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Biomedical subjects

G Pawelec

Publications and source records attributed to G Pawelec.

At least 73 records · Page 4Linked to original sources
PubMed1996

BCR/ABL leukemia oncogene fusion peptides selectively bind to certain HLA-DR alleles and can be recognized by T cells found at low frequency in the repertoire of normal donors.

Chronic myelogenous leukemia (CML) is characterized by the t(9;22) translocation that results in chimeric genes encoding bcr/abl fusion proteins. Junction-spanning sequences represent unique tumor-specific moieties that might be exploited therapeutically. We investigate here the binding of synthetic bcr/abl peptides to various HLA-DR alleles and their recognition by T cells from normal donors and CML patients. A 23-mer b3/a2 peptide bound very strongly to isolated HLA-DRB1*1101 (Dw5) and relatively strongly to DRB1*0301 (Dw3) and DRB1*0402 (Dw10) molecules, as estimated using a competition assay. It failed to bind to several other DR alleles, including three different DR4 alleles. In contrast, a 23-mer b2/a2 peptide bound only to the DRB1*0301 (Dw3) allele. Peripheral blood mononuclear cells from normal donors were sensitized in vitro against the b3/a2 peptide. After four repetitive stimulations, T cells responding to the peptide were found at low frequency in 5 of the 11 donors tested. Three of the five were HLA-DR11+, and all three of the DR11+ donors tested were found to respond. T cells recognizing bcr/abl peptides were not identified in any of the CML patients studied, regardless of HLA type. Finally, even peptide-reactive T-cell lines from normal donors were not stimulated by native CML cells in the absence of exogenous peptide. These results show the presence of low-frequency major histocompatability complex class II-restricted bcr/abl-responses in the normal T-cell repertoire of donors with certain HLA types, but suggest that unmodified tumor cells cannot be recognized by such peptide-sensitized T cells.

Amino Acid Sequence↗
PubMed1996

Interleukin 10 protects activated human T lymphocytes against growth factor withdrawal-induced cell death but only anti-fas antibody can prevent activation-induced cell death.

Interleukin 10 (IL-10) is a pleiotropic T cell-derived cytokine best known for its negative regulatory effects on T cell immunity. It inhibits responses indirectly by downregulating expression of major histocompatibility complex (MHC) molecules and co-stimulatory molecules such as CD80 on antigen presenting cells as well as directly via its effects on responding cells. On the other hand, IL-10 has been shown to protect activated T cells against apoptosis caused by withdrawal of the major growth factor, IL-2, and allow proliferation of T cells in the absence of IL-2. However, we show here that this IL-10-dependent, IL-2-independent proliferative response is short-lived, and that IL-10-responsive T cells cannot multiply in its presence. Moreover, inclusion of exogenous IL-10 in clonal cultures propagated with IL-2 results in suppression of their growth. These findings, together with the observation that IL-10 fails to protect T cells against activation-induced cell death (a fas/fas-ligand-dependent phenomenon blocked only by certain antagonistic anti-fas reagents), suggest that the negative regulatory effects of IL-10 outweigh the upregulated proliferation observed on some T cell clones (TCC) in the absence of IL-2.

Antibodies↗
PubMed1996

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts; studies of normal polyclonal T cells and T lymphocyte clones derived early after allogenic bone marrow transplantation.

T cell clones (CD4+CD8-TCR alpha beta+gamma delta- derived from bone marrow transplant recipients were stimulated with phytohaemagglutinin (PHA) +interleukin-2 (IL-2) in the presence of irradiated (50 Gy) peripheral blood mononuclear cells (PBMC) derived from acute Leukaemia patients (leukaemic PBMC containing more than 95% blast cells). Leukaemic PBMC could function as accessory cells during mitogenic T cell activation resulting in both T cell proliferation and a broad T cell cytokine response [IL-3, IL-4, IL-10, granulocyte/macrophage-colony-stimulating factor (GM-CSF) tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) secretion]. Blockade of IL-1 effects by adding IL-1 receptor antagonist together with PHA + IL-2 + leukaemia blasts increased T cell proliferation, whereas IL-6-neutralizing antibodies did not alter T cell proliferation. A qualitatively similar T cell cytokine response and a similar cytokine profile (highest levels detected for GM-CSF and IFN gamma) were detected when normal polyclonal T cell lines were stimulated with PHA in the presence of non-irradiated leukaemic PBMC. When leukaemic PBMC derived from 18 acute myelogenous leukaemia patients were cultured with PHA and cells from a polyclonal T cell line, increased concentrations of the T cell cytokines IFN gamma and IL-4 were detected for all patients. We conclude that T cell activation resulting in proliferation and a broad cytokine response can take place in the presence of excess acute myelogenous leukaemia blasts.

