Biomedical subjects
Publications and source records attributed to G Pawelec.
The characteristics of natural killer (NK) cells and their role in immunosurveillance of cancer is reviewed. Evidence derived from in vitro and in vivo experiments is in favor of an important role of NK cells in host resistance to leukemia, as illustrated by studies of untreated patients, patients treated with biological response modifiers and by bone marrow transplantation.
A majority (42/62) of TCR2+ interleukin 2-dependent human T lymphocyte clones was found concordantly to express not only the CD28 co-receptor structure at the cell surface but also its ligand B7. Interactions between CD28 and B7 can have important consequences for T cell activation, particularly in providing "signal 2" to prevent the induction of anergy caused by stimulation via the antigen receptor only ("signal 1"). However, despite the expression of co-receptor and ligand on the same cell surface, it remained possible to induce hyporesponsiveness in T cell clones either when using CD3 antibodies to deliver signal 1 or when using other T cell clones as stimulators. Thus, the potential for intraclonal autostimulation via CD28/B7 is apparently insufficient to prevent downregulation of responsiveness in these two systems.
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Cytokine-dependent AML cell proliferation was investigated in 16 patients. Dipyridamole and R-verapamil caused a dose-dependent inhibition of AML cell proliferation, and for both drugs the degree of inhibition was similar when testing various haematopoietic growth factors or growth factor combinations (IL3, G-CSF, GM-CSF, G-CSF + GM-CSF, TNF-alpha + GM-CSF). TNF-alpha alone increased AML cell proliferation for five patients, whereas four patients showed unaltered or decreased proliferation. Independent of this TNF-alpha effect, R-verapamil inhibited proliferation for all AML patients in the presence of TNF-alpha, whereas dipyridamole caused only a weak inhibition.
Because Cyclosporine A (CsA) is often reported to inhibit only the proliferation of quiescent T-cells and to have much less or no effect on activated proliferating T-cells, the effects of CsA and FK506 on long-term in vitro T-cell proliferation were investigated for alloreactive T-cell clones and clones derived from a leukaemia patient early after allogeneic bone marrow transplantation. Drug effects were tested in the presence of exogenous IL2 or IL2 + IL4 during weekly T-cell activation/restimulation. In the presence of either drug, cloned T-cells were able to respond to a reduced number of restimulations (mitogenic activation, allostimulation) and could undergo fewer cell divisions/population doublings. These effects were seen in the presence of both excess IL2 or IL2 + IL4.
We designed a primer for the PCR directed against a highly conserved sequence of the TCR V beta gene. The V beta-universal primer, in combination with a constant region-specific primer, enabled us to amplify TCR beta cDNA of allo-HLA class-II-reactive T-cell clones by PCR without prior knowledge of their V beta sequences. The amplified TCR cDNA was purified by agarose gel electrophoresis and subjected to direct sequencing. In nine of ten T-cell clones analyzed, direct TCR sequencing gave readable sequence ladders, including two-thirds of V beta, junctional, and J beta regions. One T-cell clone gave an unreadable mixed-profile sequence ladder, indicating that this clone expressed more than one major TCR beta transcript. Even in this case, however, it was possible to determine two different TCR beta sequences separately using sequence primers specific to one of the 13 J beta segments deduced from the mixed ladder. Thus, direct sequencing utilizing the single V beta-universal primer enabled a simple, rapid, and reliable sequence determination of TCR beta cDNA of all T-cell clones analyzed.
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The clinical course of a 56-year-old female patient with Sweet's syndrome (SS) preceded by a myelodysplastic syndrome (MDS) is described. During the acute phase of the disease with high remittent fever, painful skin lesions and maximal leucocytosis IL-6 and G-CSF serum levels were extremely high, while TNF-alpha was only slightly elevated and gamma-interferon and IL1-beta were not increased. On clinical improvement IL-6 serum levels rapidly fell, whereas G-CSF values already slightly elevated before the manifestation of the disease slowly declined. High G-CSF levels triggered by a yet unknown factor could explain the leucocytosis, neutrophilic dermatosis and skin lesions in SS, while IL-6 probably induced the associated clinical symptoms of fever and pain.
