Primary mixed lymphocyte responses to HLA-DP

Combinations of peripheral blood lymphocytes, matched or mismatched for HLA-DP, were analyzed in primary one-way mixed lymphocyte culture experiments. Proliferative responses as correlated with tritiated thymidine uptake were assessed over a kinetic range of 5-15 days. A proliferative response was observed between DP-mismatched combinations, whereas combinations matched for DP and all other HLA alloantigens did not elicit significant proliferation. Optimal responses were observed 9 days after the combination of 1 x 10(5) responder and stimulator cells. Responses were blocked by anti-DP monoclonal antibodies. These studies demonstrate the complexity of the primary mixed lymphocyte culture system and suggest that DP alloantigens should be considered when anomalous responses are obtained.     

HLA-DP incompatibilities induce significant proliferation in primary mixed lymphocyte cultures in HLA-A, -B, -DR and -DQ compatible individuals: Implications for allogeneic bone marrow transplantation

The major part of the proliferative response in primary mixed lymphocyte cultures (MLC) is caused by HLA-DRB1 incompatibilities. In DRB1-matched pairs the proliferation induced by HLA-DRB3, -DQ and -DP mismatches may be unmasked. In most previous studies the influence of HLA-DP incompatibilities in primary MLC has been investigated in homozygous typing cells representing only a few Dw specificities. We were interested in determining the stimulatory capacity of isolated HLA-DP mismatches, ascertained by RFLP analysis, in primary MLC in HLA-A, -B, -DR and -DQ compatible, unrelated heterozygous individuals of many different Dw specificities. Thirty-eight MLCs performed with cells from related pairs and 67 with cells from unrelated pairs were evaluated. All but nine of the MLCs were analyzed in both directions, giving a total of 201 investigated reactions. The relative responses (RR) in the three MLCs performed between DP incompatible, related pairs were all positive (RR greater than or equal to 8%). Eighty of 82 MLCs performed with cells from DP incompatible, unrelated individuals were positive, whereas 37 of 46 MLCs between DP compatible, unrelated pairs were negative (RR less than 8%) (p less than 10(-10)). The magnitude of the RR was influenced by the number of DP mismatches. Thus, the mean RR was approximately twice as high in MLCs in which responder and stimulator cells differed by two DP antigens (mean RR 60.5%) compared with reactions with only one DP mismatch (mean RR 35.4%) (p less than 10(-3)). RFLP-defined HLA-DP incompatibilities predict a positive primary MLC in HLA-A, -B, -DR and -DQ matched individuals with a high degree of accuracy (98%).(ABSTRACT TRUNCATED AT 250 WORDS)     

HLA-DP related suppression of mixed lymphocyte reaction with alloactivated lymphocytes

We studied the influence of HLA class I and class II antigens on the suppression of the MLR induced by primed lymphocytes (PLs) alloactivated in vitro. The suppression of 14 different PLs of 83 MLRs was analyzed. The PLs were primed against (i) HLA-DP (SB) (ii) HLA-DR/DQ or (iii) both HLA-DP and DR/DQ. The suppression was analyzed with special reference to the sharing of HLA-antigens between (i) the stimulator in the MLR and (ii) the stimulator generating the PL. HLA-DP and HLA-DR/DQ antigens were equally capable of generating suppressor cells. When these cells were added to MLRs, the specific stimulators induced the strongest suppression (74%), while allogeic cells sharing class II antigens induced a slightly weaker suppression (66%). The suppression related to HLA-DP (60%) was almost identical to that related to HLA-DR/DQ (59%). The HLA-A, B, C related suppression was of the same magnitude (58%). The unspecific suppression (40%), i.e. no relation to known HLA-antigens, was significantly lower than the class II related suppression, but not significantly lower than the class I related suppression. The suppression of the MLR did not seem to be caused by cytotoxic cells, consumption of lymphokines, nor changes in the kinetics of the MLR. Thus, HLA-DP antigens can-like DR/DQ antigens - induce PLs with the ability to suppress the MLR in an HLA-class II (DP or DR/DQ) related, and possibly a class I related, as well as an unspecific fashion.     

