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Biomedical subjects

G Pawelec

Publications and source records attributed to G Pawelec.

At least 181 records · Page 10Linked to original sources
PubMed1985

Dissection of human allostimulatory determinants with cloned T cells: stimulation inhibition by monoclonal antibodies TU22, 34, 35, 36, 37, 39, 43, and 58 against distinct human MHC class II molecules.

Alloantigenic determinants causing secondary lymphoproliferative responses of primed T cells were investigated by blocking stimulation with monoclonal antibodies TU22, 34, 35, 36, 37, 39, 43, and 58 binding differentially to HLA-DR and SB or MB associated molecules. In particular, the use of cloned and functionally defined T cell responders greatly facilitated the assignment of stimulator-level inhibition caused by these antibodies. Thus, a novel way of functional "mapping" for stimulatory epitopes for sets of clones with restimulation specificities associated with HLA-D, SB, MB, or hitherto unidentified class II determinants is presented here. This considerably helps to elucidate the distinct immunoregulatory roles of the three major class II alloantigen systems thus far defined.

Antibodies, Monoclonal↗
PubMed1985

Functionally distinct human T-lymphocyte clones sharing potent suppressive activity on immunoglobulin secretion.

T-lymphocyte clones derived from populations sensitized to alloantigens in vitro were tested for their regulatory effects on pokeweed mitogen-stimulated immunoglobulin (Ig) secretion. Clones with natural killer (NK)-like cytotoxicity and/or suppressive activity for lymphoproliferative (LP) responses potently inhibited Ig secretion. Moreover, certain alloproliferative T4+ interleukin-2 (IL-2)-secreting 'helper' clones shared this strong suppressive activity on Ig secretion. The remaining clones enhanced, rather than suppressed, Ig production. Inhibition by all types of suppressive clones appeared not to be restricted by MHC products, since allogeneic HLA-mismatched donors were suppressed as efficiently as the autologous donor. Suppression was radioresistant, and was apparently not caused by absorption of IL-2, or cytotoxicity of the clones. Suppression was still detectable at plateau levels when cloned cells were added as late as 96 hr after the initiation of the cultures, suggesting an inhibitory mechanism divorced from early B-cell activation events. Thus, T-lymphocyte clones with distinct different functional activities share similar profound suppressive effects on Ig secretion in vitro.

Clone Cells↗
PubMed1984

Cloned human T lymphocytes with lymphostimulatory capacity preferentially activate suppressor cells.

A proportion of cloned T cells derived from allogeneic mixed leukocyte cultures (MLC) was found to stimulate rapid primary, but not secondary, lymphoproliferative responses of autologous as well as allogeneic peripheral blood mononuclear cells (PBMC). Of eight major histocompatibility complex (MHC) class II-specific monoclonal antibodies (mAb), the reagent TU39 (which preferentially inhibits allostimulation by SB- rather than DR- or DC-associated determinants) most strongly inhibited stimulation by these clones. MAb specific for DC or DR molecules inhibited weakly or not at all. Stimulatory, but not nonstimulatory, clones were found to be strongly suppressive when titrated directly into MLC. Suppression was no MHC restricted, was radioresistant (20 Gy) and was not abrogated by the addition of partially purified interleukin 2 to the test cultures. Transfer of PBMC cocultured with stimulatory, but not with nonstimulatory, clones into a second MLC resulted in its strong suppression, suggesting that a suppressor effector population had been "induced" by the clones. These results are consistent with the hypothesis that SB-like, rather than DR or DC, determinants present on the surface of certain activated T cells are intimately involved in the regulation of cellular immune responses by rapidly inducing suppressor effector cells in normal lymphocyte populations.

Antibodies, Monoclonal↗
PubMed1984

Strong lymphoproliferative suppressive function of PLT clones specific for SB-like antigens.

From a total of 37 different priming combinations between donors matched for HLA-A,B, and/or Dw/DR, but mismatched for SB, antigens, T cell clones strongly restimulated with concordance for SB specificities were isolated from only two. Most of the alloproliferative (PLT) clones obtained were restimulated by determinants not correlated with any currently known HLA product. Nonetheless, their stimulation was inhibited by a monoclonal antibody TU 39, which preferentially blocks stimulation by SB-, rather than by Dw/DR-associated determinants. Despite having an OKT4+, OKT-, Leu8- phenotype, and secreting Interleukin-2 after contact with stimulatory cells, these clones strongly suppressed proliferative responses of cloned PLT reagents as well as unprimed lymphocytes in mixed leukocyte cultures. They may thus represent a novel type of immunoregulatory T cell, stimulated by SB-related antigens, which despite their "helper/inducer" phenotype are able directly to suppress lymphoproliferative responses.

Antibodies, Monoclonal↗
PubMed1984

Alloproliferative human T cell clones primed and cultured in vitro lose proliferative and gain suppressive activity with age.