Adult↗
PubMed1996

Cellular immune responses to autologous chronic myelogenous leukaemia cells in vitro.

Using a modification of the autologous mixed lymphocyte/tumour cell culture (MLTC), it is demonstrated here that lymphocytes from chronic-phase myelogenous leukaemia (CML) patients (n = 58), but not from their HLA-identical siblings, proliferated upon coculture with autologous tumour cells. However, in most cases, the level of proliferation measured was low (stimulation index < 3, n = 37). This was most likely related to the amount of interleukin-10 (IL-10) released into the culture medium by the CML cells, because addition of neutralizing anti_IL-10 serum to MLTC markedly enhanced proliferative responses. In addition, supplementation of media with IL-1 alpha further enhanced proliferative responses and a combination of anti-IL-10 serum and IL-1 alpha was more effective than either agent alone. Only HLA-DR-matched CML cells, but not HLA-DR-mismatched CML cells or matched or mismatched PBMC restimulated proliferation of IL-2-dependent T cell lines derived from MTLC supplemented with IL-1 alpha and anti-IL-10 serum. The responding cells under these conditions were predominantly CD4+ and secreted IL-2, and interferon gamma; some secreted IL-4, but none secreted IL-10. These data therefore suggest the existence of an HLA-DR-restricted DTH/Th1-type of tumour-specific immunity in CML patients, which may be down-regulated in vitro by excessive secretion of IL-10 together with depressed secretion of IL-1.

Cells, Cultured↗
PubMed1996

Effects of hematopoietic growth factors on interleukin 6 secretion by blast cells derived from acute myelogenous leukemia patients.

Interleukin 6 (IL6) secretion by blast cells derived from peripheral blood of patients with acute myelogenous leukemia (AML) was characterized. IL6 secretion showed a wide variation both when AML blasts were cultured in medium alone and in the presence of exogenous cytokines. The level of IL6 secretion was significantly correlated to secretion of other cytokines (IL1 alpha, IL1 beta, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha). IL1 beta, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor and stem cell factor increased IL6 secretion, whereas IL4 caused decreased IL6 secretion together with increased release of IL1 receptor antagonist.

Adult↗
PubMed1995

Long-term culture of monoclonal human T lymphocytes: models for immunosenescence?

Human monoclonal T lymphocyte populations maintained in long-term culture by intermittent reactivation via the antigen receptor and supplied with exogenous interleukin 2 manifest finite proliferative lifespans. T lymphocytes cloned from mature peripheral T cells of adult donors were constantly lost from the time point of their first isolation up to an estimated maximum of 80 population doublings (PD) for the longest lived. T lymphocytes cloned from T cell progenitors in bone marrow, on the other hand, survived for a maximum of ca. 100 PD. One facet of the functional capacity of cells derived from these two different sources was assessed by measuring their autocrine proliferation after mitogenic stimulation. For a majority of T cell clones (TCC), autocrine proliferative capacity decreased as a function of culture age, becoming absent by 50 PD for adult-derived-TCC and by 70 PD for bone marrow-derived TCC, thereby clearly occurring prior to the end of the proliferative life spans of the clones. Limiting dilution frequency analysis showed that the number of autocrine proliferative precursors within these monoclonal populations declined with age, paralleling loss of autocrine proliferative capacity in the 'bulk' clones. Of a variety of surface structures monitored during culture ageing of TCC, the density of expression of the coreceptor molecule CD28 was found to correlate with decreasing autocrine proliferative capacity in two-thirds of the clones. Thus, at least for a fraction of monoclonal human T lymphocytes, decreasing autocrine proliferative capacity, a measure of clonal expansion, may correlate with decreasing numbers of CD28 molecules expressed on the surface and therefore presumably with the strength of costimulatory signal delivered via this important coreceptor.

Adult↗
PubMed1995

Human gamma delta T cells are resistant to induction of anergy but not to induction of cell death in vitro.