An analysis of the effect of HLA-DP in the mixed lymphocyte reaction

It has recently been reported that HLA-DP antigens may play an important role in the development of graft-versus-host disease (GVHD) following transplantations of haploidentical bone marrow as a treatment for haematological malignancies. Mixed lymphocyte culture (MLC) is routinely performed prior to bone marrow transplantation to assess the suitability of the donor, and we have therefore examined the role of HLA-DP in this test. One-way MLC chequerboard experiments were performed between 17 HLA-Dw3 homozygous typing cells (HTC) with a range of HLA-DP antigens represented, including HLA-DPw1, w2, w3, w4 and CP63. The experiments were performed on multiple occasions and each time a highly significant difference (P = less than 0.001) was observed between the Relative Responses (RR) in the HLA-DP matched responder/stimulator pairs and the HLA-DP mismatched pairs. There was, however, considerable overlap in these results with ranges in the HLA-DP-matched group RRs of 0-17%, and 0-62% in the mismatched group. Only 3.1% of the HLA-DP-matched grou had a RR greater than 5%, while 48% of the HLA-DP mismatched group had a RR greater than 5%. From these results it was calculated that a positive response (greater than 5%) has a 96% chance of being due to an HLA-DP disparity of one or two antigens. Conversely, with a negative MLC the chance of their being no HLA-DP antigen disparity was only 65%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphocyte transformation in vitro. III. Mechanism of stimulation in the mixed lymphocyte culture

In view of the correlation between the mixed lymphocyte culture and the HL-A locus, experiments were performed to determine whether the reactivity in the mixed lymphocyte culture is elicited by the HL-A antigens as such.

Fresh lymphocytes were mixed with either allogeneic fibroblasts or allogeneic lymphocytes treated with various metabolic inhibitors or by heating which abolishes their capacity to tranfsorm in vitro.

No stimulation of the normal untreated lymphocytes was observed in any of these mixed cultures.

However, the HL-A antigens 4b and 7b were not affected quantitatively by this treatment as determined serologically.

The conclusion is drawn that mixed lymphocyte culture reactivity is not merely a reaction to HL-A antigens, and the consequences of this are discussed.

     

Disparity in HLA-DR typing and mixed lymphocyte culture reactivity

Prospective HLA-DR matching has been shown to enhance renal graft survival. It has also been demonstrated that the major stimuli in the one-way mixed lymphocyte culture reaction (MLR) are the DR rather than DQ class II histocompatibility antigens. A two antigen DR match between the potential recipient and donor is usually associated with a negative MLR. The findings of two family studies are reported in which HLA-DR identity between the potential recipient and donors did not correlate with the MLR reactivity. These findings further strengthen the need for performing the MLR in conjunction with DR/DQ typing since splits have been described for some of the DR antigens which may not be detected using the currently available commercial typing trays.     

Response of agammaglobulinaemic lymphocytes in mixed lymphocyte culture

Seven-day mixed lymphocyte cultures (MLC), were established from three sex-linked agammaglobulinaemics and nine acquired agammaglobulinaemics, five males and four females, with normal individuals of the opposite sex. Phytohaemagglutinin cultures were established at the same time. Except for two females with acquired agammaglobulinaemia, all the subjects responded normally to PHA stimulation. Individual MLC response was gauged by analysis of sex chromosome constitution of the dividing cells. The three sex-linked agammaglobulinaemic patients failed to respond in MLC. The male acquired agammaglobulinaemic patients responded at variable rates or not at all while the female acquired agammaglobulinaemic cells responded at least as well as the male cells with which they were cultured. In control MLCs involving the cells of pairs of normal individuals of opposite sex, we observed, consistently, that the female cells responded to a greater extent than did the male.     

Comparison with monoclonal anti-Ia antibodies of antigenic determinants inducing lymphocyte proliferation and immune interferon production in primary and secondary murine mixed lymphocyte reactions

