Histocompatibility typing by cell mediated lympholysis (CML): workshop II technical standardization.
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Biomedical subjects
Publications and source records attributed to T Kristensen.
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A case of acquired hypogammaglobulinaemia has been examined. Total and differential leucocyte counts and distribution were normal as were the lymphocyte sub-populations when defined by surface membrane immunoglobulin, E-, EA- and EAC-rosette techniques. The proliferative response of unseparated lymphocytes to PHA and Con-A was normal while the response to PWM was clearly impaired. In contrast, testing of purified B-lymphocytes by PWM was within the normal range. The response to PWM was found to be depressed by adding the patient's T-lymphocytes to autologous B-lymphocytes as well as to B-lymphocytes from 10 normal, healthy controls. The patient's T-lymphocytes were further tested for cytotoxicity against autologous. B-lymphocytes and B-lymphocytes from the 10 control individuals. Cytotoxicity was observed against autologous B-lymphocytes and against the B-lymphocytes of 3 controls. These findings indicate the presence of peripherally circulating unspecific T-suppressor lymphocytes and cytotoxic T-lymphocytes of some immunogenetic specificity. The suppressor and cytotoxic T-lymphocytes may separately or in combination be involved in the pathogenesis of hypogammaglobulinaemia. Thus, these findings are equivocal but underline the immunologic aspects of the pathogenesis.
The in vitro polyclonal pokeweed mitogen (PWM)-induced activation of human B lymphocytes is enhanced by addition of autologous or allogeneic irradiated T cells. This model for B/T-cell cooperation may be used to define and describe the balance between T helper and T suppressor phenomena. The present study investigates the helper and suppressor capacities of mononuclear cells isolated from peripheral blood of infectious mononucleosis patients during the acute disease and the reconvalescence period. During the acute disease we found a functional lack of T helper capacity; furthermore, the T cells were able to suppress the PWM and T-cell-dependent B-cell proliferation of healthy donor cells. The suppression was non-cytotoxic; i.e. not due to destruction of the responder cells. This phenomenon of non-cytotoxic suppression was found for all seven patients studied and disappeared during the reconvalescence period, indicating that the T lymphocytosis seen in infectious mononucleosis includes an expansion of T suppressor cells.
Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells, partially purified by chromatography on DNA-agarose, was obtained as a more than 80% homogeneous preparation by isoelectric focusing in a sucrose gradient. The polymerase activity was shown to be associated with the major protein in the preparation. Results obtained by electrophoresis in the presence of sodium dodecyl-sulfate indicated that poly(ADP-ribose) polymerase consists of a polypeptide chain with a molecular weight of 130 000. Ultracentrifugation at non-denaturating conditions indicated that the active enzyme may be an oligomeric form of this polypeptide chain. The isoelectric point of the polymerase was 9.40. The effects of various additions to the assay mixture on the synthesis of poly(ADP-ribose) as well as some kinetic data, are given. It is shown that poly(ADP-ribose) is a highly efficient inhibitor of its own synthesis, and results are presented which suggest that the well-known stimulatory effect of DNA on the synthesis is due to reduction of this inhibitory effect of the product.
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Six functionally HLA-D homozygous typing cells were identified by a restricted investigation into the Hungarian inbred population of Ivád. These putative HLA-D homozygous typing cells were then tested against a highly selected Scandinavian population sample of 60 individuals previously typed by histocompatibility reference reagents. The different HLA-D specificities could thus be identified: one closely matching HLA-Dw5, another resembling the Oslo LDoH specificity, while the last seems to be unique. Only one of the typing cells thus ascertained were HLA-B homozygous and were selected on the basis of the Ivád family structure and not on the basis of serological HLA typing.
In a collaboratory study involving eight different laboratories 30 human, mixed lymphocyte culture educated cytotoxic lymphocytes (CTLs) were identified yielding reproducible cytolysis on allogenic lymphocyte target cells without detectable HLA-A, B (and C) antigenic sharing between stimulator and target cells. These CTLs were collected in one laboratory (Aarhus) and tested in parallel against a population sample of 100 unrelated, healthy Danes. The testing was only performed once and 11 CTLs did not discriminate in the population, probably due to transportation damage. On the basis of pairwise comparisons between 19 CTLs, three tentative CML-defined specificities could be recognized. These three groups may have defined monospecific traits of allelic genetic origin as judged by a mutually negative, albeit not significant, correlation and a fit to Hardy-Weinberg equilibrium. The concept of determinants other than the serologically defined HLA antigens recognized by some CTLs can thus still be maintained as can the approach to CML typing tested in this workshop.
