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Biomedical subjects

G Pawelec

Publications and source records attributed to G Pawelec.

At least 163 records · Page 9Linked to original sources
PubMed1987

Generation of CD4-positive suppressor T cells from mixed lymphocyte cultures in the presence of interleukin 2 receptor antibody TU69. An in vitro model for transplantation tolerance induction.

The specificity of a novel monoclonal antibody (moAB), TU69, directed to the interleukin 2 receptor (IL-2R) was verified by sequential immunoprecipitation with anti-Tac. TU69 cross-competed with anti-Tac in binding analyses. When TU69 was added during the sensitization of normal peripheral blood mononuclear cells (PBMC) to allogeneic HLA-class I or -class II mismatched stimulator PBMC, alloproliferative responses and specific cytotoxicity were no longer detectable and the generation of natural killer (NK)-like effector cells was partially inhibited. Remarkably, however, the generation of CD4+ nonspecific suppressor T cells in such mixed lymphocyte cultures (MLC) was not inhibited--but, in contrast, was strongly enhanced in the presence of TU69. These suppressor cells inhibited unrelated allospecific responses in vitro to background levels even at a ratio of 50:1 responder:irradiated suppressor T cell lines. Such a potent experimental suppressor system suggests a possible application of TU69 for in vivo tolerance induction after transplantation, by down-regulating allospecific effector cells and allowing the generation of tolerance to graft antigens.

Antibodies, Monoclonal↗
PubMed1987

Constitutive functional heterogeneity without detectable somatic mutation of antigen receptor genes in helper T cell clones: possible regulation by novel HLA class II "DY" determinants.

The majority of interleukin 2-dependent HLA class II alloreactive human CD4-positive helper T-cell clones (TCC) presented the phenomenon of constitutive intraclonal heterogeneity, as reflected by limiting dilution analysis of changing frequencies of autonomously proliferative cells within the monoclonal population. Moreover, at 30-35 population doublings, these TCC lost their allospecific proliferative and helper capacity (phase I) and instead acquired strong antigen-non-specific suppressive activity (phase II). The TCR was still expressed at the same level on the cell surface. As shown by stable and identical rearrangement profiles of their T-cell receptor beta- and gamma-chain genes in both phases I and II, this constitutive change of function was probably not due to somatic mutation of the genes coding for the antigen-specific receptor. Moreover, antigen specific reactivity was retained in phase II TCC by the criterion of specifically stimulated secretion of granulocyte/macrophage colony stimulating factor. The preprogrammed intra-clonal functional flexibility is explained here by invoking a novel regulatory mechanism mediated by a fourth group of HLA class II determinants. When TCC were used as stimulators with the appropriate primed responders, it could be demonstrated that phase I autonomously proliferative non-suppressive T-helper lines failed to express certain novel lymphocyte activating determinants (LADs), whereas after their acquisition of suppressive function (phase II) these LADs were present. These stimulatory moieties appeared to represent a novel class II-like structure as established by serology, immunochemistry and functional characterization employing monoclonal antibodies to block stimulation. These operationally designated "DY" determinants are primarily and thus far exclusively involved in the induction of antigen non-specific suppressor cells. It is proposed that the intra-clonal changes of T-cell function described here are mediated by a switch in the utilization of qualitatively distinct class II-like restriction elements. The functional status of the phase I TCC can be altered by a switching over to the engagement of "DY" determinants in phase II. Thus, an autostimulatory and self-maintaining suppressive network may operate in extended inductive phases of human HLA restricted T-cell responses.

Cell Membrane↗
PubMed1986

Modulation of in vitro myelopoiesis by alloreactive T cell clones.

Regulatory effects of mixed lymphocyte culture (MLC)-derived CD4+ human T cell clones on granulocyte-macrophage colony (CFU-GM) formation by normal bone marrow (BM) were studied in an initial attempt to establish an in vitro model for the negative feedback control of myelopoiesis by alloactivated T cells. This is likely to be of clinical significance in the aberrant control of haematopoiesis during some cases of graft-versus-host disease (GVHD) after allogeneic BM transplantation. Whilst 5 such alloproliferative clones generally failed to suppress CFU-GM, the majority of clones with natural killer (NK)-like activity, or those with suppressive activity in MLC, regularly and strongly suppressed in this system, reinforcing the view that certain T cells may have potent negative regulatory effects on haematopoiesis.