The nature of Ia antigens inducing lymphocyte proliferation and immune interferon (IFN-γ) production in primary and secondary murine mixed lymphocyte cultures was studied by means of monoclonal anti-Ia antibodies. In primary mixed lymphocyte cultures, both lymphocyte proliferation and IFN-γ production were found to be controlled by Ia determinants coded for by the I-A subregion. E alloantigen-recognizing antibodies displayed consistent stimulation of both lymphocyte functions, instead of inhibition, suggesting a regulatory mechanism of the E molecule on lymphocyte activation. In secondary mixed lymphocyte cultures fewer Ia determinants seemed to be involved in lymphocyte proliferation, since only antibodies directed against specificities Ia.17 and Ia.19 were found capable of blocking lymphocyte proliferation. In contrast, IFN-γ production, although significantly decreased by antibodies against specificities Ia.17 and Ia.19, was not completely abrogated, even by a mixture of all anti-Ia antibodies. The data indicate that (a) in primary mixed lymphocyte reaction lymphocyte proliferation and IFN-γ production are triggered by the same Ia specificities coded by the I-A subregion and (b) in secondary mixed lymphocyte reaction, whereas lymphocyte proliferation is restricted to fewer determinants, IFN-γ production shows a lower threshold of activation, being induced also by determinants ‘silent’ in primary mixed lymphocyte reaction.     

Purification and characterization of a mouse T cell replacing factor from primary mixed lymphocyte culture supernatant

The purification and chemical characterization of a T cell replacing factor (TRF), which was induced in the supernatant of primary mixed lymphocyte cultures for 24 hr under serum-free conditions, has been carried out. This TRF was sensitive to proteolytic enzymes, trinitrobenzene sulfonic acid and sodium metaperiodate, while it was stable in buffers of a wide pH range (pH 5–10) and to freezing and thawing treatment. After purification of the TRF by DEAE Sephadex A-50 ion-exchange chromatography, preparative disc electrophoresis and gel filtration on Sephadex G-100, the final product revealed only one band after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Chemical analyses of the purified TRF showed that it was a protein, abundant in serine, valine, glycine, glutamic acid, almost devoid of basic amino acids and that it contained approximately 10% carbohydrate. Furthermore, the purified TRF described in this paper was found to consist of two identical subunits of molecular weight of 21,000 ± 3000 daltons.     

Generation of immunoglobulin secreting cells in mixed lymphocyte culture

The nature of the cellular interactions and role of the HLA system in the generation of immunoglobulin secreting cells in primary and secondary mixed lymphocyte cultures were investigated. The B lymphocyte response to alloantigen stimulation as measured by a Protein A reverse hemolytic plaque assay, consisted of polyclonal activation with production of IgG, IgM, IgA secreting cells detectable as early as day 4 in a primary and by 24 hr in a secondary mixed lymphocyte culture. B cell activation was shown to be dependent upon collaboration with T helper cells. A disparity at the HLA D/DR region between responding and stimulating cell populations was required for the induction of T helper cells. However, once activated, T helper cells could collaborate with autologous or allogeneic B lymphocytes and, without additional antigen, trigger immunoglobulin production. The mixed lymphocyte culture may now be considered a model of B cell as well as T cell activation.     

Recalcified plasma as nutrient additive in mixed lymphocyte culture

Animal or human blood protein is a costly but necessary additive to tissue culture. This supplemental protein is provided by the addition of pooled serum or heparinized plasma to standard tissue culture media. Many blood centers store CPDA-1 anticoagulated plasma, a form that does not provide optimal support of mixed lymphocyte culture (MLC). The optimal amount of CaCl2 (1 ml of 1 M CaCl2/100 g) added to citrate plasma and the use of glass vessels result in a completely clotted product that is comparable in MLC support to commercially available pooled human serum. Laboratories that have access to CPDA-1 plasma can replace the growing demand for serum with recalcified plasma without sacrificing quality.     

β-Lactamase-producing bacteria in mixed infections

Beta-lactamase-producing bacteria (BLPB) can play an important role in polymicrobial infections. They can have a direct pathogenic impact in causing infections, as well as an indirect effect through their ability to produce the beta-lactamase. BLPB may not only survive penicillin therapy themselves, but can also protect other penicillin-susceptible bacteria from penicillin by releasing free beta-lactamase into their immediate environment. This phenomenon occurs in upper respiratory tract, skin, soft tissue, surgical and other infections. The in-vitro and in-vivo clinical evidence supporting the role of BLPB in the increasing failure of penicillin to resolve such infections, and the implications of this phenomenon for the management of infections, are discussed.     

Human Ia-like DRw lymphocyte antigens stimulating activity in primary mixed lymphocyte reaction.