A condition of hyporesponsiveness can be induced in certain mature human alpha beta (TCR2) cells relatively easily by their stimulation in the absence of costimulatory signals (signal 1 alone). This state of "anergy" has been implicated in tolerance to self and transplanted organs as well as tumors and may represent an important regulatory component of immune responsiveness. Little is known about whether the same condition applies to gamma delta (TCR1) cells. We therefore undertook to investigate anergy induction in TCR1 cell clones using several approaches known to induce this state in TCR2 cells. First, TCR1 clones were found not to be anergized by culture on immobilized CD3 monoclonal antibody (mAb), while the majority of TCR2 clones were anergized. Second, blocking of autocrine proliferation (stimulated in TCR1 or TCR2 clones by mitogen in the presence of accessory cells) using CTLA-4-lg, a soluble B7 family counterreceptor resulted in anergy induction in TCR2 cells but not TCR1 cells, although experiments with CHO cells transfected with B7-1 (CD80) genes confirmed that these TCR1 clones were responsive to costimulation with B7. Third, blocking mitogen-induced proliferation with anti-IL 2 receptor antibodies and anti-IL 2 antisera resulted in anergy induction in TCR2 but not TCR1 cells. Fourth, stimulation with the calcium ionophore ionomycin also anergized TCR2 but not TCR1 cells. In all four systems, but especially in the latter, stimulation by signal 1 alone resulted in high levels of cell death (> 50%) which was similar for both TCR1 and TCR2 cells. Therefore, these data may reflect a high level of resistance to tolerance induction (manifested as proliferative anergy) but not to clonal deletion (manifested as stimulation-dependent cell death) on the part of TCR1 cells.

Abatacept↗
PubMed1995

Interleukin 10 is a human T cell growth factor in vitro.

Although interleukin (IL)-10 inhibited lymphocyte proliferation in mixed lymphocyte cultures (MLC) and blocked stimulation of alloreactive T cell clones (TCC) by peripheral blood mononuclear cells (PBMC), the cells surviving culture with IL-10 showed enhanced viability. A minority of IL-2-dependent T cell lines, moreover, incorporated tritiated thymidine when cultured with IL-10 alone; their proliferation with IL-10 was dose-dependent, prevented by addition of neutralizing antisera to IL-10 but not to IL-2 and/or IL-4 and observed both shortly (4 days) and later (7-10 days) after T cell allostimulation. Examination of the proliferative responses to IL-10 of a panel of TCC revealed heterogeneity of responsiveness: whereas only one of five CD8+ TCR2 (T cell receptor alpha, beta)-TCC proliferated with IL-10, three of five CD4+ TCR2-TCC proliferated, one of them strongly. In contrast, all three TCR1(gamma delta)-TCC tested responded to IL-10, albeit rather weakly. These results therefore suggest that in addition to its well-established inhibitory action on T cell activation, IL-10 may also exert positive influences on clonal expansion of subsets of preactivated T cells.

CD4-Positive T-Lymphocytes↗
PubMed1995

In vitro effect of r-verapamil on acute myelogenous leukemia blast cells: studies of cytokine secretion and cytokine-dependent blast proliferation.

The in vitro effect of the dextroisomer r-verapamil on blast cells derived from patients with acute myelogenous leukemia (AML) was studied. R-verapamil caused a dose-dependent inhibition of AML blast proliferation in the presence of stem-cell factor, leukemia inhibitory factor, interleukin 4, interleukin 6, and interleukin 10 when these cytokines were tested both alone and in different combinations. R-verapamil also inhibited the growth of clonogenic AML blast cells. The antiproliferative effect was not specific for AML blast cells, because r-verapamil also inhibited cytokine-dependent proliferation of blast cells derived from patients with acute lymphoblastic leukemia. The inhibitory effects of r-verapamil and anti-IL1 serum were additive, suggesting that the antiproliferative effect of r-verapamil does not depend solely on inhibition of IL1-mediated effects. Although r-verapamil inhibited spontaneous AML blast proliferation, for a majority of patients it caused only minimal, if any, inhibition of spontaneous cytokine secretion (IL1 alpha, IL1 beta, TNF alpha, IL6) by AML blast cells. Thus, although inhibition of IL1 effects may contribute in certain patients to the antiproliferative effect of r-verapamil, mechanisms other than IL1 inhibition seem to be more important in mediating the effects of r-verapamil.

