Article

Quantitative assay of antigenic disparity at HL-A—The major histocompatibility locus in man

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Abstract

We have extended the method of one-way stimulation in mixed leukocyte culture tests as previously described to quantitate different degrees of stimulation. To demonstrate that the amount of stimulation is immunogenetically meaningful, siblings and parents in families in whom genotyping on the basis of leukocyte antigen data was possible were tested. The prediction that cells of siblings differing from the responding sibling by both alleles at HL-A, stimulate more than do cells of siblings differing by only one allele, was realized in every case. One exception, with cells of a parent, is discussed. It is stressed that the differences measured here are probably fairly strong ones in the majority of cases, and that lesser differences cannot yet be detected reproducibly.

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... However, little is known about cell cycle kinetics, and quantitative aspects of the cellular proliferative response upon stimulation. When cultures are harvested (at the appropriate time after stimulation), the uptake of [3H]thymidine ([3H]TdR)I by responding, dividing lymphocytes varies with the concentration of the stimulant (mitogen [5-7], or allogeneic cells [8,9]). The exact cellular mechanism responsible for this variable response remains unexplored. ...
... However, little is known about cell cycle kinetics, and quantitative aspects of the cellular proliferative response upon stimulation. When cultures are harvested (at the appropriate time after stimulation), the uptake of [3H]thymidine ([3H]TdR)I by responding, dividing lymphocytes varies with the concentration of the stimulant (mitogen [5][6][7], or allogeneic cells [8,9]). The exact cellular mechanism responsible for this variable response remains unexplored. ...
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Being able to track donor reactive T cells during the course of organ transplantation is a key to improve the graft survival, to prevent graft dysfunction, and to adapt the immunosuppressive regimen. The attempts of transplant immunologists have been for long hampered by the large size of the alloreactive T cell repertoire. Understanding how self-TCR can interact with allogeneic MHC is a key to critically appraise the different assays available to analyze the TCR Vβ repertoire usage. In this report, we will review conceptually and experimentally the process of cross-reactivity. We will then highlight what can be learned from allotransplantation, a situation of artificial cross-reactivity. Finally, the low- and high-resolution techniques to characterize the TCR Vβ repertoire usage in transplantation will be critically discussed.
... In this case, part of the difficulty in demonstrating an additive response may stem from the use of nonsaturating concentrations of antigen in the culture. In support of the present concept, Albertini and Bach (24), using human donors, demonstrated greater magnitudes of response when mitomycin C-treated leukocytes possessing two different strong H isoantigens determined by two different alleles, rather than one, at the HL-A locus were employed as stimulatory cells. ...
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... Bone marrow transplantation (BMT) represents a quintessential cell therapy approach wherein healthy stem cells are used to reconstitute diseased tissue. Longterm success with BMT was first achieved in 1968 when infants with X-linked lymphopenic immune deficiency and Wiscott-Aldrich syndrome were treated with allogeneic transplantation (Albertini and Bach, 1968;Gatti et al., 1968). With advances in histocompatibility testing and donor registries, BMT continues to develop into technique with better survival rate and less toxic effects (such as graft-versus-host-disease). ...
... (1) determination of immunodeficiencies by testing the in vitro lymphocyte responsiveness of patients with diseases such as Hodgkin's disease (Jackson, Garret & Craig, 1970;Han & Sokal, 1970), chronic lymphocytic leukaemia (Han, 1971), thymic hypoplasia (Steele et al., 1972), combined immunodeficiencies (Ra'dl et al., 1971), subacute sclerosing panencephalitis (Lischner, Grover & DeForrest, 1971), etc.; (2) drug sensitivity testing (Halpern, Ky & Amache, 1967); (3) reactivity to tumour cells (Nagel & Holland, 1970); and (4) histocompatibility testing using mixed leucocyte cultures (Bain, Vas & Lowenstein, 1964;Bach & Hirschorn, 1964;Dausset, Ivanyi & Ivanyi, 1965;Albertini & Bach, 1968;Polet, 1972). This increased use of short-term lymphocyte cultures has pointed to the need for the development of new techniques and standardization of culture methods. ...
