- Email: [email protected]
HLA-G1 and HLA-G2 act as ligands recognized by a yet-unknown natural killer inhibitory receptor(s)
382
25 June 1997 - Poster presentations
NK cells
Previous studies have shown that soluble HLA-I molecules and HLA class I derived peptides contribute to allograft tolerance in animal transplantation models. Different mechanisms of recognition have been proposed and the specificity of this interaction requires further analysis. We study the possible immunoregulatory role of HLA class I derived peptides on NK cell activity. Peptides corresponding to high conserved regions of HLA class I molecules have been synthesized by standard methods. Synthetic peptides consisting of residues 77-83 of the a-l helix of polymorphic HLA class I molecules were used in a dimeric form to enhance their activity. NK clones of different specificities were used as effector cells in a conventional cytotoxicity assay. We present and discuss the effect of these HlAderived peptides on NK mediated lysis of target cells bearing different HIA class I alleles and the specificity of this recognition.
P.1.09.03 HLA-Gl and HLA-G2 act as ligands recognized by a yet-unknown natural killer inhibitory receptor(s) Rachel Marchal, Nathalie Rouas-Freiss, Marek Kirszenbaum, Jean Dausset I, Edwrdo D. Camsella ‘. Service de Recherches en H6mato_lmnwnologie, Cohmissariar d f’fnergie Atomique-DRM-DSV H6pital Saint-Louis, 7,avenue Claude-Vellefaux, 75475 Pads c6dex 10, France, ’ Fondation Jean Dausset, 27, rue Juliet&Dodu, 750 10 Paris c&ax, Fence HLA-G is a nonclassical major histocompatibility complex class I antigen selectively expressed on extra-villous trophoblast cells at the fetal-maternal interface that mediates protection of the fetus from maternal nature1 killer (NK) cell cytolysis. The receptors on NK cells that recognize HlA-G, namely NK inhibitory receptor 1 (NKIRI) and NKIRP were recently identified. These NKlRs consist of two immunoglobulin superfamily domains (~56) that are capable of discdminating between two dimorphic groups of HLA-C molecules. In order to further characterize the immunological functions of HLA-G prcducts, we studied the protective roles of the HLA-Gi and HLA-02 isoforms with respect tothe NK lytic activity of a clone that expresses none of the known NKlRs capable of binding HLA-G. For this purpose, HlA-Gl and HlA-GP cDNAs were transfected into the HlA class l-negative human K562 cell line, a known reference target for NK lysis. The HLA-Gl protein, encoded by a full-length mRNA, presents a structure similar to that of classical HIA class I antigens. The HLA-GP protein, deduced from an alternatively spliced transcript, consists of the rrl domain linked to the (r3 domain. We used a T-cell leukemia NK like clone that expresses no NKIR known to interact with HLA-G as effector cells. In this study we demonstrate that: when transfected into the K562 cell line, both HLA-Gl and HLA-GP abolish lysis by the T-cell leukemia NK-like clone, due to interaction between the HLA-G isoform on the target cell surface and a membrane receptor on the clone. Since NKIRI and NKIR2, known to interact with HLA-G, were undetectable on this clone, we conclude that a yet-unknown specific receptor(s) for HLA-Gl and HLA-GP is present on these cells.