Alloreactive human T lymphocyte clones were found to lose antigen-stimulated proliferative capacity and their ability to secrete interleukin 2 (IL 2) after a critical period in tissue culture. Instead, they gained the previously absent capacity to suppress lymphoproliferative (LP) responses in the presence or absence of exogenous IL 2. Such "ex-PLT" suppressive clones continued to grow perfectly well, retaining IL 2 and filler cell dependency, apparently normal karyotypes, and their OKT4+, OKT8- phenotypes. At least two suppressive mechanisms were demonstrated: (1) the "induction" of suppressive effectors in normal peripheral lymphocytes, and (2) a direct suppressive activity on lymphocyte proliferation shown by their ability to inhibit restimulation of cloned lymphocytes lacking suppressor cell precursors. The consistent "differentiation" from IL 2-secreting "helper" status to nonspecific suppressive status may represent a novel immunoregulatory phase in the long-term differentiation of normal human T cells.

Cell Differentiation↗
PubMed1984

The new HLA-DRw6- and 8- associated HLA-Dw HAG specificity defined by homozygous typing cell 9W 1802. Analysis with primed lymphocyte typing clones.

Intrafamilial primary mixed lymphocyte culture (MLC) typing established that an HLA-A, B, C homozygous, DP heterozygous donor HAG was homozygous for HLA-Dw and behaved as a homozygous typing cell (HTC). Both haplotypes of the HTC were HLA-DR identical, but could not be assigned a clear DR specificity, giving reactions with sera containing antibodies against DRw6, DRw8 and TA10. MLC checkerboard studies failed to assign the HTC HAG specificity to any established or provisional cluster, suggesting that it defined a new Dw specificity. Primed lymphocyte typing (PLT) clones derived from intra-familial priming against either HAG haplotype displayed heterogeneous reactivity patterns. One clone was restimulated only by family members and unrelated donors positive for Dw HAG. Other clones were restimulated by determinants associated with either Dw8 or Dw6. Blocking of stimulation with monoclonal antibodies against different class II molecules suggested that while stimulatory determinants associated with Dw HAG and Dw8 were classifiable as HLA-D related, those associated with Dw6 were of a DP-like nature.

Antibodies, Monoclonal↗
PubMed1983

Homogeneity of the HLA-linked SB2 and SB3 specificities demonstrated by cloned alloreactive T cells.

The HLA-linked "SB" antigens comprise a new segregant series of B-cell alloantigens mapping between HLA-DR and glyoxylase. They can be detected by secondary proliferative responses of lymphocytes primed against HLA-A, B, C, DR, MB- and MT-compatible stimulators. To asses genetic complexity of the SB-gene region, alloreactive cloned T-cell lines were derived from four reagents detecting specificities designated SB2 and SB3. In two families, products detected by seven different clones segregated with the HLA haplotypes bearing the SB2 or SB3 specificities as recognized by the uncloned reagents. There were no indications that the cloned cells differed from the oligoclonal reagents in their fine specificity. In contrast to previous results with an SB4-associated specificity, in population studies of 25 SB2-positive and 23 SB3-positive donors, no evidence could be found for subtypes of either specificity. Thus, even at the level of recognition by cloned T-cells, both SB2 and SB3 appear to be remarkably homogeneous in the population.

Clone Cells↗
PubMed1983

Cyclosporin A inhibits interleukin 2-dependent growth of alloactivated cloned human T-lymphocytes.

The effect of cyclosporin A (CsA) on the growth of mixed leukocyte culture-derived human cloned T-cells with different functions has been studied. Interleukin 2-dependent clones with allospecific cytotoxic, natural killer-like cytotoxic, lymphoproliferation suppressive, or primed lymphocyte typing function, were used as test cells in short term (36 h) tritiated thymidine incorporation assays. CsA inhibited growth of these cells in a dose-dependent manner, and the lowest concentration of CsA tested (0.1 microgram/ml) was already inhibitory for the majority of clones. Differences between clones in their susceptibility to CsA as measured in this assay did not correlate with their defined functions. Long term (2-3 week) growth of cloned cells cultured in the presence of CsA was also depressed in a dose-dependent manner; moreover, in the case of a suppressor cell clone, as well as a natural killer-like cytotoxic, and a primed lymphocyte typing clone, function of cells surviving after preculture with CsA was suppressed too. It may be important that NK-like effector function and growth of an NK-like T-cell clone showed more resistance to the inhibitory action of CsA than a PLT clone. These data show that previously alloactivated continuously proliferating Interleukin 2-dependent human T-cell clones are fully susceptible to CsA-mediated inhibition of their function as well as their proliferation. Thus, CsA can exert a strong immunosuppressive effect not only by preventing initial activation of T-cells.