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Studies were designed to provide some explanation for the unexpectedly large proportion (2%) of parental rat strain peripheral blood lymphocytes that are reactive in the mixed lymphocyte interaction (MLI) to a strong homologous transplantation isoantigen(s) present on cells from an F1 donor. The possibilities considered involve nonspecific activation and multispecific reactivity on the part of the responding cells. The essential findings of this study were: (a) In 3-way mixed cultures, lymphocytes obtained from tolerant animals were not "recruited" to proliferate in the presence of cells from normal, isologous donors which were in the process of responding to F1 cells bearing the tolerance-inducing antigens. With the use of chromosome markers and normal and tolerant parental strain donors of different sexes, the responsive cells were identified and proved to be derived from the normal and not the tolerant donor. (b) The magnitude of the proliferative response is increased additively when potentially reactive cells are exposed to two antigen systems simultaneously. On the other hand, doubling the "gene-dosage" of the genetic determinants of the H isoantigens employed had no effect on the responding cells. (c) A state of induced immunologic tolerance to one H isoantigen system did not alter the response capacity of cells from such a donor to an alternative antigen system. (d) Mixed cultures of heterologous cells from human and rat donors displayed a proliferative response which was less than that of homologous mixed cultures from human or rat donors. Prior sensitization of rat donors with human cells, however, greatly increased the mitotic activity of rat lymphocytes stimulated with human cells. These results suggest that the large number of responsive cells in the MLI do not include a significant number recruited or activated in some nonspecific manner. Rather, they appear to be fully specific in their response capacities so that a given lymphocyte does not react to a multiplicity of different antigens. The degree of proliferation depends on the number of different antigen systems presented to the responding population and not on the number of genetic determinants or "gene dosage" of a given isoantigen system. Finally, on a cell-for-cell basis, the peripheral blood lymphocyte population contains more cells reactive to histocompatibility isoantigens within the species than to heterologous antigens of a different species.
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Chapter
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This chapter deals with the new and exciting developments in MLC (multi-level cell) typing in human histocompatibility studies. The genetics, specificity, and biological implications of human mixed-lymphocyte culture reaction are also discussed. The use of homozygous cells from specific delineates at least six different distinct MLC antigens that can be recognized by B-cell-specific alloantisera and clearly relate to the Ia antigens of the murine system. Certain disease associations and the genes involved in the complement components, appear more closely linked to the MLC genes than to the other components of the HLA (human leukocyte antigen) system. The MHC (major histocompatibility complex) in man HLA is defined by four distinct loci: (1) HLA-A, (2) B, (3) C, and (4) D. Three of these loci code for alloantigen are readily detectable by serological methods (HLA-A, B, and C). The fourth locus (HLA-D) controls lymphocyte responses in the in vitro mixed-lymphocyte culture reaction (MLR). Three important characteristics shared by the genes or gene products of each locus are also discussed. The study of genetic control of immune response and that of clinical transplantations emphasizes the significance of the HLA-D segment of the HLA complex. The chapter summarizes the major achievements in the clarification of the genetic control and polymorphism of the serologically detectable HLA-A, B, and C determinants.
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The mixed lymphocyte reaction has been optimised with Ficoll--Triosil separated lymphocytes: 100,000 responding cells with 100,000 mitomycin-C-blocked stimulating cells, in 0.2 ml of TC 199 with 10% AB-negative known normal serum in Cook microplates. After incubation at 37 degree C for 120 h, 2 muCi of [3H]thymidine was added, harvesting after a further 15 h at 37 degree C. A pool of stimulating cells, freshly separated from 5 normal subjects (usable up to 36 h if kept at 4 degree C), produced a Gaussian normal range of increment in CPM from 17,000--88,000, with a between-batch coefficient of variation for triplicates of 22%. For tests against stimulating cells from a single donor a transformation index above 1.64 was significant.
