LD Antigens Research Articles

Cytolytic T lymphocyte responses to metabolically inactivated stimulator cells: II. Effect of a soluble “costimulator” factor(s) in primary and secondary mixed leukocyte culture

The ability of a concanavalin A-stimulated spleen cell supernatant (“costimulator”) to overcome the effects of impaired CD and LD antigen presentation by metabolically inactivated stimulator spleen cells was examined in the primary and secondary cytolytic T lymphocyte (T c) response. (i) Cells inactivated by ultraviolet irradiation or mild glutaraldehyde treatment, which were unable to stimulate primary cytolytic activity on their own, generated near maximal responses in the presence of costimulator. The 30-fold lower efficiency of splenic membrane fragments as antigen in primary MLC with the supernatant indicated that the damage to immunogenicity caused by membrane isolation was not equivalent to that caused by uv light and glutaraldehyde, as has previously been assumed. (ii) Comparison of the relative effects of antigen and costimulator demonstrated that costimulator played the dominant regulatory role in primary MLC, increasing sensitivity to suboptimal antigen doses 10- to 30-fold; neither antigen nor the supernatant appeared preferentially to control the strength of the secondary response. (iii) Metabolically inactivated adult and untreated neonatal spleen cells failed to release costimulator activity in response to concanavalin A. However, the ability of the neonatal cells to induce a primary cytolytic response suggested that costimulator production by the stimulator cells themselves is not essential for primary T c activation, and supports the hypothesis that the lack of primary immunogenicity of inactivated spleen cells reflects their failure to induce costimulator production by the responder population.

Identification of a BALB/c H-2Ld gene by DNA-mediated gene transfer.

Gene transfer and immunoselection were used in the identification of a BALB/c genomic clone containing an H-2Ld gene (clone 27.5). Transformation of thymidine kinase-negative C3H mouse L cells with the cloned 27.5 DNA together with the herpes simplex virus tk gene produced transformants expressing Ld molecules detected by radioimmune assay with monoclonal hybridoma antibodies to Ld antigens. The foreign Ld gene products expressed by cloned mouse L cell transformants were shown to be virtually indistinguishable from BALB/c spleen Ld molecules by two-dimensional electrophoretic analysis of H-2Ld immunoprecipitates. These results indicate that the genomic clone 27.5 contains a functional BALB/c H-2Ld gene and demonstrate the usefulness of this approach for identifying the gene products encoded by cloned genes which are members of a multigene family. Furthermore, the ability to place cell-surface recognition molecules on the surfaces of foreign cells provides a powerful opportunity for functional analyses of these molecules.

Flow microfluorometric analysis of H-2L expression.

Abstract The cell surface expression of H-2L, a major transplantation antigen, was compared by flow microfluorometry to the expression of products of H-2K and H-2D loci, using monoclonal antibodies. By this methodology, the ontogeny and tissue distribution of Ld antigens were found to be indistinguishable from those of the K and D antigens. In a reciprocal blocking assay, using fluorescein-labeled test reagents, it was shown that monoclonals anti-H-2.65 and anti-H-2.64 did not inhibit the binding of each other. These results suggest that the alloantigenic determinants H-2.64 and H-2.65 are located at distinct sites on Ld molecules. Quantitative comparisons using the fluorescein-labeled monoclonal reagents indicated that Ld molecules are expressed at 2- to 3-fold lower levels on the cell surface compared with K and D molecules. These findings give new credence to a "3-locus" model for the major histocompatibility complex of man and mouse, where H-2L and HLA-C share several homologies that are unique and distinguish them from the other histocompatibility loci.

Immunochemical evidence in two haplotypes for at least three D region-encoded molecules, D, L, and R.

Sequential immunoprecipitation analyses have defined a new transplantation antigen, designated R, which in addition to the 2 previously isolated D and L molecules is encoded in the D region. All 3 of these gene products are 45,000 m.w. glycoprotein, and each bears a unique combination of specificities as recognized by monoclonal and/or conventional anti-H-2 sera. Three D region products, D, L, and R, have now been isolated from soluble antigens of both the H-2d and H-2q haplotypes. The resulted reported here also indicate that the loss-mutant BALB/c-H-2dm2 fails to express both Ld and Rd antigens. Further chemical comparisons of the primary structure of R molecules with D and L molecules will now be necessary to determine whether R antigens are the products of a unique gene, as opposed to a glycosylation or conformational variant of D and/or L molecules. In either case, the discovery of 3 D region-encoded gene products in certain haplotypes raises new questions about the evolution and regulation of expression of H-2 loci.

