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Quantitative assay of antigenic disparity at HL-A—The major histocompatibility locus in man
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.
... 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. ...
Lymphocytes, stimulated with different doses of plant mitogens or allogeneic cells, incorporate varying amounts of [3H]thymidine. Theoretically, this may be due to different numbers of responding cells, to earlier proliferative response of these cells, and/or to their more or less rapid transit through the cell cycle. Dog peripheral blood lymphocytes were stimulated in vitro with different doses of phytohemagglutinin (PHA), or with allogeneic lymphocytes. After their synchronization by incubation with hydroxyurea (4 mM), the mean durations of the cell cycle, and of the different cell cycle phases were constant and unrelated to strength or type of stimulation. PHA-stimulated lymphocyte cultures were maintained in the presence of colchicine, to prevent clonal proliferation of responding lymphocytes. DNA uptake in this setting, attributed to first generation responders, was related to the strength of proliferative lymphocyte response in control cultures without colchicine. Furthermore, cell proliferation occurred earlier with greater stimulation. It is concluded that higher [3H]thymidine uptake in vitro by stimulated lymphocytes is due to greater numbers of responding cells, which are triggered into proliferative response earlier, and not to a more rapid transit of the responding cells through the cell cycle.
... Using a mixed lymphocyte reaction (57), it has been shown that 1-10% of T cell in peripheral blood can be activated (58). As mentioned before, the number of HLA mismatches between donor and recipient is a primary driving force that mobilized a larger fraction of T cells than nominal antigens. ...
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. ...
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.
... 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. ...
Use of lymphocyte cultures for in vitro studies such as pretransplant histocompatibility testing has established the need for standardization of this technique. A microculture technique has been developed that has facilitated the culturing of lymphocytes and increased the quantity of cultures feasible, while lowering the variation between replicate samples. Cultures were prepared for determination of tritiated thymidine incorporation using a Multiple Automated Sample Harvester (MASH). Using this system, the parameters that influence the in vitro responsiveness of human lymphocytes to allogeneic lymphocytes have been investigated.
One-way mixed lymphocyte cultures were established between related cell donors A (haplotype designated ) and B (). The cells from A, proliferating in response to stimulation by mitomycin treated cells from B, were eliminated from the culture by a hot pulse of 3H-thymidine. A marginal response was observed when the remaining cells from A reencountered additional stimulating cells from B, or cells from an HL-A identical sibling to B. In addition, the remaining responding cells were virtually incapable of responding to secondary stimulation by family member C (), who shared one haplotype (b) with individual A and the other haplotype (c) with the individual stimulating cell donor B. The MLC secondary stimulation response to family member D (), who differed from A by both haplotypes, but shared one haplotype with B, was reduced to approximately 50% of control values. In other experiments it was found possible to completely eliminate the response of A () to D () by using a combination of stimulating cells from related donors B () and C () in the initial hot pulse MLC.Separate populations of responding cells reactive to antigenic products of each major histocompatibility system haplotype is a likely explanation of these observations.
A 2-year-old boy with the Wiskott-Aldrich syndrome has been treated by bone-marrow transplantation. An initial attempt to transplantation without extensive preparation of the recipient failed. Subsequently the recipient was prepared with a 4-day course of cyclophosphamide followed by bone-marrow transplantation from a sister identical at HL-A??? the major histocompatibility locus in man. 6 weeks after transplantation the patient is chimaric and has shown no evidence of graft-versus-host disease.