Bone Marrow Cells↗
PubMed1986

Dissection of suppressor cell generation in vitro.

Suppressor cells (SC) that nonspecifically inhibited lymphoproliferative (LP) responses were found after culturing peripheral blood mononuclear cells: with suppressor T-cell clones, in mixed lymphocyte cultures (MLC), and with recombinant interleukin 2 (IL-2), but were not found after culture in medium alone. A monoclonal anti-IL-2 receptor (R) antibody (MoAb), TU69, which blocked LP responses of IL 2-dependent T-cell lines, also blocked SC induction by T-cell clones, but completely failed to inhibit SC generation in MLC or with IL-2. This suggests that the IL-2R epitope defined by TU69 was not involved in SC induction in the latter systems. MoAb against HLA-DQ (TU22), -DR (TU34, SG157), -DP (B7/21), or DR and DP (TU43, 58), all of which were able to block stimulation of appropriately specific clones, did not block SC induction in any of the three systems studied. In contrast, the broadly reactive moAb TU39, which binds at least DR and DP but also has additional reactivity for determinants tentatively designated "DY," blocked SC induction by T-cell clones and in MLC. Finally, an anti-HLA class I MoAb, W6/32.HL, greatly decreased SC generation in MLC, but not with rIL-2 or T-cell clones. Thus, the induction of nonspecific SC was dissected into three pathways involving: class I and TU39-defined but not DR, DQ, or DP determinants (in MLC) which was independent of the IL-2R epitope bound by TU69; only TU39-defined determinants (with T-cell clones), which were IL 2R dependent; and, neither class I, class II nor TU39-defined determinants (induction by rIL-2), which was also TU69+ IL-2R independent.

Antibodies, Monoclonal↗
PubMed1986

Gamma ray-induced mutants as a tool for the production and characterisation of monoclonal antibodies against HLA-alloantigens.

To simplify the screening procedure for murine monoclonal antibodies specific for polymorphic HLA determinants, spleen cells from a mouse immunized with the human cell line BJAB-B95.8.6 were fused with NS1 mouse myeloma cells, and hybridoma supernatants were screened for their reactivity on BJAB-B95.8.6 and two gamma ray-induced HLA-loss mutants of this line. The use of these HLA-loss mutants allowed the rapid identification of two new allospecific MOABs designated TU160 and TU161. Serological as well as biochemical studies revealed TU160 to be specific for HLA-A2, and TU161 for HLA-B13 molecules, respectively. Both MOABs were determined to be antibodies of the IgG class and were able to precipitate their antigens from lysates of radioactively labeled cells.

Animals↗
PubMed1986

Acquisition of suppressive activity and natural killer-like cytotoxicity by human alloproliferative "helper" T cell clones.

To investigate potential functional changes in alloantigen-specific proliferative CD3+, CD4+, CD8-, Leu-8-, interleukin 2 (IL 2)-secreting noncytotoxic in vitro primed human helper T cells, a set of 12 clones was studied sequentially throughout their finite life spans. Clones surviving to greater than 30 population doublings (PD) retained their requirements for exogenous IL 2 and filler cells for continued growth, but lost the ability to proliferate and to secrete IL 2 when specifically restimulated. This was not accompanied by changed surface marker phenotypes or acquisition of abnormal karyotypes, but was accompanied by the acquisition of MHC-unrestricted potent radioresistant suppressive activity for lympho-proliferative responses. Suppression was not caused by absorption of IL 2, secretion of interferons, de novo mycoplasma contamination, or cytotoxic activity. At least two suppressive mechanisms were demonstrated: 1) the induction of suppressor effectors in naive lymphocyte populations, which required cell to cell contact and could be inhibited by certain monoclonal antibodies against MHC class II determinants; and 2) a direct effect on responding lymphocytes, shown by suppressive activity on cloned PLT-active reagents. Moreover, the majority (75%) of originally allo-proliferative clones also acquired a previously absent cytotoxicity against natural killer (NK) cell-susceptible, but not NK-resistant, target cell lines. This modulation of function from specific alloproliferative, IL 2-secreting nonsuppressive status to strong nonspecific suppressive and NK-like cytotoxic status represents a novel functional activity of human T helper lymphocytes under conditions of clonal propagation.

