Mixed Leukocyte Culture Test Research Articles

A 50-year retrospective: cell-mediated immunity and the major histocompatibility complex

HE AREA in which I was most involved in transplantation is best summarized by focusing on the cellmediated immune responses to antigens of the major histocompatibility complex (MHC). Many of these reminiscences are discussed in the text of my Medawar Lecture, which is also included in this volume. In the period around 1960, one could appreciate from the work of Gowans, Brent, Medawar and others that lymphocytes were both the cells that expressed the transplantation antigens and that responded to those antigens. Thus, in an attempt to develop an in vitro model of the then-called homograft (now, allograft) reaction, I mixed lymphocytes of two individuals in the hope that the antigens on the cells of each would stimulate the lymphocytes of the other to form blasts and divide. This was the result obtained and was the initiation of the mixed leukocyte culture (MLC) test, 1 from which we then developed a one-way MLC test, 2 in which the stimulating cells of the potential donor were treated with either mitomycin-C or x-irradiation. 3 The one-way MLC test has been used as a histocompatibility test to match donor and recipient and has become the basis for in vitro studies of alloimmunity since. I found that one of four sibling pairs failed to stimulate each other in MLC, and there was a single polymorphic locus that controlled reactivity in MLC. 4 With Bernard Amos, we were able to show that the MLC test was controlled by the same locus that controlled the serologically defined antigens being studied by Amos, Payne, van Rood, Dausset, and others. We suggested that this was the major histocompatibility complex in humans based on skin graft experiments that each of us had performed and on the observations that in other species the MHC controlled

6.1#66 - Analysis of relative proliferative responses in mixed leukocyte culture (MLC) tests in relation to outcome of renal allografts from living related donors

6.1#66 - Analysis of relative proliferative responses in mixed leukocyte culture (MLC) tests in relation to outcome of renal allografts from living related donors

Human Marrow Transplantation: An Immunological Perspective

Publisher Summary The first successful marrow transplants were carried out with identical twins as donor for patients with acute leukemia. Later development of the one-way mixed leukocyte culture (MLC) test and alloantisera for HLA typing made it possible to select MHC-identical allogeneic donorhecipient pairs. At the same time, tolerable preparative regimens of total body irradiation (TBI) and chemotherapy capable of providing sufficient immunosuppression for engraftment of allogeneic marrow were developed, and post-transplant immunosuppressive regimens allowing at least partial control of graft-versus-host disease (GVHD) were identified. Collectively, the developments have made it possible for marrow transplantation to evolve from a last-resort experimental therapy reserved for patients with end-stage leukemia or aplastic anemia to become the first-line treatment of choice for an increasingly large number of conditions. The indications for marrow transplantation depend on the underlying disease and the degree of genetic disparity between the donor and recipient. The indications for marrow transplantation are more limited if an HLA-identical sibling is not available. Because of the increased risk of complications—such as, rejection or GVHD—marrow transplantation from a partially compatible donor should not be recommended for patients with chronic myelogenous leukemia (CML), in chronic phase or acute nonlymphocytic leukemia (ANL) in first remission, where there is a good prognosis for the immediate future or where the disease may be cured by conventional therapy. Apart from the core discussion on immunological perspective of human marrow transplantation, this chapter focuses on the four principal immunologic aspects of human marrow transplantation: hematopoietic engraftment, GVHD, immunologic reconstitution, and the graft-versus-leukemia effect.

Regulation of lytic function by recombinant IL2 and antigen.

The study of signals involved in the generation and maintenance of cytotoxic T lymphocytes (CTLs) has been facilitated by the availability of various techniques based on the mixed leukocyte culture (MLC) test (Bain et al. 1964; Bach and Hirschhorn 1964). In addition to the proliferative response in an MLC, CTLs are generated (Hayry and Defendi 1970; Hodes and Svedmyr 1970; Solliday and Bach 1970).KeywordsTissue Culture MediumLymphoblastoid Cell LineLytic FunctionMixed Leukocyte CultureStimulator RatioThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Freezing of canine lymphocytes for use in mixed leukocyte culture and cell-mediated lympholysis tests.