Unidirectional mixed lymphocyte culture (MLC) reactions were investigated to determine the feasibility of assessing in vitro histocompatibility in two species of marmosets. When lymph node cells from white-lipped marmosets were mixed in allogeneic or xenogeneic MLCs, there was an enhanced incorporation of 14C-thymidine in comparison to autologous mixtures. Peak response was at 5 days. In contrast, cotton-top marmoset allogeneic mixtures showed weak responses with no enhanced label uptake when cultured for a period of 8 days. When blood lymphocytes were cultured in the same medium used to assay histocompatibility with lymph node cells, there was little difference between the values for MLCs and responding cells alone. This might be explained by the fact that marmoset blood is known to transform blastically and proliferate myeloid elements in vitro. Modification of the culture medium produced significant responses in xenogeneic MLCs with blood lymphocytes; however, allogeneic MLC values were not significantly different from tolerant cotwin MLC values. Lymph node MLC response with cells from a kidney graft recipient that died on day 302 postsurgery failed to show specific sensitization to donor cotwin antigens
Xenogeneic mixed leucocyte cultures composed of human, chimpanzee, baboon, goat, sheep, pig, and dog cells were set up with a variety of plasma culture supplements. The characteristics of the human leucocyte response to xenogeneic cells was similar to its response to allogeneic cells. Peak response to xenogeneic stimulation occurred on the same culture day as the peak response to allogeneic stimulation. Similar numbers of xenogeneic and allogeneic cells produced peak stimulation of cells from any one individual. There was, however, a wide variation in the response of human lymphocytes to both allogeneic and xenogeneic cells. A factor in the plasma supplement specific for responding or stimulating cells inhibited the mixed leucocyte response in some combinations and could be removed by absorption techniques.
Sera and IgG fractions from normal subjects as well as patients with rheumatoid arthritis and systemic lupus erythematosus were added to mixed leukocyte reactions (MLC) in an attempt to study the effects on cell-to-cell interactions in such systems. Seven and seven-tenths percent of 52 RA sera and 26% of 27 SLE sera showed suppression of MLC reactivity. MLC suppression appeared to be localized to IgG serum fractions separated by DEAE chromatography. This suppressive effect showed a strong correlation with presence of lymphocytotoxic antibody in individual sera.
Addition of mercaptoethanol to the culture medium has permitted a mixed leukocyte interaction (MLI) to be achieved with mouse spleen cells in a chemically defined protein-free medium. Results which confirm those obtained in culture medium supplemented with heterologous sera are: 1) the maximum incorporation of isotope occurred between 60 and 72 h of culture; 2) the index of stimulation was 3–15; 3) the number of responder and stimulator cells required for maximum incorporation was 3 x 106 – 4 x 106 and 4) immunization resulted in a higher level of incorporation that occurred earlier than the maximum response of normal cells. Other results obtained confirm previous reports which are in apparent conflict: i.e., 1) stimulation of parental strains (P1) by F1 hybrids (P1 x P2) may be either equal to or one-half that induced by the other parental strains (P2); and 2) F1 hybrids may or may not be stimulated by the parental strains. These latter two results were found to be dependent on the strains used. Other advantages of the system are discussed in terms of the elimination of artifacts that occur in medium containing serum and the usefulness in examination of molecular events occurring early in immune reactions.
A Danish family with twelve members and with two first cousin marriages was studied. Two inbred siblings (designated J) were found to be homozygous for HL–A2,27 (AJ) and therefore also presumably homozygous for all or most of the major histocompatibility complex. When used as stimulators in one-way MLC, these two individuals elicited a very low response in five members of the family possessing this same HL–A haplotype, which was clearly disjunct from the response by family members not possessing the HL–A2,27(AJ). When J cells were used as stimulators against cells from 27 unrelated individuals, the responses against J – expressed relative to the median response of a given responder against several unrelated persons – fell into two clearly disjunct groups. One group of seven gave low responses, from 13% to 41%. The individuals of this group are designated J-like. The rest gave responses from 83 % to 132 %. In one family with three children where the father was J-like and the mother was not J-like, the trait J-like segregated in coupling with the HL–A determinants. It is postulated that these specifically low responding, or J-like, individuals possess the MLC allele j, for which the individuals J are homozygous. Among the 27 unrelated individuals tested, there was no obvious association between the MLC allele j and any particular HL–A antigen. Another inbred HL–A homozygous individual did not stimulate the family members at a low level in compatible combinations and is thus presumably not homozygous at the MLC locus.