Primed lymphocyte testing in pigs: Conditions for the generation of primed cells and their response characteristics

Pig peripheral blood lymphocytes, typed for LD antigens by negative reactions to homozygous cells in primary MLRs and for SD antigens by complement-mediatedlymphocytotoxicity, were sensitized in vitro against X-irradiated lymphocytes. These “primed” cells were rechallenged with lymphocytes possessing a range of LD and SD phenotypes and were found to respond by rapid proliferation specifically to LD antigens of the type expressed by the original stimulating cell, but not to SD antigens. Third party LD antigens, not expressed by the original sensitising cell, or by the responder, caused a significant proliferation, but primed cells could be rendered operationally monospecific by reducing the number of responding cells until the third party reaction became negligible. Conditions fot the generation and testing of these primed pig cells are described here.

Cell-free factor substitute for "signal 2" in generating cytotoxic reactions.

AbstractCytotoxic T lymphocytes in the responding cell population recognize allogeneic target antigens on ultraviolet light (uv)‐treated stimulating cells in a mixed leukocyte culture. However, under these conditions no cytotoxic response is generated unless the responding cells are also stimulated with x‐irradiated or mitomycin C‐treated cells differing by the H‐2 I region‐determined LD antigens. We refer to the signal given to the cytotoxic T lymphocyte by its target antigen as signal 1, and to the help which the cytotoxic T lymphocyte receives presumably from the LD‐responsive T helper cell as signal 2.We have developed a method in which different cell populations are separated by an agar layer to demonstrate that a soluble “factor(s)” can both potentiate a weak cytotoxic reaction as well as allow the development of cytotoxicity against uv‐treated stimulating cells. These results support the two‐signal model in which a “factor”, presumably produced by helper T cells responding to LD antigens, can give signal 2 to the cytotoxic T lymphocyte that has recognized target determinants.

Genetic basis of memory in cell-mediated immune response.

The present experiments were undertaken to delineate the role of LD and SD antigens in terms of memory in cell-mediated immune response (CML). Using recombinants that differ for either the H-2K or H-2D determinants (SD-different) and other recombinants differing for the central region of H-2 (LD-different), we have investigated this question. The results indicate that priming to SD determinants leads to a more rapid and higher response when cells are restimulated in vitro with either the same SD antigen alone or the same SD plus an LD antigen and tested on target cells that possess either the SD antigen alone or both the LD and SD antigen. Antigens in the central regions of H-2, which includes LD, serve as the stimulus both for the helper T cells and for the cytotoxic T lymphocytes. Priming with a high dose of LD antigens leads to memory in the cytotoxic T lymphocyte recognizing the cytotoxic target antigens coded by the central region of H-2. Nevertheless, memory could not be detected when priming was done under the same conditions as for SD antigens. Moreover, a primed helper T-cell response does not facilitate the development of increased CML on restimulation in vitro with the LD antigen used for priming and a new SD antigen. Thus, it seems that memory in CML is in the cytotoxic T lymphocyte and not in the proliferating helper cells.

Genetics of transplantation: the major histocompatibility complex.

A genetic region called the major histocompatibility complex of MHC (which plays an important role in the control of graft survival) has been defined in a large number of different species. Several different loci of the MHC have been described, including loci coding for transplantation antigens. These antigens can be divided into two categories: first, the SD antigens that were originally defined serologically and that appear to function as targets for the killer lymphocytes involved in the rejection of a graft and second, the LD antigens that were originally defined by lymphocyte response in the mixed leukocyte culture test and that stimulate helper T lymphocytes. In addition, there are genes for other antigens (referred to as immune response-associated or Ia antigens) and genes that control the ability of an animal to respond immunologically to antigenic stimuli, the immune response or Ir genes. There is evidence for epistatic interaction between these genes in that immune recognition of LD and SD antigens lead to a more pronounced development of cytotoxic (killer) cells than does stimulation by either antigen alone. In addition, the genetic control of immune responsiveness appears, at least in some systems, to reside in two very closely linked genes that may function better in the cis than in the transposition. This latter finding suggests a possible explanation for the strong linkage disequilibrium found between genes of the MHC. A number of new tests have been described to define the antigens of the MHC. Results of these tests have been used for studies of transplantation immunology but in addition to study the very strong associations that exist between some of the MHC antigens in man and various diseases.

Generation of cytotoxic lymphocytes in vitro against autologous human leukaemia cells.