Article
It now appears unequivocal that three markers exist in a linkage group in chromosome 6 of man: HLA-A, HLA-B and PGM3 (Fig. 1.) Tentatively, two other HLA loci and one Ir gene have been mapped close to HLA-B. The probable map order is HLA-A - HLA-C - HLA-B - HLA-D - Ir. The biological functions of these loci are unknown. However, HLA-A, B and C are important in allograft rejection. Other closely linked loci (HDR, CML) appear to be important in the first events of the allograft rejection (first set) and in generation of killer cells. HLA-D might be important in cellular recognition and graft-versus-host reactions (matching at HLA-D decreases the incidence and severity of graft-versus-host disease), and the Ir genes in the defense against infections. HLA-B and HLA-D loci are important markers in studies of disease susceptibility. HLA-B locus antigens HLA-B27 and HLA-B8 are frequently associated with arthritic or autoimmune disorders. HLA-D determinants have been found in association with multiple sclerosis and C2 deficiency (HLA-DW2); juvenile diabetes and Addison's disease (HLA-DW3) and adult type of rheumatoid arthritis (HLA-DW4).
To compare microtitre plates with flat-bottomed and round-bottomed wells and to standardize a method for mixed lymphocyte culture (MLC), the effects of cell number, culture time, 3H-thymidine concentration and labelling time were studied. On both plates, allogeneic cells induced increased RNA synthesis beginning at about 30 hours and increased DNA synthesis beginning at about 50 hours, if suitable cell numbers were used. On plates with flat-bottomed wells, 1.5 X 10(5) responding and stimulating cells per well had near-exponential growth on day four and five, often through day six; on plates with round-bottomed wells the corresponding cell number was 0.25-1.0 (optimally 0.5) X 10(5). Near these cell numbers, the response depended closely on the number of responding cells. On plates with flat-bottomed wells, stimulating cells had a dose-dependent effect on the response, whereas on plates with round-bottomed wells, increasing the stimulating cell dose did not consistently strengthen the response. Both plate types proved suitable for MLC experiments; plates with round-bottomed wells have the obvious advantage of requiring smaller cell numbers. 3H-thymidine (spec, act 2000 mCi/mmol) self-suppressed its incorporation, which increased only slightly or even decreased if labelling time exceeded 12-18 hours. Relative responses remained virtually unaltered, however, with 3H-concentrations of 0.5 and 2.0 micronCi/ml and with labelling times of 8 and 24 hours.
Article
Suspensions of isolated peripheral lymphocytes were frozen to --95degreesC using a programmed freezing apparatus and dimethyl sulphoxide as a cryoprotective agent. In comparisons among fresh cells, frozen cells, and cells stored in storage medium, freezing was found to be the best method of storage with retention of almost the same immunocapacity as fresh cells and with the same coefficients of variation for results after stimulation with phytohemagglutinin, pokeweed mitogen, concanavalin A, purified protein derivative, and allogenic cells in mixed lymphocyte cultures as was obtained with fresh cells. Blast transformation was found to be dependent on the number of cells in the cultures, the amount of [14C]thymidine added, and the amount of phytohemagglutinin used by independent of the amounts of pokeweed mitogen and concanavalin A. The maximum responses for normal lymphocytes and lymphocytes from uremic patients after stimulation with phytohemagglutinin occurred simultaneously, but after stimulation with allogenic cells maximum response was obtained earlier for "uremic" than for normal lymphocytes. It was concluded that frozen-stored lymphocytes are suitable for in vitro quantitative measurements of the cellular immune response in an immunological sequential study provided that the above mentioned factors are well defined.
Article
A migration inhibition factor (MIF) assay to detect cellular immunity to tumor antigens in man is described. This assay utilizes a 48-hr incubation period of lymphocytes with the tissue homogenate and subsequent assay of the supernate for inhibition of the migration of guinea pig peritoneal macrophages. With this method MIF is not demonstrable in the supernate of lymphocytes cultured with histoincompatible tissue homogenates and the assay can be applied to homologous as well as autologous tumor homogenates. It can thus be used to detect tumor immunity in patients and their healthy relatives or contacts.
Article
The evidence is reviewed that a single genetic system, the major histocompatibility locus in man, HL-A, determines most of the antigens measured by presently available leukocyte isoantisera, and also controls reactivity in one-way mixed leucocyte culture tests. Studies in 12 families are presented to support this conclusion. Some interesting exceptions to the general typing-MLC tests correlation are presented and discussed.
Article
A single locus with 15 or more alleles controls reactivity in mixed leukocyte culture tests. Genes at this locus also control most of the specificities measured by cytotoxic antiserums to leukocytes. Both of these tests can be used to predict survival of skin grafts. It is proposed that this is the major histocompatibility locus in man.