Gravida and related cord blood lymphocytes were studied in one-way mixed lymphocyte culture to find out if tolerance or sensitization existed between these organisms. Maternal lymphocytes were consistently stimulated by both related cord blood and adult allogeneic lymphocytes, and the kinetics of the reactions did not suggest that any specific tolerance or sensitization existed toward fetal histocompatibility antigens. Cord blood lymphocytes were stimulated by related maternal lymphocytes in approximately one-half the tests, and also seemed less reactive toward adult allogeneic cells. However, the temporal kinetics of the reaction did not suggest any sensitization or tolerance toward maternal lymphocytes. The findings suggest that the success of the human pregnancy in terms of cell-mediated immunity does not depend upon intrinsic cellular maternal-fetal tolerance.
In the mixed-leukocyte reaction (MLR) the variance in the peak responses by cells from normal individuals is small when pools of stimulating cells from three donors are selected to include representatives of each of four groups of crossreacting HL-A specificities. In contrast, the peak responses of cells from normal individuals to pools of stimulating cells from three donors chosen at random showed a larger variance. The largest variance was present when the stimulating cells came from a single donor.
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.
Publisher Summary This chapter discusses the cytotoxic effects of lymphoid cells in vitro . The chapter discusses the complex problem of different types of cytotoxic effects of lymphoid cells. These outstanding workers in the field have managed to present a cohesive picture of the various effects on the target cells. The role of “nonspecific” factors is particularly well clarified. The interrelationships among contact lysis, release of pharmacologically active substances, and the terminal components of the complement system are given in the chapter for special consideration. In an in vitro model, it is shown that lymphoid cells from sensitized donors destroy tissue culture cells carrying the antigen to which the cell donor is sensitized. This type of cytolytic reactions is encountered in a great variety of immune situations, comprising all those mentioned in the chapter. The cell that initiates in vitro cytotoxic reaction is assumed to be the sensitized lymphocyte, equipped with its own recognition sites for antigen on the cells that are destroyed. Although this may be true in many situations, it now seems clear that “normal” lymphoid cells can become cytotoxic to other cells by a variety of pathways. The study of the various pathways by which lymphoid cells can become cytotoxic has been helpful for the understanding of effector role of these cells in cell-destructive reactions in general.
Human lymphocytes proliferate in vitro in response to foreign histocompatibility antigens that are present on allogeneic lymphocytes. Within a population of immunocompetent lymphocytes there are specific subpopulations that respond to allogeneic cells from different individuals. A means of selectively eliminating such subpopulations is suggested.
Post-transplant administration of a soluble donor spleen extract (SSE), in combination with a limited period of immunosuppression, prolonged baboon hepatic allograft survival. The extract was a soluble glycoprotein and was extracted from donor spleen cell membranes with Freon 113, followed by purification by gel exclusion chromatography. SSE was able to combine with and absorb out antidonor alloantibody from a polyvalent alloantiserum. SSE did not stimulate allogeneic lymphocytes in culture, and increasing concentrations of SSE progressively inhibited transformation of allogeneic lymphocytes. SSE also inhibited the stimulation of allogeneic lymphocytes by irradiated lymphocytes autologous with SSE. In an animal surviving for more than 7 months after transplantation, evidence of enhancement was the demonstration by mixed cell agglutination (MCA) of alloantibodies in its serum directed against 5 of a random panel of 10 baboons, despite the continued presence of a viable graft. At 200 days the lymphocytes of this animal in culture were not stimulated by stored pretransplanted donor liver cells, but were well stimulated by indifferent liver cells and lymphocytes.
