly and that the characteristics of such stimulation are similar to those observed in mixed lymphocyte cultures. Strong stimulation by tissue cells does not always correlate with the expression of serologically detectable Ia-like antigens, but appears to be controlled by the major histocompatibility complex. There is evidence that certain tissue cells possess lymphocyte stimulatory properties not shared by lymphocytes. Preliminary data suggest that such tissue cell specific stimulation is not controlled by the major histocompatibility complex, though more detailed genetic analysis is required.

Animals↗
PubMed1979

Cyclosporin A: an immunosuppressive agent preferentially active against proliferating T cells.

The in vitro action of the immunosuppressive agent Cyclospirin A has been investigated using porcine cells. Lymphocyte proliferation induced in response to transplantation antigens and phytohaemagglutinin (PHA), was inhibited by this agent at doses that failed to inhibit mitogenisis in response to sheep anti-pig IgM, growth of kidney cell monolayers, and leukocyte migration.

Animals↗
PubMed1978

Modification of lymphocyte responsiveness in vitro by lambda carrageenan compared with colloidal silica and depletion of surface adherent cells.

The effects of the immunosuppressive sulphated polygalactan lambda carrageenan on in vitro models of allograft immunity were compared with the effects of removing macrophages (surface adherent and/or phagocytic cells) by established methods. Carrageenan depressed the primary mixed lymphocyte reactions, but not to the same extent as the removal of macrophages. 2-Mercaptoethanol restored the response. Secondary mixed lymphocyte reactions and responses to phytohaemaglutinin were depressed by carrageenan but not by the removal of macrophages, and in these systems 2-mercaptoethanol failed to restore the responses of carrageenan-treated cultures. In contrast, cell-mediated cytolysis by presensitized lymphocytes was not affected by carrageenan or by colloidal silica. Carrageenan depressed cell-mediated cytolysis only if it was present during the sensitization of the effector cells. We conclude that carrageenan can have two dose-related effects in vitro: one on the macrophage and one on the responding lymphocyte.

Animals↗
PubMed1977

Is there a significant gene dose effect in the primed lymphocyte test?

Pig lymphocytes typed for products of the MHC were primed in vitro against allogeneic lymphocytes. When rechallenged with a panel of different cells, specific 'typing' responses were distinguishable which were not obscured by high non-specific responses or by a restimulator gene dose effect. A gene additive effect was observed in both primary MLR and in the primed lymphocyte test (PLT).

Alleles↗
PubMedDate not supplied

Class-specific effects of selenium on PWM-driven human antibody synthesis in vitro.

The influence of inorganic and organic forms of selenium (Se) on human antibody production was studied in a Pokeweed Mitogen (PWM)-driven in vitro system. Mitogen-stimulated peripheral blood mononuclear cells (PBMC) of eight healthy donors were cultured with different Se compounds at concentrations between 10(-3) and 10(-9) M. At high Se levels (10(-3)-10(-4) M), IgM and IgG production of all donors were strongly inhibited owing to reduced cell viability. However, in five of eight donors, low levels of Se enhanced IgG secretion. This was most effective in the presence of inorganic Se, whereas selenomethionine and selenocystine were less effective. In contrast to IgG, IgM synthesis was significantly reduced by low Se levels in five donors. No significant correlation between donor serum Se levels and antibody production in vitro was found. The addition of low levels of Se to PBMC, stimulated with PHA or PWM, showed no effect on proliferation, whereas a high concentration (5 x 10(-3) M) of sodium selenite and selenocystine suppressed proliferation owing to reduced cell viability. Thus, the present results show that Se supplementation can enhance human antibody production and, moreover, suggest some selectivity of Se action on human immune responses that may result in increased switching from IgM to IgG production.

Antibody Formation↗
PubMedDate not supplied

Prevention of human helper T-cell clone activation by cyclosporin A also blocks secretion of granulocyte/macrophage colony stimulating activity.