P.1.09.04 Peripheral blood lymphocyte subpopulations analysis in unexplained primary recurrent spontaneous abortions C. Psarra’, Ch. Papastetiades I, V. Kapsimali ‘, K. Tarassi‘, S. Dendtinos *, G. Kallipolitis*, Th. Athanassiadis’, J. Economidou ‘, D. Aravantinos *. ’ Department of lmmunol~-HMocompatibili& Evangelismos Hospital, Athens, Greece, *Recurrent Spontaneous Abortion Center; A’ Obstetric and Gynecofogic Clinic, University of Athens, “Alexandra” Maternity Hospital, Athens, Greece Introduction: Different immune mechanisms have been implicated in the pathogenesis of unexplained primary recurrent spontaneous abortions (UPRSA). Recently, there is a lot of evidence indicating that immunologic effector cells, especially suppressor (CD6 positive) T cells and natural killer (NK) cells, may be involved in the early fetal loss. The aim of this study was to evaluate the contribution of peripheral blood lymphocyte subpopulations in the pathogenesis of UPRSA. Materials and Methods: Peripheral blood lymphocyte subpopulations were determined with flow cytometty using the method of direct immunofluorescence in whole blood, in 45 women with UPRSA and 30 healthy controls. The HLA class I and II antigens and the presence of lymphocytotoxic panel reactive antibodies and antipatemal antibodies were tested with classical serological techniques in these 45 couples with UPRSA, in 143 couples without UPRSA and in 105 healthy individuals as wntrols for HLA, as well. Ftesutts~The mean and the standard deviation of the mean of B cell (CD20), NK cell (CD3-CDl6/58+) and T cell subpopulation percentages are presented in the table. It is obvious that in the group of women studied as a whole, the percentages of lvmohocvte subwoulations were not sianificantlv different from the normal conirois. N&ertheie& 6 women (13%.) iave shown increased and 2 (4%)
n
Women v&h UPRSA COfllRJlS
CD2+
CD3+
CD3+CD4+
CDS+CDB+
CD3CD16/56+
45
62.5f4.5
75.6f5.4
30
64.0f6.0
75.4i5.9
47.5dc6.6
25.1f6.7
6.3f4.6
46.7f6.7
25.1f6.6
7.7i4.6
CD*
9.7 f 3.9 10.6f4.6
decreased percentages of NK cells, 10 (22%) have shown low percentage of CD3+CD8+ T cells and 2 (4%) very low percentage of CD20+ B cells. Women with the above mentioned disturbances have not shown characteristic findings concerning the sharing of HLA antigens with their husbands, HLA anti-paternal antibodies or lymphocytotoxic panel reactive antibodies in comparison with the control groups. Conclusion: It seems that the abnormalities of peripheral blood lymphocyte subpopulations concern some women with UPRSA suggesting that there is a diversity in the immunologic mechanisms contributing in the pathogenesis of UPRSA.
activity of tissue and blood lymphocytes P.1.09.05 Cytolytic of primates exposed to external gamma-irradiation O.F. Melnikov, MB. Sambur. Department of Pathophysi~ogy and Immunology, Research Institute of Otofaryngo~ Kyiv, Ukraine The success of immunologic surveillance against tumors and virus infections to a greate extent is related to natural killer (NK) and antybodydependent cellular cytotoxic (ADCC) effector cells cytolytic activity, their resistance to various pathogenic agents. The purpose of the work was to investigate the functional activity of palatine tonsils and blood NK and ADCC in primates (Papio gamadtyads) in dynamics of fractionated external Caesium-137 irradiation with a help of radiometric method (Cr-Bl-test). Stimulation of NK and ADCC cytolytic activity of blood cells rather than cells of palatine tonsils was observed in range of external gamma-irradiation dose up to 37.5 cGy. The doses of 62.5 and 100 cGy caused acute, abrupt impairment of the functional activity of blood as well as tissue NK and ADCC. Significant difference was marked between the velocities of restoration of cytolytic activity levels of tissue and blood NK and ADCC in 2.5 months following irradiation. The obtained data suggest that tissue NK arecomparatively more resistant to the effect of external gamma-irradiation.