16 patients underwent renal transplantation from a sibling donor who was prospectively determined to be ABO compatible and HL-A identical with the recipient. Unidirectional mixed leukocyte reactions were performed; in each instance, lymphocyte stimulation in either direction was not observed. The plasma creatinine 10-68 months after transplantation in these 16 patients ranged between 0.9 and 1.9 mg/100 ml. The creatinine clearance ranged from 48 to 113 ml/min, and the blood urea nitrogen (BUN) ranged between 12 and 35 mg/100 ml. Urine protein excretion varied from 0.11 to 1.86 g/day. Six patients exhibited no detectable clinical episodes of acute rejection; they were treated with azathioprine alone and each of them demonstrated normal or near normal renal histology when biopsy specimens were obtained more than 6 months after transplantation. Nine patients experienced acute rejection episodes that required the use of steroid therapy. The severity of these rejection episodes was variable; they included a mild reduction in renal function with an immediate steroid-induced restoration of function and eventual discontinuance of steroid therapy to severe reduction in function requiring prolonged and moderate doses of steroids without return to normal renal function. Renal histological observations in this group ranged from mild to marked cellular and structural changes which fit the criteria of the rejection. One patient demonstrated a gradual loss of renal function with heavy proteinuria. Biopsy of this allograft demonstrated the recurrence of original disease, i.e., lobular glomerulonephritis. The marked variability in the clinical course and allograft morphology in these 16 patients could be explained by antigenic differences at non-HL-A loci. The presence of minor histocompatibility loci has been well documented in other mammalian species and they are most certainly present in man. The need for their identification and definition is stressed.
A review aiming to give a short survey of comparative aspects of major histocompatibility systems (MHS) in different species would certainly be much too lengthy if it did not limit itself to selected aspects. The main object of this review is simply to point out that, in all species which have been examined in sufficient detail, the genetic systems show remarkable similarities. The second aim of this review is to try to point out the possible general significance and/or the common evolutionary forces which could have led to the development of these genetic complexes. This review goes into many hypotheses and presumptions, a number of them being the expression of the author’s personal conception, which may serve as a working hypothesis. We shall give more space to recently published data. These data will be discussed in detail, altough some of them are not yet fully understood or confirmed, as they may serve for alternative explanations.
The major histocompatibility locus in man has been named HL-A and the mixed lymphocyte reaction (MLC) has been felt to be an in vitro measure of the locus. In this report we propose a more complex histocompatibility system and term this the HL-1 chromosomal region. Results suggest that the MLC locus is linked to HL-A and not a product of the HL-A locus itself. Studies in a family in which recombination between the genes determining the antigens of the first and second HL-A loci were observed suggested that the locus controlling this reaction was closely linked to the second HL-A locus; differences involving second-series antigens provoked strong stimulation but first-locus antigenic differences did not. Linkage of immune response genes (Ir) is suggested by the correlation of HL-A with mean skin allograft survival times (MST) in families. The skin graft survival times were not always predictable by the degree of MLC stimulation. The absence of absolute correlation of allograft survival with either HL-A or MLC suggests that a genetic factor (Ir) linked to HL-A rather than HL-A itself might provide primary control of delayed-type hypersensitivity and that HL-A, MLC, and Ir comprise a series of closely linked loci that make up the majority transplantation region in man-HL-1.
Die gemischte Leukocytenkultur (MLC) ist ein wichtiger Test in der immunologischen Spenderauswahl. Sie ist besonders geeignet, um identische Lebendspender innerhalb der Familie zu finden. Als Abstoungsreaktion in vitro gibt sie einen guten Hinweis auf zu erwartende Immun-reaktionen in vivo. Die Methodik und ihre Vor- und Nachteile werden beschrieben, ihre Anwendungsmglichkeit bei verschiedenen Transplantationen wird, auch anhand eigener Beispiele, diskutiert.The mixed leucocyte culture is an important test in the immunologic donor selection. It is particularly suited to find identical living donors within the family. The in vitro homograft reaction serves as a good indicator for the immunological reaction in vivo. The method and its advantages and disadvantages are described; its scope of application in the field of transplantation is discussed, including also the author's own observations.
The generation of an immune response is a complex process requiring multiple interactions and cellular events. The genetic elements participating in immune recognition and response appear to serve as permissive signals, the “green lights” of the immune network. Their interaction provides the basis for coordinated immune function. We summarize here recent information on the nature of these inherited elements and discuss the implications of these findings for genetically determined disease susceptibility.