A satisfactory and reproducible technique of cryopreservation of canine lymphocytes for use in mixed leukocyte culture (MLC) and cell-mediated lympholysis tests has been developed. Dimethyl sulfoxide was used as the cryopreservation agent. Cells were frozen to -50 C at a controlled rate (-1 C/min) and stored at -169 C. The best preservation was obtained with a concentration of 10 x 10(6) lymphocytes/ml in Waymouth's MB-752/1 medium supplemented with 30% dog serum (MB-30). Before use in MLC or cell-mediated lympholysis tests, lymphocytes were rapidly thawed at 40 C and then rapidly diluted with MB-30 at room temperature. For best responses in MLC, one fresh component (either stimulating or stimulated cells or serum) was needed. The magnitudes of responses of frozen lymphocytes to phytohemagglutinin or in MLC were similar to those seen with fresh cells, but peak responses occurred usually 24 hr after those seen with fresh cells.

Mixed Leukocyte Culture Incompatibility Index for Donoh-Recipient Selection in Kidney Transplantation

The mixed leukocyte culture test has been applied to selection of histocompatibility non-identical donor-recipient pairs for renal transplantation. The clinical course of transplant recipients who have histocompatibility mismatches but low mixed leukocyte culture stimulation is similar to that of mixed leukocyte culture and histocompatibility identical recipients. Living donor-recipient pairs with high mixed leukocyte culture stimulation had no better graft survival than cadaver recipients.An incompatibility index derived from mixed leukocyte culture may aid in the selection of satisfactory non-identical living related donors and may help avoid use of immunologically unsatisfactory living donors.

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.

Correlation of the relative response index with marrow graft rejection in patients with aplastic anemia.

Thirty-four patients with severe aplastic anemia were treated with cyclophosphamide or total body irradiation and an infusion of marrow from a genotypically HLA-identical sibling donor. Engraftment occurred in all patients but rejection was noted in 12. Mixed leukocyte culture (MLC) tests undertaken before grafting to determine donor-recipient histocompatibility were analyzed retrospectively. The response of patient cells to sibling cells in mixed culture was compared to the response of patient cells to cells of an unrelated individual and expressed as a relative response index (RRI). Of the 12 patients who showed positive RRI to donor leukocytes in pregraft MLCs, 9 rejected their grafts, wheras of 23 patients with negative RRI only three rejected their grafts (P LESS THAN 0.005). The results suggest that the MLC test can detect individuals who are most likely to reject their HLA identical sibling graft.

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.

Genetic aspects of canine mixed leukocyte cultures.

Recent data stress the importance of matching donor and recipient of an organ graft for both the serologically defined (SD) and lymphocyte-defined (LD) determinants. To allow experimental evaluation of the effect of these SD and LD structures in a noninbred experimental animal, mixed leukocyte culture tests were performed between SD identical and nonidentical dogs to clarify the LD system in these animals. The results of these experiments can be summarized as follows: (a) In the dog there is a LD locus distinct from the known SD loci, which in all probability is localized outside the first (SD-1) series locus on the chromosome. (b) The crossing-over frequency between the SD and LD loci on the chromosome is low. (c) Studies in SD identical unrelated dogs and random unrelated dogs show an apparent high linkage disequilibrium between SD and LD loci. (d) The LD system in dogs is polymorphic.

Treatment of Established Human Graft-Versus-Host Disease by Antithymocyte Globulin

Abstract Forty successful marrow grafts have been carried out since April 1972 for the treatment of severe refractory aplastic anemia or acute leukemia. All patients were HL-A identical and nonreactive in mixed leukocyte culture tests with the marrow donor. Nineteen of the patients developed moderately severe to severe graft-versus-host disease (GVHD) and were treated with rabbit or goat antihuman antithymocyte globulin (ATG). Under ATG therapy, 12 of the 19 showed complete resolution of GVHD, five showed improvement of most organ systems involved, and two showed no change except for improvement in skin lesions. Six of the 19 became long-term survivors. Five of the six are alive between 276 and 629 days after grafting, and one died on day 346 with chronic respiratory failure. Of the remaining 13 patients, 11 died with interstitial pneumonitis of predominantly viral etiology, and two died with fungal and bacterial infections. In conclusion, the present study demonstrates the relative effectiveness of ATG in reversing human GVHD. Death with interstitial pneumonitis was the single most serious impediment to successful treatment of GVHD by ATG.