P.1.09.06 IL-2 and IL-12 enhanced NK lytlc actlvlty in the centenarians E. Matiani ‘**, A. Meneghetti ‘, A. Taroui ‘, L. Cattini ‘, A. Facchini ‘t2. ’ Laboratorio di lmmunof~ia e Genetica, IstWo di Ricema Co&ii/a Puni, IOR, Bologna, Italy; * Dipatimento di Medicina lntema e Gasbtmntemlogia, University of Bolqna, /My
Introduction: NK cells are a subset of lymphocytes that mediates spontaneous cytolytic activity against virus-infected and neoplastic cells. Despite the progressive increase in the number of peripheral NK cells found in the elderly people, these data did not correlate wkh aconesponding increase in lytic activity, on the contrary a decreased function of circulating NK cells from old subjects was observed after a sorting procedure or after limiting dilution and cloning of NK cell precursors. The aim of the study was to evaluate whether the exposure to 11-12, a stimulatoty cytokine for NK cells, was able to increase NK lytic activity in old subjects and these effects were compared to those mediated by 11-2. Materialsand Methods:We studied two groups of healthy subjects: 21 old subjects (91-106 years) and 6 young subjects (26-33 years). Peripheral blood lymphocytes were separated by density gradient centrifugation. Phenotype characterization was performed using CD3, CD16 and/or CD56 FITC or PE monoclonal antibodies and flow cytometry analysis. NK calls, purified by negative selection using magnetic beads were incubated with IL-2 (100 U/ml) and/or IL-12 (1 @ml) for 4 and/or 18 hours. Untreated NK cells were used as controls. Lytic activity was determined by the release of 51 Cr in a micm-cytotoxicity test. Resultcl: Comparative analysis of the dose-response curves of iytic activity before or after IL-2 and/or IL-12 cytokines treatment showed a similar increase in both groups of subjects. Nevertheless the increment of cytolytic activity after IL-2 stimulation was more evident than after the IL-12 one, the time-course kinetic of activation was similar for both cytokines. In addition IL-12 was able to activate lysis with a concentration significantly lower than IL-2 one. Concluslon: In conclusion, we demonstrated that NK cells maintain their ability to respond to IL-2 and IL-12 cytokines, also in centenarians and that IL-12 was effective also at low doses. Supported by grants from University of Bologna, Italy and performed under the aegis of EUCAMBIS (BMHI-CT94-1209)
25 June 1997 - Poster presentations
NK cells
Previous studies have shown that soluble HLA-I molecules and HLA class I derived peptides contribute to allograft tolerance in animal transplantation models. Different mechanisms of recognition have been proposed and the specificity of this interaction requires further analysis. We study the possible immunoregulatory role of HLA class I derived peptides on NK cell activity. Peptides corresponding to high conserved regions of HLA class I molecules have been synthesized by standard methods. Synthetic peptides consisting of residues 77-83 of the a-l helix of polymorphic HLA class I molecules were used in a dimeric form to enhance their activity. NK clones of different specificities were used as effector cells in a conventional cytotoxicity assay. We present and discuss the effect of these HlAderived peptides on NK mediated lysis of target cells bearing different HIA class I alleles and the specificity of this recognition.