Cell Division↗
PubMed1983

Age-related loss of function of alloactivated interleukin 2-propagated human primed lymphocyte typing clones.

Human lymphocytes alloactivated in vitro were cloned by limiting dilution in the presence of filler cells and interleukin 2 (IL 2)-containing supernatants of phytohaemagglutinin-stimulated lymphocytes. Clones with allospecific proliferative reactivity (PLT clones), measured by tritiated thymidine (3H-TdR) incorporation, were selected for extensive IL 2-dependent expansion. The cloned lines had finite lifespans, ranging from an estimated minimum of 28 to greater than 65 doublings. Function as PLT reagents, however, was retained in all cases for only an estimated 30 cell doublings. This apparent cessation of function was not caused by loss of the ability to metabolize thymidine, since lines continuing to grow for greater than 30 doublings still incorporated 3H-TdR in the presence of IL 2. An altered requirement for stimulating antigen (number of stimulating cells), or altered response kinetics, did not contribute to loss of PLT function. Exogenous IL 2 added during restimulation to responders previously 'rested' overnight without IL 2 did not restore the response. Thus, under present experimental conditions, functional lifespans of cloned PLT reagents appear fixed at approximately 30 cell doublings.

Cell Survival↗
PubMed1982

Constitutive interleukin 2 production by the JURKAT human leukemic T cell line.

Interleukin 2 (IL2) is a lymphokine produced from phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells and characterized biologically by its ability to maintain the in vitro proliferation of activated T cells. In a search for a convenient alternative source of biologically active human IL2, cells from the five established T cell lines, MOLT4, HSB2, CCRF-CEM, RPMI1301 and JURKAT were cultured at high concentrations for 18-36 h (induction cultures), and their cell-free supernatants thereafter screened on IL2-dependent cultured human and mouse T cells. MOLT4, HSB2, RPMI1301, and CCRF-CEM all failed to produce detectable levels of IL2. Of the three JURKAT cell lines obtained from different sources, one, designated JMN, produced high levels of IL2 activity. A second, JM, failed to produce any IL2, while the third, JHAN, produced intermediate levels. Stimulation of the IL2-producing JMN or JHAN variants with PHA, the phorbol diester 12-0-tetradecanoyl-phorbol-13-acetate (TPA), or both PHA and TPA together, resulted in an apparent increase of IL2 activity in the culture supernatant when assayed by a short-term tritiated thymidine incorporation test. However, both PHA and TPA added directly to the test cells caused substantial thymidine incorporation. Moreover, the nonproducer line JURKAT-JM could not be converted to an IL2 producer by stimulation with PHA, TPA, or both. When JMN supernatants were used to support actual long-term growth and cloning of T cells in limiting dilution, the constitutively produced IL2 was superior to that produced after PHA and/or TPA stimulation. Addition of TPA, but not of PHA, to lectin and TPA-free JMN IL2 resulted in a decreased ability of such supernatants to support clonal T cell growth, suggesting that TPA had a growth-inhibiting effect. These results show that the continuously growing JURKAT-JMN cell line could provide a suitable source of mitogen-free human IL2.

Animals↗
PubMed1982

Alloactivated long-term cultured human T lymphocytes express both HLA-DR and SB antigens but lack lymphocyte stimulation capacity.

Cell populations obtained from mixed leukocyte cultures of 6- or 10-day duration were found specifically to restimulate primed lymphocytes detecting HLA-linked SB as well as HLA-D-associated antigens. After expansion in vitro (9-75 days) with medium containing interleukin 2, the cultured cells expressed the T lymphocyte markers detected in indirect immunofluorescence by monoclonal antibodies Lyt-3, OKT3, OKT4, OKT8, and had high levels of HLA-DR antigens. In addition, they were shown in cell-mediated lymphocytotoxicity specifically to express SB antigens of the donor B cell type. Despite their positivity for DR and SB antigens, such cultured T cells failed to restimulate either SB- or D-specific secondary lymphocyte proliferation. Homogeneous cloned populations of cultured T cells also lacked lymphocyte stimulation capacity. In contrast, B cell lines, which also expressed DR and SB antigens, were potent stimulators of both SB- or D-directed proliferation. These data show that the activated T lymphocytes which express both HLA-DR and SB antigens are by themselves unable to stimulate lymphocyte proliferation.

Cells, Cultured↗
PubMed1982

Immunohistology of graft-versus-host disease mediated skin lesions and its correlation to a large granular lymphocyte surface phenotype and function.