Secretion of different cytokines may be an important T-cell effector mechanism for bone marrow engraftment, graft versus host disease and graft versus leukaemia effects after allogeneic bone marrow transplantation (BMT). Cytokine secretion and autocrine proliferative capacity of T-cell clones derived from leukaemia patients 3-6 weeks after allogeneic bone marrow transplantation were investigated. Only a minority of post-transplant T-cell clones (23/120; 19%) was capable of undergoing autocrine proliferation. By contrast, 21/65 (32%) normal control clones from the marrow donors derived under the same conditions were autocrine proliferative. All clones were interleukin-2 (IL-2) responsive. A majority (12/17; 71%) of autocrine proliferating post-transplant clones secreted detectable IL-2. Compared with control clones, CD4+ T-cell clones derived early after BMT produced decreased levels of interleukin-4 (IL-4) and interleukin-6 (IL-6), whereas secretion of interleukin-3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) showed no significant difference. The small number (n = 8) of posttransplant CD8+ clones showed decreased production of IL-3, IL-4 and IL-6 compared with control clones, but normal secretion of GM-CSF. Neither CD4+ nor CD8+ T-cell clones secreted interleukin-7 (IL-7).
Secretion of the potentially antileukaemic cytokines IFN-gamma and TNF-alpha was investigated for CD4+ and CD8+ TCR alpha beta + T-cell clones derived from 4 leukaemia patients 3-6 weeks after allogeneic BMT. We investigated cytokine secretion in response to the activation signal accessory cells+phytohaemagglutinin+Interleukin 2. All clones derived after BMT were capable of IFN-gamma and TNF-alpha secretion, and both for CD4+ (n = 96) and CD8+ (n = 8) T cells quantities of IFN-gamma and TNF-alpha were significantly correlated with one another. When comparing the overall results for posttransplant and normal T-cell clones derived from 2 bone marrow donors (n = 65), both CD4+ and CD8+ TCR alpha beta + T-cell clones showed increased IFN-gamma production, and CD4+ but not CD8+ clones showed a decreased TNF-alpha secretion. The results suggest that noncytotoxic T cells derived after allogeneic BMT can produce IFN-gamma and TNF-alpha and may thus be capable of mediating antileukaemic effects.
The human histocompatibility leucocyte antigen (HLA) class II-associated invariant chain comprises at least four polypeptides. One of these is expressed as a membrane-bound subunit and has been designated as CD74. We investigated the expression of CD74 on B- and T-cell types utilizing monoclonal antibodies (mAb) specific for the c-terminus of CD74. All B cells tested expressed CD74 regardless of their HLA phenotypes. High and low CD74 expressors were provisionally assigned based on quantitative variations found in some haplotypes. A mutant cell with selective deletions of class II loci DR and DQ but with retention of DP and additional class II heterodimers synthesize normal amounts of CD74. In contrast, however, a mutant with a total class II deletion had diminished expression of CD74, suggesting that at least one of the class II isotypes is essential for full constitutive expression of CD74. Major histocompatibility complex (MHC) class II negative T-cell lines and CD3+ T cell isolated from peripheral blood did not express CD74. However, allostimulated T cells which express de novo induced class II determinants also did not express CD74. Taken together, these results show that CD74 is expressed on B cells and unexpectedly is not synthesized on de novo-induced class II positive T-cell clones.
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Perturbations of hematopoietic regulation ranging from transient granulocytopenia to rare cases of bone marrow failure are associated with infections due to hepatitis A virus (HAV). In an attempt to elucidate the pathogenetic mechanisms we had previously established that HAV has a direct suppressive effect on human bone marrow progenitors (CFU-GM, -GEMM, BFU-E). These studies were extended to long-term bone marrow cultures (LTBMC): Inoculation of bone marrow mononuclear cells with HAV did not interfere with the establishment of an adherent stromal layer, nor did the inoculation of already established layers cause any morphologically recognizable changes to the stroma. In contrast, a significant and progressive decline of the CFU-GM content in the culture supernatants was demonstrated. HAV antigen was detected by APAAP stain in a subpopulation of stromal cells, and sequential estimations of virus titers in the supernatants provided evidence for viral replication in primary bone marrow cultures. Interferon-gamma and tumor necrosis factor-alpha levels of infected cultures did not differ from those of uninfected controls. These findings argue for a direct suppression of (pre-) CFU-GM by HAV in a model system (LTBMC) lacking an immune defense which would limit viral replication.