P.1.09.03 HLA-Gl and HLA-G2 act as ligands recognized by a yet-unknown natural killer inhibitory receptor(s) Rachel Marchal, Nathalie Rouas-Freiss, Marek Kirszenbaum, Jean Dausset I, Edwrdo D. Camsella ‘. Service de Recherches en H6mato_lmnwnologie, Cohmissariar d f’fnergie Atomique-DRM-DSV H6pital Saint-Louis, 7,avenue Claude-Vellefaux, 75475 Pads c6dex 10, France, ’ Fondation Jean Dausset, 27, rue Juliet&Dodu, 750 10 Paris c&ax, Fence HLA-G is a nonclassical major histocompatibility complex class I antigen selectively expressed on extra-villous trophoblast cells at the fetal-maternal interface that mediates protection of the fetus from maternal nature1 killer (NK) cell cytolysis. The receptors on NK cells that recognize HlA-G, namely NK inhibitory receptor 1 (NKIRI) and NKIRP were recently identified. These NKlRs consist of two immunoglobulin superfamily domains (~56) that are capable of discdminating between two dimorphic groups of HLA-C molecules. In order to further characterize the immunological functions of HLA-G prcducts, we studied the protective roles of the HLA-Gi and HLA-02 isoforms with respect tothe NK lytic activity of a clone that expresses none of the known NKlRs capable of binding HLA-G. For this purpose, HlA-Gl and HlA-GP cDNAs were transfected into the HlA class l-negative human K562 cell line, a known reference target for NK lysis. The HLA-Gl protein, encoded by a full-length mRNA, presents a structure similar to that of classical HIA class I antigens. The HLA-GP protein, deduced from an alternatively spliced transcript, consists of the rrl domain linked to the (r3 domain. We used a T-cell leukemia NK like clone that expresses no NKIR known to interact with HLA-G as effector cells. In this study we demonstrate that: when transfected into the K562 cell line, both HLA-Gl and HLA-GP abolish lysis by the T-cell leukemia NK-like clone, due to interaction between the HLA-G isoform on the target cell surface and a membrane receptor on the clone. Since NKIRI and NKIR2, known to interact with HLA-G, were undetectable on this clone, we conclude that a yet-unknown specific receptor(s) for HLA-Gl and HLA-GP is present on these cells.
P.1.09.04 Peripheral blood lymphocyte subpopulations analysis in unexplained primary recurrent spontaneous abortions C. Psarra’, Ch. Papastetiades I, V. Kapsimali ‘, K. Tarassi‘, S. Dendtinos *, G. Kallipolitis*, Th. Athanassiadis’, J. Economidou ‘, D. Aravantinos *. ’ Department of lmmunol~-HMocompatibili& Evangelismos Hospital, Athens, Greece, *Recurrent Spontaneous Abortion Center; A’ Obstetric and Gynecofogic Clinic, University of Athens, “Alexandra” Maternity Hospital, Athens, Greece Introduction: Different immune mechanisms have been implicated in the pathogenesis of unexplained primary recurrent spontaneous abortions (UPRSA). Recently, there is a lot of evidence indicating that immunologic effector cells, especially suppressor (CD6 positive) T cells and natural killer (NK) cells, may be involved in the early fetal loss. The aim of this study was to evaluate the contribution of peripheral blood lymphocyte subpopulations in the pathogenesis of UPRSA. Materials and Methods: Peripheral blood lymphocyte subpopulations were determined with flow cytometty using the method of direct immunofluorescence in whole blood, in 45 women with UPRSA and 30 healthy controls. The HLA class I and II antigens and the presence of lymphocytotoxic panel reactive antibodies and antipatemal antibodies were tested with classical serological techniques in these 45 couples with UPRSA, in 143 couples without UPRSA and in 105 healthy individuals as wntrols for HLA, as well. Ftesutts~The mean and the standard deviation of the mean of B cell (CD20), NK cell (CD3-CDl6/58+) and T cell subpopulation percentages are presented in the table. It is obvious that in the group of women studied as a whole, the percentages of lvmohocvte subwoulations were not sianificantlv different from the normal conirois. N&ertheie& 6 women (13%.) iave shown increased and 2 (4%)
n
Women v&h UPRSA COfllRJlS
CD2+
CD3+
CD3+CD4+
CDS+CDB+
CD3CD16/56+
45
62.5f4.5
75.6f5.4
30
64.0f6.0
75.4i5.9
47.5dc6.6
25.1f6.7
6.3f4.6
46.7f6.7
25.1f6.6
7.7i4.6
CD*
9.7 f 3.9 10.6f4.6
decreased percentages of NK cells, 10 (22%) have shown low percentage of CD3+CD8+ T cells and 2 (4%) very low percentage of CD20+ B cells. Women with the above mentioned disturbances have not shown characteristic findings concerning the sharing of HLA antigens with their husbands, HLA anti-paternal antibodies or lymphocytotoxic panel reactive antibodies in comparison with the control groups. Conclusion: It seems that the abnormalities of peripheral blood lymphocyte subpopulations concern some women with UPRSA suggesting that there is a diversity in the immunologic mechanisms contributing in the pathogenesis of UPRSA.