Skin lesions of five patients presenting with acute and chronic Gvhd after bone marrow transplantation were analyzed on frozen tissue sections using selected monoclonal antibodies against various T-cell determinants and HLA-antigens in order to define immunological phenomena characteristic for cutaneous damage seen in various GvHD states. Four of these patients showed marked increase of a certain T-cell subpopulation positive for HLA-D region products in the upper dermis. A considerable number of these T-cells seemed to show cytotoxic reactivity on the basal cell layers of the epithelium and to correlate with the appearance of an OKT4-, OKT8+, HLA-DR+ T-cell subset in the peripheral blood presenting natural killer (NK) cell like activity on various targets. Ia antigen expression on keratinocytes observed in one patient with chronic GvHD could result from a rapid and irregular turnover of epidermal cells affected by continuous stimuli of these T-cells. Further immunological studies on skin biopsies of patients with different states of GvHD and autoimmune diseases may lead to valuable diagnostic criteria for an early and accurate assessment of various skin lesions in patients after bone marrow transplantation.

Bone Marrow Transplantation↗
PubMed1982

Cloned human T-cell lines with allospecific or natural killer-like cytotoxicity.

Cloned human T-cell lines were produced by limiting dilution of lymphocytes alloactivated in mixed leukocyte cultures, followed by expansion of clonal progeny with interleukin 2. Selected clones were analyzed for cell-mediated cytotoxicity (CTX) against a variety of targets susceptible or resistant to lytic attack by natural killer (NK) cells. Clones with classical alloantigen-restricted lytic capacity for normal lymphoid targets were found to be distinct from those mediating natural killer-like CTX against NK-susceptible cell line targets. This was established by direct CTX assays and by cold target cross-competition experiments. Moreover, clones with NK-like activity displayed heterogeneous patterns of lysis on different target cell lines in direct CTX, suggesting a clonal distribution of receptors for NK-like target antigens amongst these cultured human NK-active T-cell clones.

Clone Cells↗
PubMed1982

Population studies of HLA-linked SB antigens and their relative importance in primary MLC typing. Analysis of HLA-D homozygous typing cells and normal heterozygous populations.

SB phenotyping was undertaken on 96 HLA-D homozygous typing cells (HTCs) and 129 normal unselected heterozygous donors in the German population, using Interleukin-2-propagated primed lymphocyte typing (PLT) reagents. The results showed that the SB antigens in the normal population behave as a system of alleles at a single locus in Hardy-Weinberg equilibrium (p approximately equal to 0.20). Estimated gene frequencies in the German population appeared to be significantly different (p less than 0.002) from the North American Caucasian population: the principal differences were increased frequencies of the specificities SB1 and SB4, and decreased frequencies of blanks. Of HLA heterozygous donors 41% typed for two distinct SB specificities; 57% typed for one; and 2% were blank. In the HTC group, 20% typed for two specificities; 68% typed for one; and 12% were blank. Thus, a significant proportion of HLA-D homozygous test cells were, nonetheless, heterozygous for HLA-linked SB antigens. Performance of checkerboard mixed leukocyte cultures (MLCs) between 16 SB typed HLA-Dw3 HTCs, however, did not indicate that the observed mutual or one-way responses were influenced in any simple way by SB antigens; neither heterozygosity nor assumed homozygosity for SB antigens appeared to influence the frequency of MLC typing responses of HLA-Dw3-positive donors on these HTCs. These results add further confirmation of the genetic and functional independence of the SB gene product(s) and the HLA-D/DR gene product(s).

Genes, MHC Class II↗
PubMed1982

Discrete PLT clones detecting HLA-D and new HLA-non-D encoded cell surface determinants.

Clones of alloreactive T-lymphocytes were isolated from 6-day-old mixed leukocyte cultures (MLCs) by limiting dilution in the presence of filler cells (either pooled irradiated peripheral blood mononuclear cells (PBMCx) or autologous PBMCx) followed by expansion of clonal progeny with Interleukin 2 (IL 2). Using allogeneic IL 2, produced by stimulating pooled PBMC with phytohaemagglutinin (PHA), in conjunction with pooled filler cells for the limiting dilution cloning, over half the clones obtained had primed lymphocyte typing (PLT) function. The majority of these discriminated priming cell HLA-D type with fine specificity. However, clones which were restimulated by cells of specificities other than the priming HLA-D type were also found, and, moreover, some ("wild" clones) were not restimulated by the original priming cells. Antigens responsible for restimulating these clones included hitherto undetected HLA-encoded products (different from HLA-A, B, C, D, DR, MT, MB or SB). Such "wild" clones were also obtained after cloning procedures in which autologous filler cells were used. Autologous IL 2, depleted of mitogenic PHA, and used in conjunction with autologous filler cells, also failed to prevent generation of such clones. These results indicate that although the PLT-functional lymphocyte population alloactivated by a single HLA-DR/Dw specificity expressed by HLA-D homozygous stimulating cells consists of a majority of clones exclusively recognising specific priming-type HLA-DR/Dw products, it also contains clones recognising different HLA-DR/Dw specificities, as well as clones recognising lymphocyte stimulating determinants hitherto undetected by serological or cellular methods.

Clone Cells↗