The streptokinase molecule (415 AA) was cleaved at methionine 237, 347 and 370 yielding four polypeptide fragments. Human HLA-class II restricted streptokinase-specific T cell clones and cell lines (CD2+, CD3+, CD4+, CD8-, TCR alpha beta+, TCR gamma delta-) recognized antigenic epitopes on all four fragments AA 1-236, AA 238-346, AA 348-369 and AA 371-415. T cell clones recognizing fragment AA 1-236 were restricted by at least two different HLA-class II elements, this indicating that more than one antigenic epitope can be recognized on this fragment. In addition, two streptokinase-specific T cell clones recognized only the intact molecule and none of the molecular fragments. These two clones probably recognized an antigenic epitope including one of the methionine residues used for molecular cleavage. We conclude that T cell proliferative responses to streptokinase are determined by recognition of at least five different antigenic epitopes distributed along the entire streptokinase polypeptide chain.
CD4+ TCR alpha beta+ T-cell clones were prepared from three CML-patients 4-6 weeks after allogeneic bone marrow transplantation and the effects of theophyllamine and verapamil on clonal growth then assessed. Both drugs inhibited PHA-stimulated autocrine proliferation of clones as well as proliferation of clones dependent on exogenous growth factors. Inhibition was seen when using different accessory cells (PBM or BCL) during T-cell activation, and both for IL2- and IL4-dependent proliferation of previously activated T-cells. The isomer R-verapamil inhibited PHA-stimulated proliferation as well as IL2- and IL4-dependent T-cell proliferation. The drugs also inhibited proliferation of CD8+ T-cells. Although the T-cell clones were functionally heterogeneous and were derived from different patients and priming cultures, both drugs inhibited all clones investigated. However, the degree of inhibition varied between different clones.
The antifungal azole drugs itraconazole (itra; R51,211), fluconazole (flu; UK-49,858), ketoconazole (keto) and micronazole (mico) have been investigated for their suppressive influence on the gene expression of the immunoregulatory cytokines IL2, IL4, IL9, GM-CSF, TNF-alpha, IFN-gamma, as well as both chains of the IL2 receptor in human PBMC and of the cytokines in the human keratinocyte cell line HaCat 17.5. The results obtained in Northern blot analysis were compared with the effects of the established immunosuppressant drug CSA and the new immunosuppressive drug FK 506, as well as the cytokine TGF-beta, which is also immunosuppressive. While 1 microgram/ml CSA and 0.1 microgram/ml FK 506 completely suppressed PHA-stimulated accumulation of mRNA for IL2, IL4, IL9, GM-CSF, TNF-alpha and IFN-gamma in PBMC, flu, keto and TGF-beta failed to inhibit any (except TNF-alpha blocked by TGF-beta). Itra and mico did suppress accumulation of mRNA, but unlike CSA and FK 506, only at high doses (10 micrograms/ml) and after extended incubation (24 h). None of the drugs nor TGF-beta suppressed the expression of the IL2R-alpha and IL2R-beta genes or TNF-alpha-stimulated cytokine gene expression in keratinocytes. Itra and mico, 1 mg/ml (achievable serum level), caused only slight inhibition of the cytokines in PBMC after 6 and 24 h of incubation. These results demonstrate that the mode of action of the azoles is different from CSA and FK 506.(ABSTRACT TRUNCATED AT 250 WORDS)
In an attempt to explore T-cell functions shortly after allogeneic bone marrow transplantation more fully, IL2- and IL4-dependent proliferation was assessed on CD4+ TCR alpha beta+ T-cell clones derived 4-6 weeks after transplantation. Both allogeneic pooled peripheral blood mononuclear cells and Epstein-Barr virus-transformed B-cell lines (BCL) could function as accessory cells (AC) for PHA activation of T-cell clones. Although minimal clonal proliferation was seen when the T-cell activation signal was BCL+PHA+IL4, a majority of the clones could undergo IL4-dependent proliferation after previous activation with AC+PHA+IL2. For certain clones, IL4 also showed an additive effect with IL2. Thus, IL4 was a growth factor for a majority of the investigated posttransplant T-cell clones, and in vivo modulation of IL4-dependent T-cell functions may thus become a future therapeutic possibility to enhance graft-versus-leukaemia effects in bone marrow transplant recipients.