activity of tissue and blood lymphocytes P.1.09.05 Cytolytic of primates exposed to external gamma-irradiation O.F. Melnikov, MB. Sambur. Department of Pathophysi~ogy and Immunology, Research Institute of Otofaryngo~ Kyiv, Ukraine The success of immunologic surveillance against tumors and virus infections to a greate extent is related to natural killer (NK) and antybodydependent cellular cytotoxic (ADCC) effector cells cytolytic activity, their resistance to various pathogenic agents. The purpose of the work was to investigate the functional activity of palatine tonsils and blood NK and ADCC in primates (Papio gamadtyads) in dynamics of fractionated external Caesium-137 irradiation with a help of radiometric method (Cr-Bl-test). Stimulation of NK and ADCC cytolytic activity of blood cells rather than cells of palatine tonsils was observed in range of external gamma-irradiation dose up to 37.5 cGy. The doses of 62.5 and 100 cGy caused acute, abrupt impairment of the functional activity of blood as well as tissue NK and ADCC. Significant difference was marked between the velocities of restoration of cytolytic activity levels of tissue and blood NK and ADCC in 2.5 months following irradiation. The obtained data suggest that tissue NK arecomparatively more resistant to the effect of external gamma-irradiation.
P.1.09.06 IL-2 and IL-12 enhanced NK lytlc actlvlty in the centenarians E. Matiani ‘**, A. Meneghetti ‘, A. Taroui ‘, L. Cattini ‘, A. Facchini ‘t2. ’ Laboratorio di lmmunof~ia e Genetica, IstWo di Ricema Co&ii/a Puni, IOR, Bologna, Italy; * Dipatimento di Medicina lntema e Gasbtmntemlogia, University of Bolqna, /My
Introduction: NK cells are a subset of lymphocytes that mediates spontaneous cytolytic activity against virus-infected and neoplastic cells. Despite the progressive increase in the number of peripheral NK cells found in the elderly people, these data did not correlate wkh aconesponding increase in lytic activity, on the contrary a decreased function of circulating NK cells from old subjects was observed after a sorting procedure or after limiting dilution and cloning of NK cell precursors. The aim of the study was to evaluate whether the exposure to 11-12, a stimulatoty cytokine for NK cells, was able to increase NK lytic activity in old subjects and these effects were compared to those mediated by 11-2. Materialsand Methods:We studied two groups of healthy subjects: 21 old subjects (91-106 years) and 6 young subjects (26-33 years). Peripheral blood lymphocytes were separated by density gradient centrifugation. Phenotype characterization was performed using CD3, CD16 and/or CD56 FITC or PE monoclonal antibodies and flow cytometry analysis. NK calls, purified by negative selection using magnetic beads were incubated with IL-2 (100 U/ml) and/or IL-12 (1 @ml) for 4 and/or 18 hours. Untreated NK cells were used as controls. Lytic activity was determined by the release of 51 Cr in a micm-cytotoxicity test. Resultcl: Comparative analysis of the dose-response curves of iytic activity before or after IL-2 and/or IL-12 cytokines treatment showed a similar increase in both groups of subjects. Nevertheless the increment of cytolytic activity after IL-2 stimulation was more evident than after the IL-12 one, the time-course kinetic of activation was similar for both cytokines. In addition IL-12 was able to activate lysis with a concentration significantly lower than IL-2 one. Concluslon: In conclusion, we demonstrated that NK cells maintain their ability to respond to IL-2 and IL-12 cytokines, also in centenarians and that IL-12 was effective also at low doses. Supported by grants from University of Bologna, Italy and performed under the aegis of EUCAMBIS (BMHI-CT94-1209)