The introduction of mixed leukocyte culture testing (MLC) [5,13] combined with the establishment of a one-way method of stimulation in MLC [6], allowed the correlation of proliferative response in MLC with results of serological studies and the suggestion that reactivity in MLC is controlled by the major histocompatibility complex (MHC) in man [4] now known as HLA. That the chromosomal region of HLA which controls the strongest stimulatory determinants for MLC (now known as the HLA-D region) was different from the class I encoding genes was already suggested in two early studies [2, 7]; definitive evidence in this regard came from the studies by Yunis and Amos [46].
Allogeneic lymphocytes mixed in vitro ("mixed cultures") undergo a series of changes characterized by increased synthesis of RNA, protein, and DNA resulting in morphological transformation (1-3), provided that the lymphocytes originate from individuals who are incompatible at the major human histocompatibility locus. Syn- thesis of DNA occurs 2-3 days after mixing and mitoses appear from the 3rd to the 5th day. Similar cellular changes occur in lymphocytes stimulated by phytohemagglutinin (PHA)* (4). This growth stimulating effect of PHA has been investigated by means of cytophotometric techniques (5-7). Fluorescence measurements were carried out on in- dividual fixed leukocytes stained with acridine orange (AO) which binds to the nega- tively charged phosphate groups in nucleoproteins (8). A few minutes after adminis- tration of PHA, a pronounced increase in fluorescence intensity was found, due to an increased AO binding to the deoxyribonucleoprotein (DNP) complex of the stimulated cells. No change in the cellular amounts of nucleic acids occurred during this time (5, 6). It was concluded that the increased AO binding was due to an unscreening of pre- viously masked phosphate groups in DNA, most probably as a result of weakened bonds between the DNA and the protein moiety in the DNP complex. Such DNP changes might be of importance for the later synthesis of RNA and blast transforma- tion (5-7).
The mechanism of growth stimulation in allogeneic lymphocytes mixed in vitro was studied at the cell level by means of cytophotometric
techniques. A pronounced increase in fluorescence intensity of fixed and acridine orange (AO) stained lymphocytes was observed
as soon as after 1–3 hr in mixed culture. No increase in the amount of DNA took place during this time. The higher fluorescence
intensity was due to an increased accessibility of AO binding sites in the deoxyribonucleoprotein (DNP) complex, most probably
as a result of weakened bonds between the DNA and the protein moiety in the DNP complex. Similar DNP changes have been found
in other systems of growth stimulation and may be one prerequisite for later induction of cellular synthetic processes.
Increased AO binding only occurred when the lymphocyte donors were incompatible at the major histocompatibility locus (HL-A);
there was no change in AO binding in cases of HL-A identity. The AO binding reaction probably reflects a specific recognition
of HL-A antigens, whereas other antigenic discrepancies between the individuals do not seem to cause an analogous response.
Positive associations between the antigens D(R)3 and D(R)4 and negative associations of D(R)2 have been reported with insulin dependent or type I diabetes mellitus. It has been suggested that susceptibility factor(s) associated with the D(R)3 and D(R)4 haplotypes and a resistance factor associated with the D(R)2 haplotype may be involved in the pathogenesis of the disease. We propose a hypothesis herein which attempts to unify these findings based on our present understanding suggesting (a) the existence of multiple antigenic determinants associated with any one D(R) haplotype and (b) the sharing of “single” D region encoded determinants between what we now refer to as different D(R) haplotypes. The hypothesis, in its simplest form, focuses on a single D region determinant which can be found associated at different frequencies with the various D haplotypes as potentially explaining the findings.