Antigens, Surface↗
PubMed1985

Heterogeneity of human natural killer recognition demonstrated by cloned effector cells and differential blocking of cytotoxicity with monoclonal antibodies.

Monoclonal antibodies (MoAb) against cell surface determinants were employed to investigate the specificity of natural killer (NK)-like lysis by cloned human effector cells recognizing only K562, only HSB2, or both K562 and HSB2 target cells. MoAb W6/32.HL, TU39, YD1/48.HLK, and anti-Tac failed to inhibit lysis despite the expression of antigens bound by these MoAb on the effector cell surface. MoAb OKT3 moderately (less than or equal to 50%) blocked lysis of K562 and HSB2 targets, whereas MoAb 13.1, which binds T200 molecules, strongly (up to 95%) blocked lysis of K562, but not HSB2, targets. MoAb 13.1 inhibited lysis by clones which killed only K562, as well as lysis by those which killed both HSB2 and K562. In the latter case, however, only lysis of K562 was inhibited. Taken together, these results may suggest the existence of multiple receptor specificities on a single NK-active clone.

Antibodies, Monoclonal↗
PubMed1985

HLA-DR-, MB- and novel DC-related determinants restrict purified protein derivative of tuberculin (PPD)-stimulated human T cell proliferation.

Class II major histocompatibility complex determinants restricting recognition of tuberculin antigens (purified protein derivative; PPD) were studied by using monoclonal antibodies (mAb) to block lymphoproliferative responses. Anti-class II mAb were shown to exert inhibitory effects at the level of the antigen-presenting cells, without inducing suppressive lymphocytes or macrophages. Using panels of HLA-typed antigen-presenting cells and nonalloreactive proliferative T cell lines, derived by limiting dilution, restriction elements for PPD responses appeared to correlate with the donor's HLA-DRw6 specificity (one clone), MB1 (one clone), MB3 (one clone), or no established class II (or class I) specificity (three clones). mAb TU22, reacting with nonpolymorphic DC-like determinants, strongly inhibited stimulation of all clones except that restricted by DR antigens, suggesting the DC-like character not only of the MB1- and MB3-associated, but also of the unassigned, restriction elements of these cloned lines. In contrast, stimulation of the DR-restricted line was strongly inhibited by DR/SB-specific mAb which only weakly inhibited the stimulation of clones restricted by DC-like determinants. These results suggest that clonally distributed PPD-reactive proliferative lymphocytes from a single donor may be restricted by at least three different class II determinants (HLA-DR, MB, or a second, novel, DC-related molecule).

Antibodies, Monoclonal↗
PubMed1985

Function of alloproliferative T lymphocyte clones correlates better with their class II recognitive specificity than with their cell surface phenotype.