Adolescent↗
PubMed1995

Cytokine modulation of interleukin 1 and tumour necrosis factor-alpha secretion from acute myelogenous leukaemia blast cells in vitro.

Spontaneous secretion of interleukin 1 (IL-1) alpha, IL-1 beta and tumour necrosis factor-alpha (TNF-alpha) from acute myelogenous leukaemia (AML) blasts showed significant correlation, and detectable levels of all cytokines were seen for a majority of patients. IL-3 and granulocyte/macrophage colony-stimulating factor increased secretion of IL-1 alpha, IL-1 beta and TNF-alpha for a majority of AML patients, whereas IL-4 decreased cytokine secretion. The effect of IL-6 and stem cell factor on cytokine secretion varied between different patients. A wide variation in IL-1 alpha, IL-1 beta and TNF-alpha secretion between different patients was seen both for spontaneous secretion and in the presence of all cytokines.

Adult↗
PubMed1995

Secretion of leukaemia inhibitory factor after allogeneic bone marrow transplantation: a study of CD4+ and CD8+ TCR alpha beta+ T-cell clones derived from four leukaemia patients.

CD4+ and CD8+ TCR alpha beta+ T-cell clones were derived from 4 leukaemia patients early (4-6 weeks) after allogeneic bone marrow transplantation. Leukemia inhibitory factor (LIF) secretion in response to the activation signal accessory cells (AC) + phytohaemagglutinin (PHA) was investigated for each individual clone. Only a minority of CD4+ TCR alpha beta+ T-cell clones secreted LIF in response to AC + PHA, whereas most T-cell clones were capable of LIF secretion when exogenous interleukin 2 was added together with AC + PHA. LIF secretion could also be demonstrated for CD8+ TCR alpha beta+ posttransplant T-cell clones. Thus, posttransplant CD4+ and CD8+ TCR alpha beta+ clonogenic T-cells are capable of LIF secretion, and LIF secretion may be a T-cell effector mechanism in graft versus host disease, graft versus leukaemia effects or posttransplant haematopoietic reconstitution.

Bone Marrow Transplantation↗
PubMed1995

Relative roles of natural killer- and T cell-mediated anti-leukemia effects in chronic myelogenous leukemia patients treated with interferon-alpha.

Potential anti-leukemia effects mediated by T cells or by natural killer (NK) cells were investigated in chronic myelogenous leukemia (CML) patients treated with interferon-alpha. Therapy-associated modulation of T cell and NK reactivity was monitored for one year from initiation in autologous mixed lymphocyte-tumor cell reactions and cytotoxicity directed against autologous CML cells, respectively. During the course of IFN-therapy, NK activity against autologous CML cells increased steadily, whereas T cell reactivity fluctuated randomly. Despite the high level of T cell reactivity to autologous tumor cells in short-term (6 days) culture, 1) they failed to respond to synthetic peptides corresponding to the bcr/abl fusion sequence of the patient, and 2) only one proliferative T cell clone (TCC) was isolated which specifically recognized HLA-DR-matched CML cells. This TCC appeared not to recognize synthetic peptides corresponding to the bcr/abl fusion sequence of the patient; the antigen to which it responds remains unknown. To assess potential immunogenicity of bcr/abl peptides, it was attempted to sensitize T cells from normal donors in vitro. Of 109 cell lines obtained from seven different donors, eleven showed peptide-dependent proliferation. Therefore, although these results show that it is possible to isolate apparently CML-specific T cells from patients, as well as to prime T cells against tumor-specific peptide in vitro, the frequency of such T cell-mediated reactivity appears low and its relevance to anti-leukemic effects questionable. On the other hand, the strong time-dependent enhancement of natural killing of autologous CML blasts during IFN-alpha treatment, a phenomenon not observed for T cell reactivity, suggests that natural immunity may be more important in controlling disease.

Amino Acid Sequence↗
PubMed1995

Effects of interleukin 10 on blast cells derived from patients with acute myelogenous leukemia.