Serologically defined and lymphocyte-defined components of the major histocompatibility complex in the mouse.

The mixed leukocyte culture (MLC) test is an in vitro model of the recognition phase of the homograft response. For the most part, activation in MLC is dependent on differences of the major histocompatibility complex (MHC). Our present studies in the mouse suggest that activation is primarily associated with differences of genetic regions of the MHC other than those which control the serologically defined (H-2) antigens. These differences do not lead to cytotoxic or agglutinating antibody formation despite extensive immunization; we have called these differences lymphocyte-defined (LD) differences. The strongest stimulation in MLC is associated with differences of the Ir region. It is possible that the Ir product is the T cell receptor and that it is this same molecule which can act as the stimulatory agent in MLC. Other possibilities are discussed.

Mixed lymphocyte culture reactivity and H-2 histocompatibility loci differences

The relationship between the major histocompatibility systems as defined serologically and by the mixed leukocyte culture (MLC) test is one which has received attention in both man (1) and mouse (2). In man, studies within families suggest that cells of siblings who inherit the same HL-A chromosomes from their parents and thus are serologically identical at HL-A do not stimulate in the MLC test. Cells of siblings who inherit different HL-A chromosomes do stimulate. This, combined with the finding that cells of all of several hundred unrelated pairs stimulate in the MLC test, suggests that a single genetic system controls reactivity in MLC, that this system is highly polymorphic and either the same as that coding for the HL-A antigens or very closely linked to the HL-A (1). Similarly, differences at the H-2 system in the mouse result in MLC activation (2). The H-2 system in the mouse can be divided into two separate regions, H-2K and H-2D (3). Recently Rychlikova et al. (4) studied a series of mouse recombinant strains that differed only for the H-2D region or only for the H-2K region and other inbred strains that differed at both the H-2D and the H-2K regions. Their findings suggested that differences for H-2K alone or for both H-2K and H-2D resulted in MLC activation. Differences for the H-2D region alone. however, did not result in MLC activation in their study. We have repeated these studies using a number of recombinant strains tested in

Responsiveness of germfree mice in mixed leukocyte culture. Reexamining the nature of the responding unit.

Conventional (cv) and germfree (gf) mice are able to give a good proliferative response to allogeneic cells in the mixed leukocyte culture (MLC) test, while the response to xenogeneic stimulating cells has been in question. Previous studies by others have suggested only a low MLC response in cv animals and none in gf ones. We have found that both cv and gf animals can give a good MLC response to xenogeneic as well as allogeneic cells. These findings are of importance for our understanding of both MLC stimulation and response.

Third party mixed-leukocyte culture test: a potential new method of histocompatibility testing.

The disparity index is an expression of histocompatibility difference between two siblings with identical human leukocyte antigen (HL-A) type who are nonreactive in mixed leukocyte culture (MLC) test. This index is derived from a third-party MLC test. The disparity index was found to be correlated with the severity of graft-versus-host reaction in bone marrow transplantation between HL-A-identical siblings. The disparity index may prove to be a useful means of predicting the severity of graft-versus-host reaction and the outcome of bone marrow graft when HL-A- and MLC-matched sibling donors are studied. The third-party MLC test is offered as a new method of histocompatibility testing. It may provide a useful model for the study of immunogenetics relative to so-called weaker histocompatibility determinants in man and experimental animals.