Article
In order to study the cumulative effect of mouse histocompatibility antigens, donor-host combinations were utilized which differed at 2, 3, 4, and multiple histocompatibility loci. Double difference pairs involving the Y-linked and one of several autosomal loci were produced by grafting from a male of one strain to a female of its congenic resistant partner strain. Double difference pairs were also produced by grafting between strains each having a single distinct autosomal difference from a common congenic resistant partner. Triple-difference pairs were produced by using a male donor and a female recipient, each having a different single autosomal histocompatibility difference from a common congenic resistant partner. A quadruple-difference pair was produced by using a male donor and a female recipient, one having a double autosomal histocompatibility difference from a common congenic resistant partner. The inbred strains C57BL/10 and 129 were utilized to study multiple gene differences. Grafts exchanged between such double, triple, quadruple, and multiple-difference pairs showed a cumulative effect if the discrepancies between the median survival times of the constituent pairs were not large. Conversely no cumulative effect was shown if the discrepancies between the median survival times of the constituent pairs were large. It made no difference if the pairs differed at non-H-2 loci or at a non-H-2 locus and the H-2 histocompatibility locus. In the absence of strong histoincompatibilities, otherwise weak and insignificant immunogens became signifi- (C) Williams & Wilkins 1966. All Rights Reserved.
Article
The evidence is reviewed that a single genetic system, the major histocompatibility locus in man, HL-A, determines most of the antigens measured by presently available leukocyte isoantisera, and also controls reactivity in one-way mixed leucocyte culture tests. Studies in 12 families are presented to support this conclusion. Some interesting exceptions to the general typing-MLC tests correlation are presented and discussed.
Article
We have developed an improved method for the mixed leukocyte culture test. Control values, as determined by rates of incorporation of thymidine, are very low, allowing evaluation of low levels of stimulation in homologous cell mixtures. One-way stimulation is assayed by treating the cells of one individual with mitomycin C; treated cells cannot respond (incorporate thymidine) but can still stimulate homologous untreated cells to do so.
Article
A procedure was described for the separation of lymphocytes, polymorphonuclear (PMN) leukocytes and monocytes on Garvin’s glass bead columns. Dextran-sedimented leukocyte suspensions were added to columns and incubated at 37 C. Lymphocytes were eluted with fresh serum. The columns were then washed completely free of erythrocytes, platelets and lymphocytes while PMN leukocytes and monocytes continued to adhere. PMN leukocytes and monocytes were released from the glass with EDTA. Monocytes appeared in the effluents somewhat later than the PMN leukocytes, permitting a separation. A fresh serum, heat labile, Ca+ +- and Mg+ +-requiring PMN leukocyte and monocyte adherence promoting factor was demonstrated and found to be essential to the procedure. The viability of column-separated cells was shown by their non-staining with trypan blue, motility, phagocytic ability, oxygen consumption, and survival or development in tissue culture. In tissue culture, macrophages developed only from monocytes, whereas Nowell’s blast-like, dividing, phytohemagglutinin cells were produced only in cultures containing lymphocytes.
The cumulative effective of histocompatibility antigens. Transplantation. 4.'605. 8. Rabinowitz, Y. 1954. Separation of lymphocytes, polymorphonuclear leukocytes and monocytes on glass columns, including tissue culture observations
  • R J Graft
  • W K Silvers
  • R E Biuingham
  • W H Hildermann
Graft, R. J., W. K. Silvers, R. E. BiUingham, W. H. Hildermann, and G. D. Snell. 1966. The cumulative effective of histocompatibility antigens. Transplantation. 4.'605. 8. Rabinowitz, Y. 1954. Separation of lymphocytes, polymorphonuclear leukocytes and monocytes on glass columns, including tissue culture observations. Blood. 23:811.
Tissue alloantigens in humans. Identiticafion of a complex system (Hu-1) In Histocompatibility Testing
  • J Dansset
  • P Ivanyi
  • D Ivanyi
Dansset, J., P. Ivanyi, and D. Ivanyi. 1965. Tissue alloantigens in humans. Identiticafion of a complex system (Hu-1). In Histocompatibility Testing 1965. Munksgaard, Copenhagen. 51.