NORMAL lymphocytes cultured with allogeneic cells in one-way mixed leukocyte culture (MLC) respond proliferatively to the “lymphocyte-defined” (LD) antigens and subsequently cytotoxic cells are generated against the target antigens on the stimulating cells1. In contrast, when responding lymphocytes are cultured with stimulating lymphocytes that differ with respect to target antigens but not LD antigens, proliferative and cytotoxic responses are not easily generated; the addition of a second stimulating cell differing from the responding cell with respect to LD antigens in a ‘three-cell’ experiment allows the generation of cytotoxic lymphocytes directed against target antigens1–4. Consistent with results of others5–9, we have detected low but significant MLC responses after culturing remission lymphocytes with irradiated autologous leukaemia cells; however, cytotoxic lymphocytes were not generated. Using the ‘three-cell’ approach, where remission lymphocytes, irradiated leukaemia cells and irradiated allogeneic stimulating cells were cocultured, we have generated lymphocytes specifically cytotoxic for autologous human leukaemia cells.

Cellular basis of neonatal induction of in vitro tolerance.

The LD and SD antigens of the major histocompatibility complex subserve differential roles in the induction of the proliferative phase in mixed lymphocyte culture and in the cytotoxic reaction seen in cell-mediated lympholysis. The present study suggests that they also behave differently in the neonatal induction of tolerance. SD antigens appear to induce tolerance in the cytotoxic T lymphocytes very effectively, whereas LD antigens (or the cytotoxic targets coded by genes in the I and/or S regions) are relatively ineffective in this regard. LD antigens presented neonatally are effective at inducing tolerance in the proliferating helper cells.

Secondary cell-mediated lympholysis: importance of H-2 LD and SD factors.

Lymphocytes stimulated in mixed leukocyte cultures and left for 13-17 days, i.e. beyond their peak proliferative and cytotoxic reactivities, can be restimulated to give a secondary-type rapid and strong proliferative and cytotoxic response when confronted with cells of the original sensitizing cell donor. We have concerned ourselves primarily with the requirements of restimulation for the presence of LD and/or SD stimuli on the restimulating cells. (a) The low level cell-mediated lympholysis (CML) associated with LD differences in a primary CML can be restimulated to give a secondary-type response by those same LD antigens. (b) If the original sensitizing cells differ from the responding cells by both LD and SD antigens, restimulation with only the LD antigens, or third-party cells presumably carrying cross-reactive LD antigens, can restimulate the secondary CML responses directed against the SD antigens on the original sensitizing cells. (c) The presence of SD antigens on the restimulating cells that are cross-reactive with the primary sensitizing SD antigens (as determined in a primary CML) leads to the preferential activation of cytotoxic T lymphocytes reactive to those antigens although maximum cytotoxicity is still directed at cells carrying the original sensitizing SD antigens. A model to explain these results is presented.

On the significance of HL-A and LD antigens in myasthenia gravis.

Annals of the New York Academy of SciencesVolume 274, Issue 1 p. 451-460 ON THE SIGNIFICANCE OF HL-A AND LD ANTIGENS IN MYASTHENIA GRAVIS* Ritva Pirskanen, Ritva Pirskanen Department of Neurology University of Helsinki Helsinki, FinlandSearch for more papers by this author Ritva Pirskanen, Ritva Pirskanen Department of Neurology University of Helsinki Helsinki, FinlandSearch for more papers by this author First published: May 1976 https://doi.org/10.1111/j.1749-6632.1976.tb47706.xCitations: 17 † Supported in part by grants from the Finnish Cultural Foundation, the Finnish Medical Foundation, and the Paulo Foundation. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume274, Issue1Myasthenia GravisMay 1976Pages 451-460 RelatedInformation

Collaboration between in vivo responses to LD and SD antigens of major histocompatibility complex.

STUDIES using mixed leukocyte culture (MLC) and cell mediated lympholysis (CML) tests, have suggested a possible differential role for LD and SD antigens associated with the major histocompatibility complex (MHC, H–2 in mouse) in allograft reactions1–3. LD differences are primarily responsible for stimulating the proliferative response in MLC; the SD antigens are the major targets in CML1. The presence of LD differences on the stimulating cell in MLC enhances the cytotoxic response against the SD antigens. The cellular basis for this potentiation of CML is that two separate subpopulations of T lymphocytes are differentially reactive to LD and SD antigens1,3–5. A proliferating helper cell responds primarily to LD differences whereas cytotoxic T lymphocytes are more strongly activated by the SD antigens. Lafferty et al.7 and Talmage et al.10 have shown that allogeneic thyroids transplanted under the kidney kapsule are rejected less rapidly if they are first cultured in vitro. Injection of peritoneal exudate cells from the thyroid donor into the recipient speeds up rejection. They suggest that the peritoneal exudate cells sensitise the recipient and that the sensitised cells can invade and reject the thyroid. A further refinement in the interpretation of their data might suggest that either in addition to the mechanism they propose or as an alternative to it the peritoneal exudate cells provide an LD stimulus to the recipient's proliferating helper T cells, and that these cells either by themselves or through a secreted helper factor potentiate the development of cytotoxic T lymphocytes directed at the SD antigens on the graft. Our experiments reported here are consistent with this model.