Cyclosporin A (CsA) blocks stimulation and growth of alloreactive T helper cell clones (Th-TCC), even in the presence of exogenous Interleukin-2 (IL-2). To examine whether this might reflect a generalised inhibition of cytokine production by these cells, their production of granulocyte/macrophage colony-stimulating factors (GM-CSF), thought not to be involved in the autocrine proliferation of the clones themselves, was investigated. Contrary to the prediction that only pathways relevant to T cell clonal expansion would be blocked by CsA, it was found that this immunosuppressive substance exerted a profound inhibitory activity on GM-CSF secretion, even in the presence of exogenous IL-2. Although a higher concentration of CsA was required to block GM-CSF secretion than to block proliferation, the former was not due to toxic effects on the cells, or permanently switching off the genes for GM-CSF, since upon further cultivation of the clones without CsA, alloantigen-specific responsiveness was restored. These results therefore show that clonal human T helper cell populations are sensitive to inhibition of GM-CSF secretion by CsA. Part of the immunosuppressive mechanism of action of CsA may therefore reside in this activity, since blockade of GM-CSF secretion by T helper cells would influence functional activities of antigen presenting cells.

Clone Cells↗
PubMedDate not supplied

Decreased proliferative capacity and increased susceptibility to activation-induced cell death in late-passage human CD4+ TCR2+ cultured T cell clones.

The growth characteristics in vitro of interleukin 2 (IL 2)-dependent human CD4+ alpha beta-T cell receptor-positive helper T cell clones (TCC) were studied in relation to alterations in surface phenotype, cytokine responsiveness, and susceptibility to activation-induced cell death (AICD). TCC derived from peripheral blood T cells had finite lifespans averaging 33 population doublings (PD) with a recorded maximum lifespan of 80 PD (n = 208). First analyses of the TCC were undertaken at ca. 25 PD, at which time all cells of all TCC expressed high intensity CD45RO and low intensity CD45RA, as well as high intensity CD95 (fas) and MHC class II antigens. The expression of these molecules remained elevated throughout the proliferative lifespan of the clones, but for those TCC which were initially CD28+ (the majority), the density of expression of the latter was diminished in most late-passage clones. Concomitant with this, late-passage cells showed reduced responsiveness to CD28-mediated costimulation by CHO transfectants expressing human CD80 compared to early-passage cells. Additionally, the level of expression of IL 2R gamma c and IL 7R chains was commonly reduced, as was the response to IL 2 and IL 7. Despite unchanged levels of fas expression on TCC with time, late-passage cells were more susceptible to AICD than early, passage cells. These observations further document functional and phenotypic alterations in long-term cultured human T helper cells, which may be considered as biomarkers of immunosenescence. This may contribute to an improved understanding of the mechanisms underlying depressed T cell function in old age.

Animals↗
PubMedDate not supplied

Generation of chronic myelogenous leukemia-specific T cells in cytokine-modified autologous mixed lymphocyte/tumor cell cultures.

Chronic myelogenous leukemia (CML) may be amenable to cell-based adoptive immunotherapy, as suggested by the graft-versus-leukemia effect of bone marrow transplantation and the therapeutic benefit of donor leukocyte infusions. Specific adoptive immunotherapy without bone marrow transplantation might be more effective and less cost-intensive. Professional antigen-presenting cells, the dendritic cells, from patients with CML are derived from the malignant clone and may stimulate antileukemia T-cell responses. Autologous T cells may also be able to recognize tumor antigens on CML cells directly. Here, the authors show that CD4 and CD8 T-cell responses to autologous CML cells can be generated in vitro rapidly and effectively by performing modified autologous mixed lymphocyte/tumor cell cultures (MLTC) in serum-free medium in the presence of cytokines known to support dendritic cell differentiation. MLTC-sensitized T cells secreted large amounts of the type 1 cytokine interferon-gamma, as well as interleukin (IL)-2. However, they also secreted a variety of other cytokines, including the type 2-subtype cytokine IL-13 but not the classic type 2 cytokines IL-4, IL-5, and IL-10. Monoclonal populations of CML-specific CD4 cells could be derived from these lines in limited numbers but showed markedly enhanced reactivity. This suggests that CML-specific T cells are relatively rare in these autologous MTLC-derived sensitized populations, but that their isolation and propagation would yield much more potent antitumor effector cells for use in adoptive immunotherapy without the need for bone marrow transplantation.

CD4-Positive T-Lymphocytes↗