Alloproliferative primed lymphocyte typing (PLT) clones recognizing determinants associated with HLA-DR/Dw, SB, MB, or novel "SB-like" gene products were screened for their ability to suppress lymphoproliferative responses in primary and secondary mixed lymphocyte cultures (MLC), and for their surface marker phenotypes. Two nonsuppressive HLA-D specific PLT clones were OKT4-, OKT8+ whereas all others possessed an OKT4+, OKT8-, Leu-8- phenotype. All clones secreted interleukin 2 (IL2) after specific stimulation. The eight PLT clones specific for "SB-like" antigens strongly suppressed MLC, whereas only one of 35 DR/Dw-specific, and none of 20 SB-specific PLT clones did so. Suppressive activity of such PLT clones was not restricted by major histocompatibility complex products, was radioresistant (20 Gy), and was not caused by absorption of IL2 or by cytotoxicity of the cloned cells. Suppressive clones exerted their effects directly on proliferating T cells, as assessed by their ability to prevent growth of cloned PLT cells stimulated by B cell lines, and their ability to block primary MLC even when added 96 h after the start of the 144-h culture. Culture supernatants from suppressive, but not from nonsuppressive, PLT clones also strongly and nonspecifically inhibited lymphoproliferative responses. The suppressive factor(s) was not dialyzable, not sensitive to pH 2 or heat treatment and not cytotoxic. Thus, all T cell clones proliferating against novel "SB-like" but not SB antigens, as well as rare clones specific for D region determinants, possess powerful nonspecific suppressive activities dissociated from their "helper-related" OKT4+, OKT8-, Leu 8-, IL2-secreting phenotypes.

Antigens, Surface↗
PubMed1985

Refinement of HLA gene mapping with induced B-cell line mutants.

The lymphoma cell line BJAB.B95.8.6 was gamma-irradiated to induce mutations of major histocompatibility complex (MHC) encoded genes. Cloned "wild-type" cells were phenotyped HLA-A1, A2, B13, B35, Bw4, Bw6, Cw4, DR5, DRw52, DQw1, DQw3, DPw2, DPw4, GLO1 1, PGM3 2-1, and ME1 0 and possessed two apparently normal chromosome 6s prior to mutagenesis. Loss mutants were selected 5 days after 3 Gy gamma-irradiation employing three complement-fixing monoclonal antibodies specific for HLA-A2 (TU101) and Bw4 (TU48, TU109). Fifteen independently arising mutants were isolated and cloned. Typing with monospecific alloantisera and cell-mediated lympholysis revealed the presence of HLA-A1, B35, Bw6, Cw4, DR5, DRw52, DQw3, and DPw4 specificities on all mutant clones. HLA-A2, B13, and Bw4 were absent. Mutants differed in their expression of class II antigens. One group retained DQw1 and DPw2, another was DQw1-, DPw2+, and a third was DQw1-, DPw2-. Karyotyping of the "wild-type" line and selected mutant clones showed that the loss of HLA specificities correlated with deletions which map the HLA-A and -B loci directly to the distal part of the 6p21.33 region and the class II genes to the region 6p21.33 (proximal) to 6p21.31 (distal) on the short arm of chromosome 6.

Antibodies, Monoclonal↗
PubMed1985

Homozygous typing cell-defined HLA-Dw specificities correlate better than serologically defined HLA-DR specificities with restriction elements for influenza virus-specific proliferative human T lymphocyte clones.

Human T lymphocyte clones with specific proliferative response to influenza A virus were derived by limiting dilution from peripheral blood lymphocytes (PBL) after in vitro stimulation with autologous irradiated, virus-infected PBL. Four OKT3+4+8- T lymphocyte clones (TLC) that showed HLA-restricted antigen-specific proliferative responses were used for a detailed analysis of the restriction elements for antigen presentation. None of the clones showed alloreactivity and all required the presence on the antigen-presenting cell of HLA class II antigens of one or other haplotype of the donor. Restriction elements for two clones were correlated with Dw1 rather than DR1, and for two others with Dw6 rather than DRw6. These latter clones showed differential recognition of HLA-Dw6 subtypes as defined tentatively by homozygous typing cells, without relationship to putative serological "splits" of DRw6. One of the Dw6-restricted clones was specific for a Dw6.1 (now Dw18) "subtype," confirmed by family segregation analysis, the other for a broad Dw6 (Dw18 and Dw19) specificity. Studies with a panel of monoclonal antibodies against monomorphic determinants of HLA class II antigens revealed heterogeneous patterns of blocking activity, distinguishing between clones of different restriction specificity. Inhibition patterns were partly as predictable from the known activity of the monoclonal antibody in alloantigeneic PLT systems. These results provide evidence that certain structures that function as restriction elements for antigen presentation also carry alloantigeneic determinants.

Antibodies, Monoclonal↗