The intensity of the primary mixed lymphocyte reaction (MLR) seems to be influenced by at least two distinct determinants of the HLA-D region: the HLA-Dw stimulating products and the human Ia-like B lymphocyte DR (D-related) antigens. In three families, the primary MLR is significantly higher in case of HLA haploidentity, when stimulating and responding cells differ with regard to both Dw and DRw determinants, than when only Dw products are different. Thus, an additional effect of DR antigens during primary MLR is observed. The role of DRw antigens in primary MLR and the results obtained by a primed lymphocyte test which can discriminate between Dw3 and DRw3, indicate that DRw and Dw products could be distinct determinants.     

Rapid responses of lymphocytes in an optimized mixed lymphocyte culture

When adherent mononuclear cells are removed from whole blood, the separated lymphocytes respond rapidly by proliferation to foreign cocultured lymphocytes. The reaction of responding cells against a pool of foreign stimulating cells can be detected in this rapid mixed lymphocyte culture (r-MLC) within 28 h and antigenic disparity between family members, differing for only one HLA haplotype, can be measured within 36 h. These early responses show genetic specificity corresponding to those observed in a standard MLC at 96 h. The involvement of similar cell populations in the regulation of early responses to allogeneic cells and increased responses to autologous cells in r-MLC, as well as accelerated responses in secondary MLC, is discussed.     

Polymorphism of the mixed lymphocyte culture response in the rabbit

We have investigated the amount of polymorphism of the mixed lymphocyte culture response in unrelated or distantly related rabbits from 6 independent sources. Nine RLA-D alleles have been isolated in the homozygous state with evidence for at least 2 additional alleles. These estimates are similar to the mixed lymphocyte polymorphisms found in the rat and in man.     

Role of children's sex in mixed mother-child lymphocyte culture reactivity

One-way mixed mother-child lymphocyte cultures (MMCLC) 4 to 20 years after the last delivery were studied with maternal responding and children's or fathers' stimulating cells (MMFLC) in 14 multiple child families with 18 sons and 20 daughters. HLA antigen typing locus A, B, DR was performed for all family members. As reported previously for newborn cells, a significantly increased maternal response could be observed in MMCLC with male as compared to female children's stimulating cells. Although the number of cases studied was small, it seems that the increased stimulating effect of male children's cells could also be observed when MMCLC values from children of different sex, and identical A,B,DR haplotypes were compared. In contrast to this, A,B,DR haploidentical children of the same sex seem to have a similar stimulating effect on the maternal response in MMCLC. The results suggest that male children's Y-chromosome-correlated minor histocompatibility antigens may additionally stimulate the maternal immune response in MMCLC.     

Chinese hamster lymphocyte cultures. Relationship between lymphocyte proliferation,cell concentration,culture time and culture area

Different numbers of Chinese hamster lymphocytes were cultured in microtiter plates with flat-, round- and V-bottomed wells for different culture times. The smallest number of cells could be stimulated in plates with V-bottomed wells. At least 25,000–50,000 cells and a longer culture time than for round- or V-bottomed plates were required for maximal stimulation in flat-bottomed plates.For a given well conformation the optimal day of culture is earlier with higher and later with lower cell concentrations.The optimal culture time for a given number of cells is shortest in V- and longest in flat-bottomed plates.The amount of PHA producing the highest thymidine incorporation for a given number of cells depends upon the well conformation. It decreases with lower cell concentration and increases with longer culture time.     

Alloresponses of cord blood cells in primary mixed lymphocyte cultures

The aim of this study was to compare the alloreactive responses against HLA antigens of cord blood cells with those of adult peripheral blood cells. In primary mixed lymphocyte cultures and bulk cell-mediated lympholysis experiments cord blood cells demonstrated significantly decreased proliferation and cytotoxicity. Experiments analyzing the specificity of anti-HLA cytotoxic T lymphocytes (CTL) revealed that cord blood (CB) CTL reacted only partially with third-party cells expressing the stimulating HLA antigens. Lower frequencies of IL-2 producing helper, cytotoxic T-cell precursors and IL-4 producing CB cells were found, whereas the frequencies of IFN-gamma producing cells, as determined by ELISpot experiments, were equivalent to the frequencies of adult IFN-gamma producing cells. Our results imply that, although CB cells have significantly decreased proliferative and cytotoxic alloresponses in bulk mixed lymphocyte cultures, their IFN-gamma production is comparable with that of adult mononuclear cells. Preserved production of IFN-gamma may be a risk factor for the development of graft-versus-host disease and should be taken into consideration when evaluating the possibility for stem cell transplantation with HLA-mismatched CB.