Cancer is associated with increased consumption of both platelets and fibrinogen within tumor tissue, and the turnover rate of these factors is related to the type and extent of disease. Thrombocytopenia is the major cause of bleeding; it occurs when the disease involves marrow or spleen, thus either limiting platelet production or increasing sequestration. Although platelet function inhibitors prevent tumor induced consumption of both platelets and fibrinogen, these agents would not be expected to provide helpful therapy for bleeding problems, because they cause platelet dysfunction and because platelet consumption is a less prominent cause of the thrombocytopenia than the impaired platelet production or increased pooling. Anticoagulation is similarly not useful, because it only interrupts fibrinogen consumption and therefore does not affect bleeding due to thrombocytopenia. Patients with cancer associated venous thromboembolism are benefited by the antithrombotic effect of anticoagulant therapy, but not by platelet function inhibitors. Clearly, thrombocytopenic bleeding, in addition to consumption associated with malignancy, is most effectively treated with antitumor therapy to reduce or remove the underlying lesion. In the actively bleeding patient, interim management consists of platelet transfusion rather than manipulation of the hemostatic system with antithrombotic drugs.
In order to utilize the mixed lymphocyte reaction (MLR) as an assay of T-lymphocyte competence, pools of target lymphocytes obtained from different individuals are used to increase the magnitude and decrease the variation of the in vitro response. We evaluated variations in MLR response due to variations in target cell populations. Response increased with an increased target/responder cell ratio. Peak response occurred with a target/responder cell ratio of between 1:1 and 1:4. Response to a pool of lymphocytes from different individuals increased as the number of individuals contributing to the pool increased. Peak stimulation occurred with three to four different donors to the target cell pool. Stimulation produced by pooled target cells resulted in a higher mean index of stimulation and decreased variation of response as compared to stimulation produced by target cells from individual donors. Stimulation produced by pooled target cells was approximately equal to the sum of the stimulation produced by each of the target cell populations acting alone. These findings indicate a practical method of modifying the MLR as a test of T-lymphocyte function.
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.
Several normalization procedures have been applied to single MLC-typing experiments in order to neutralize experimental variation and hence to secure assignment of HLA-D phenotypes. The present paper concerns a statistical description and isolation of experimental and biological factors in repeated MLC-typing experiments. The statistical model used gives a meaningful description of the variables in MLC as judged by its application to MLC-typing data of HLA-identical siblings. Subsequent testing of the model in an HLA-A, -B,-C and -D-genotyped family material provides evidence that the level of typing response to selected homozygous typing cells may be influenced by factors coded for by the homologous HLA-D region. It is, however, concluded that such evidence must be accepted with caution due to the (for the present statistical purpose) suboptimal design for the HLA-D typing experiments and hence the relative insensitivity of the method. A general method for locating erroneous triplicates is devised.
When human lymphocytes are cultured for 9 to 14 days with stimulating cells of a family member differing by a single HL-A
haplotype they become "primed" to recognize specific HL-A LD (mixed lymphocyte culture) antigens. These primed lymphocytes
respond specifically and rapidly when "restimulated" with cells of a person that contain the same LD antigens as those of
the priming haplotype. Specific HL-A LD antigens can be detected within 24 hours by this primed LD typing.
The mixed lymphocyte reaction (MLR) between donor and recipienTlymphocytes has been measured by the macrophage electrophoretic mobility (MEM) test and the modified (MOD-MEM) test. Its value as a measure of compatibility has been assessed by comparison with conventional HL-A serotyping and with the outcome of renal transplantation.
Thirty-six living donor/recipient pairs and 59 cadaver donor/recipient pairs for transplantation have been studied. Whilst uniovular twins gave lymphocyte interactions, measured as macrophage slowings of about 1 %, the slowing produced by paired allogeneic lymphocytes ranged from 2 % to 26 % depending on the number of HL—A matches.
The test measurement of lymphocytic interaction was significantly correlated with histocompatibility measured by HL—A serotyping, in both living and cadaver donors. One way MEM-MLR showed the dominant role of the second HL—A sublocus in mixed lymphocyte reactivity. The long term success of the renal graft correlated with the pre-transplant initial reaction between donor and recipienTlymphocytes.
The test has advantages in the field of human histocompatibility assessment since no particular reference to individual antigens is made and it may be performed in a matter of hours.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.