Lymphocytes from a healthy female repeatedly giving rise to MLC-typing response against HLA--D homozygous typing cells of three different specificities were investigated for cytotoxic capacity by the direct CML technique. Testing against a panel indicated the presence of circulating cytotoxic lymphocytes with specificity towards HLA--A2. When tested against selected HLA--D homozygous typing cells, the pattern of CML reactivity closely resembled the pattern of MLC-typing responses, i.e. typing responses were mostly restricted by the presence of HLA--A2 on the stimulator cells. This pattern was also found when time course studies of Cr--51 release were performed using experimental conditions identical to ordinary MLC typing, but involving chromium-51 labeled, irradiated homozygous typing cells as targets. These studies indicate that the presence of in vivo educated cytotoxic lymphocytes among the responder lymphocytes may in some instances mimic typing responses. Such lymphocytes are thought to lyse relevant stimulator lymphocytes prior to initiation of the proliferative response.
A Hungarian random population sample was tested for six well-known and two new HLA--D specificities. HLA--D antigen and gene frequencies in the studied population agree with the frequencies observed in pooled random Caucasians, only HLA--Dw3 being significantly elevated. The incidences of Dw1 and Dw4 are, however, lower while the incidences of the Dw5 and Dw6 alleles seem to be higher in our population sample without reaching statistical significance. As for the two new specificities, the IVAD-1 specificity has a low frequency, while IVAD-3 occurs quite frequently. HLA--B and HLA--D associations seem to be different in our population sample as compared to others. In spite of the high incidence of the HLA--Bw35 antigen, no HLA--D association was found. The two new HLA--D specificities did not show association with any of the established HLA--B antigens.
In a study of lymphocyte subpopulations in peripheral blood during infectious mononucleosis the lymphocytosis was found to be of T-cell origin (i.e. E-RFC), while the number of non T lymphocytes (i.e. EA-, EAC-RFC and SmIg + ve cells) was normal in 7 out of 8 patients. Ten patients were tested for the presence of HLA-DR determinants on their B and T cells and not only B lymphocytes but also a great part (31--75%) of T cells were lysed by the anti HLA-DR testsera, indicating that HLA-DR determinants are expressed on a population of T cells in IM patients. After recovery all patients were retested and showed a normal pattern of HLA-DR typing. This indicates an increase of HLA-DR antigens on T cells or a vigorous proliferation of a small DR-positive T-cell subpopulation during the acute stage of IM.
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The genetics of five HLA-D specificities (Dw1, Dw2, Dw3, Dw4 and Dw6) have been assessed in 21 normal families with four or more children. The HLA-D traits, as defined by typing response against homozygous typing cells, normally behave as dominant characters. The data support the concept of allelic factors. The locial flaw in the basic algorithm of MLC typing (HLA-D typing), i.e. to draw positive conclusions from negative observations, has been amply reinforced in the following studies. Five assignments could not be verified genetically under the assumption of dominant traits. Homozygous lack of specific response genes is among the mechanisms proposed as a cause for the phenomenon which has not yet been fully explained. The estimated magnitude of the frequency of false assignments is approximately 10%.
HLA--A,--B,--C and--d typing and intra-familial MLC studies in 21 couples and their 91 children revealed three B, D recombinants in 165 informative haplotypes. This corresponds to a B,D map distance of 1.8 centiMorgans, which is somewhat higher than previous estimates based on intra-familial MLC chessboard experiments. All three recombinants are maternal.
A patient suffering from aplastic anaemia was treated by bone-marrow transplantation from an ABO- and HLA-identical, MLC- and CML-negative, unrelated donor. MLC and CML became positive after transplantation indicating that a cellular immune response had developed against lymphocyte determinants not recognized prior to sensitization in vivo. Whether these determinants are governed by genes of the HLA region is unknown at present.
The object of this study has been to evaluate the recipient's immunological reactivity towards donor lymphocytes in relation to rejection episodes. All recipients (20) of local necrokidneys during 1976 were immunologically monitored immediately before transplantation and subsequently twice weekly for donor-specific complement dependent lymphocytotoxic (CDC) antibodies, antibody dependent cell-mediated cytotoxicity (ADCC) and cell-mediated lympholysis (CML). Experiments were performed until graft removal or dismissal (approx. 1100 patient days). Clinical diagnosis of rejection was made independently of immunological results. All clinically suspected rejection episodes, except one, were checked by microscopy. A positive CML-test accompanied 9 out of 11 rejection episodes; the test was negative on all other occasions. Positive CDC and ADCC tests exhibited no obvious correlation with rejection episodes: positive ADCC may be more frequent in clinically uncomplicated phases. Positive CML did not generally precede clinical graft rejection. Positive CML before transplantation was observed in two cases and was followed by irreversible, accelerated acute rejections. The CML-test may prove a reliable tool in rejection diagnosis and may yield results comparable with graft biopsy without inflicting any risk on the patient.
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