The effect of interleukin 10 (IL-10) on proliferation and cytokine secretion by acute myelogenous leukemia (AML) blast cells was investigated in vitro. IL-10 inhibited spontaneous AML blast proliferation for a majority of patients, whereas in the presence of exogenous growth factors (granulocyte-stimulating factor, G-CSF; granulocyte-macrophage colony-stimulating factor, GM-CSF; interleukin 3) the IL-10 effect on blast proliferation showed a wide variation depending on the individual AML patient. IL-10 seemed to cause an irreversible inhibitory effect on AML blasts, as inhibition could also be demonstrated when IL-10 was present only during the initial preincubation of the leukemia cells. IL-10 also inhibited AML blast colony formation. However, independent of the effect on AML blast proliferation, IL-10 decrease cytokine secretion from AML blast cells for all patients, as demonstrated for IL-1 alpha, IL-1 beta, tumor necrosis factor-alpha, GM-CSF and interleukin 6. IL-10 did not inhibit development of apoptosis in AML blasts cultured in vitro. Expression of complement receptors and capability to adhere and internalize bacteria by AML blasts were not altered by IL-10.

Aged↗
PubMed1994

Requirements for stimulation or anergy induction in alloreactive human T cell clones.

The "two signal" concept for T cell activation is widely accepted. Signal 1 is commonly delivered via the antigen receptor, and signal 2 via accessory interactions. Delivery of both signals results in activation, signal 1 alone in induction of hyporesponsiveness. The nature of signal 1 in alloreactivity is not completely clear; most evidence suggests that a complex of foreign major histocompatibility complex molecules and their bound peptides is recognized. Interactions between B7 (CD80) ligand and CD28/CTLA-4 receptors are currently considered the most important sources of signal 2. Xenogeneic cells transfected with human genes provide useful stimulators for dissecting signals 1 and 2 in alloreactivity. We show here that the majority of DR-specific alloreactive T cell clones (TCC) fails to recognize Chinese hamster ovary (CHO) cells transfected with human DR, whether or not these are cotransfected with genes for CD80 or LFA-3. Stimulation was not observed even in the presence of a pool of peptides isolated by low pH release from B cell line (BCL)-derived DR molecules, or in the presence of synthetic peptides corresponding to the sequences of the three most commonly identified endogenous peptides. Lack of recognition was observed both in failure to stimulate proliferation and in failure to induce anergy. However, one TCC was identified which responded weakly to DR+ CHO cells, and for this clone, the presence of either CD80 or LFA-3 strongly enhanced proliferative responses. Anergy was not induced, even in the absence of CD80. Immobilized HLA-DR molecules purified from a BCL also failed to stimulate proliferation, but unlike the CHO transfectants, they did induce anergy. Stimulation with BCL also induced anergy if CD80-dependent interactions were blocked with soluble CTLA-4-Ig receptor. These results are consistent with the model that DR molecules expressed in the absence of appropriate peptide are simply not recognized by most alloreactive T cells, whereas DR molecules containing appropriate bound peptide are recognized as signal 1 and induce anergy. CTLA-4-Ig blocking confirms that CD80-dependent interactions can be important in preventing anergy induction, but that they are not always necessary is illustrated by the existence of a single clone which recognized DR molecules on CHO transfectants, giving very weak proliferation without CD80, and nonetheless no anergy induction.

Amino Acid Sequence↗
PubMed1994

Role of three quantitatively dominant endogenous peptides from HLA-DRB1*0401 molecules in class II specific alloreactivity.

Alloreactivity remains an important barrier to organ transplantation and is caused by T cell recognition of foreign histocompatibility antigens (HAg) in two ways: (1) indirect recognition, in which processed HAg peptides are presented by self MHC like any other foreign antigen, and (2) direct recognition, where the foreign MHC itself is recognized in contravention of the T cell recognition rule of self restriction. Whereas the role of endogenous peptides in direct MHC class I specific recognition is now established, their role in class II specific direct alloreactivity remains controversial, since no defined endogenous peptide has been shown to be required for alloreactivity. That mutations resulting in defective antigen processing impair class II specific allostimulation, however, suggests that the endogenous pathway is important for class II as well as class I alloreactivity. We attempted to establish the importance of endogenous peptides for alloreactivity by identifying common sequences of peptides bound by DR molecules of an HLA-DRB1*0401 homozygous B cell line. Peptides corresponding to three of these (calreticulin, HLA class I and an unidentified molecule) were used to restimulate established allospecific HLA-Dw4 reactive T cell clones, as well as to sensitize allogeneic T cells de novo in vitro. Xenogeneic chinese hamster ovary (CHO) cells coexpressing the relevant DR allele together with CD80 were used as antigen presenting cells. The role of CD80 could be determined on these cells because (1) they are xenogeneic and (2) they do not express B7 family members bound by CTLA-4Ig.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