An Adaptation of the Mixed Leukocyte Culture Test for Use in Evaluating Lymphocyte and Macrophage Function

Abstract The mixed allogeneic leukocyte culture reaction was adapted into a test in which lymphocyte and macrophage function in this system can be assessed individually. In this test, purified lymphocytes from the patient under study were stimulated to incorporate 3H thymidine by irradiated leukocytes from three allogeneic, control donors. Test donors' macrophage function was evaluated by stimulating purified lymphocytes from the three control donors with irradiated leukocytes from the patient under study. The macrophage content in cultures of unpurified leukocytes was also measured. The test proved to be sensitive and reproducible. This report shows that this test can identify specifically lymphocyte unresponsiveness, quantitative and functional macrophage abnormalities, and combined lymphocyte and macrophage defects.

Extraneous lymphocytic HL-A antigens in severe combined immunodeficiency disease.

Lymphocytes of three patients with severe combined immunodeficiency disease were markedly anomalous in having extraneous HL-A specificities. Data from family studies disclosed that the phenotypic specificities expressed were in excess of the predicted genotypic antigens. Contrary to the current theories of HL-A, which limit the number of antigens to four, as many as 12 specificities were found in one patient. No stimulation by the extraneous antigens was revealed by the mixed leukocyte culture (MLC) test with a sibling of this patient who was presumed to be genotypically HL-A-identical. Bone marrow transplantation from this genotypically HL-A-identical sibling resulted in heightening immunoglobulin levels and successful clinical outcome to the present time (11 months). Although the phenotyping anomaly disappeared 2 weeks after transplantation, it subsequently reappeared and has persisted for 5 months. A possible association of immunoglobulins and histocompatibility antigens with respect to synthesis and membrane sites is discussed.

LYMPHOCYTE REACTIVITY IN VITRO

SUMMARY One-way mixed leukocyte culture (MLC) tests were performed using, in all cases, mitomycin C-treated stimulating cells from healthy university students. Responding cells from four groups were tested: hospitalized patients with malignancy, age- and sex-matched hospitalized patients without known malignancy, age- and sex-matched nonhospitalized controls, and students who were younger than the persons in the other three groups. Whereas approximately 30% of the variation in response was unaccounted for by known variables, the great majority of the remaining variation could be accounted for by the number of polymorphonuclear leukocytes present in the culture. These results suggest that quantitation of MLC tests may be better after removal of polymorphonuclear leukocytes from the culture—a step we have now instituted in our laboratory.

Histocompatibility and Transplantation.

Meeting Abstracts1 May 1971Histocompatibility and Transplantation.Fritz Bach, M.D.Fritz Bach, M.D.Search for more papers by this authorAuthor, Article, and Disclosure Informationhttps://doi.org/10.7326/0003-4819-74-5-843_1 SectionsAboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail ExcerptThe success of transplantation in man depends to some degree on antigenic disparity between the donor and the recipient tissues. Two general approaches are available for study of this problem. Lymphocyte typing uses antisera-measuring histocompatibility antigens to define the phenotype of both donor and recipient; pairing is achieved by minimizing antigenic disparity. Mixed leukocyte culture tests involve the in vitro culture of lymphocytes of the donor and the recipient to study the amount of lymphocyte response (a measure of antigenic disparity) without defining the antigens responsible for the disparity. Results using these two methods have shown that a single genetic... This content is PDF only. To continue reading please click on the PDF icon. Author, Article, and Disclosure InformationAffiliations: Madison, Wis. PreviousarticleNextarticle Advertisement FiguresReferencesRelatedDetails Metrics Cited BySilicon Carbide, SiC 1 May 1971Volume 74, Issue 5Page: 843-843KeywordsAntigensGeneticsHistocompatibilityLymphocytesMajor histocompatibility antigensPhenotypesTransplantation Issue Published: 1 May 1971 PDF DownloadLoading ...

Mixed leukocyte stimulation of normal peripheral leukocytes by autologous lymphoblastoid cells.

Lymphocyte culture lines can be established by using buffy coat cells from normal or diseased individuals. Established lymphocytoblasts from a normal donor were used in the one-way mixed leukocyte culture test and were found to stimulate the peripheral lymphocytes of that donor. These lymphocytoblasts were also capable of stimulating allogeneic lymphocyte populations.