Differential function of major histocompatibility complex antigens in T-lymphocyte activation.

We have emphasised the functional dichotomy of MHC LD and LD antigens as well as the differences in cellular responses to these antigens. Perhaps in so doing we have failed to stress adequately the similarities that exist. But while the similarities (for example skin graft rejection associated with both K and I region differences) are so very clear, the differences have best allowed our progressive understanding of MHC induced cellular responses from the perspective stressed in this article. Of greatest importance to our understanding of these transplantation antigens are the potentially differential roles for the LD and SD antigens in the complex series of events that are collectively referred to as the "allograft reaction". It has been suggested that these differences may be "merely quantitative". This possibility has been discussed repeatedly in our previous reports on the distinction of LD and SD. In fact, the great bulk of biological phenomena can be reduced to quantitative differences. It would seem to us that sufficient evidence for such differential activity exists to make the LD-SD dichotomy model an heuristically valuable one for purposes of designing future experiments. We have discussed the clinical relevance of this model elsewhere. Many authors have speculated and evidence has been gathered to suggest, that cell surface antigens associated with the MHC are important in developmental and other cell interactions. Some studies have directly addressed the question of the need for MHC compatibility to allow cell interaction to proceed optimally. It thus seems most appropriate that the genetic complex with which we are dealing has been termed the major histocompatibility complex; allowing for the literal interpretation of this term this may be the genetic region that by its influence on "tissue compatibility" may control critical cellular interactions in addition to those observed in allograft reactions. It is the simple good fortune for those whose attention was focused on this complex by transplantation problems to find themselves with a panorama of biological phenomena that require extensive experimental probing and integration, hopefully ultimately leading to an understanding of the MHC in a broader context than has to date been possible.

LD antigens associated with HL-A8 and myasthenia gravis.

The finding that the SD antigen HL-A8 is associated with myasthenia gravis has raised the question as to whether or not there is also an association between some specific LD antigen and myasthenia gravis and/or HL-A8. MLC technique was used to study LD antigens in 33 myasthenic patients. The cells of a myasthenic patient who was homozygous for both HL-A and LD products were used in MLC tests as stimulators with HL-A8 bearing cells from 24 myasthenics, their 17 relatives and 16 controls and to non-HL-A8 cells from nine myasthenics and 16 controls. At least three different LD genes were found to be associated with HL-A8. One of them, called LDm' was present in 17 (63 %) of the 27 HL-A8-bearing haplotypes of myasthenics, and in nine (47 %) of the 19 HL-A8-bearing haplotypes of controls. In our study LDm was not found in the 84 haplotypes devoid of HL-A8. LDm is strongly associated with HL-A8 and through this also with myasthenia. Calculated from phenotype frequencies for HL-A8 and its association to LDm' the LDm frequencies are 9 % in the control population, 30 % in myasthenics and 48 % in young females with onset of myasthenia below 35 years. LDm had no indendent correlation with any of the clinical parameters of myasthenia gravis, even though is was found more often in HL-A8+ females with the onset of myasthenia before 35 years than in the whole myasthenic group. LDm appears to be similar to LD8a antigen.

Stimulation of mixed lymphocyte cultures and cytotoxic responses: evidence that T cells express SD but not LD antigens, whereas B cells express both.

By separating T and B cells from stimulator spleens it has been found that mixed lymphocyte response (MLR) and cytotoxic cells are generated in responder T cell populations by B but not by T stimulator cells in a wide variety of strain combinations. It is suggested that MLR-stimulating or LD antigens are expressed on B but not T cells, and that their presence is necessary in order to generate cototoxic responses to H-2K and/or H-2D (or SD) antigens which are present on both T and B cells. The finding that B cells and macrophages stimulate MLR is consistent with the hypothesis that Ia antigens, expressed on both these cell types but not on nylon column purified peripheral T cells, are the MLR-stimulating or LD determinants.

Human lymphocytes recognise mouse alloantigens.

H-2 LD antigens1 of the mouse major histocompatibility complex and the lymphocyte-activating determinants of the Mls (ref. 2) locus induce vigorous lymphocyte proliferation and enhance generation of cytotoxic effector cells in allogeneic combinations (ref. 3 and D. J. Schendel, and F. H. Bach, unpublished). These antigens do not serve well as targets for cell-mediated lympholysis (CML)—a function which in the mouse is performed by the classical H-2 determinants, the SD antigens4,5. A similar differential function of LD and SD antigens is found in human allograft reactions in vitro6.