Evaluation of the mixed lymphocyte culture (MLC) assay as a method for selecting unrelated donors for marrow transplantation

Abstract: The utility of the MLC assay as a test of HLA-D region matching and predictor of graft-versus-host disease (GvHD) was evaluated in 435 patients receiving marrow grafts from unrelated donors. Donors and recipients were phenotyped for HLA-A, B and DR antigens by serology, tested in MLC, and retrospectively genotyped for DRB1, B3, B4, B5, DQB1 and DPB1 alleles by PCR/SSOP. Of the 244 HLA-A, B, DR-identical donor-recipient pairs with evaluable MLC and DRB1 typing results available, 208 were matched for HLA-A, B and DRB1, while 36 were matched for HLA-A and B and mismatched for a DRB1 allele. Donor anti-recipient relative responses (RR) in MLC, corresponding to the GvHD vector in marrow transplantation, ranged from 7.2 to 100%, with a median of 4.0%. A comparison of reactivity in MLC between pairs matched versus mismatched for DRB1 alleles showed a significant overlap in the distribution of RRs. Using optimally-defined RR cutoffs of 4 and 16%, no correlation between MLC results and risk of developing clinically significant grades III-IV GvHD (p=0.6 and 0.5, respectively) was found when the contribution of DRB1 mismatch was accounted for. Matching for DRB1 alleles, in contrast, was a better predictor of clinically significant GvHD, with DRB1-matched transplant recipients less likely to develop grades III-IV GvHD than DRB1-mismatched recipients (p=0.14). Among the 208 patients and donors matched for DRB1 alleles, the MLC, although reactive (RR > 4.0%) in 45% of cases, did not predict GvHD. Overall, these results underscore the limitations in using the MLC to predict DRB1 matching or risk of clinically significant GvHD among patients receiving unrelated marrow grafts. The availability of DRB1 allele matching by sequence-specific oligonucleotide probes (SSOP) or by direct sequencing provides a method for donor matching that is rapid, precise and superior to the MLC for predicting clinically relevant outcome.     

Typing for human alloantigens with the primed lymphocyte typing (PLT) technique with notes on the interpretation of PLT data

A typing system for HLA-D/DR-associated PLT-defined determinants, which have been called “DP” antigens, is reported. Some of the results concerning a data interpretation system, the reproducibility of PLT results, and the correlations between the results of HLA-D, -DR, and DP typing are presented. Also, a “new” human alloantigen, EP1, not belonging to the series of DP antigens, is defined with PLT.     

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.     

Monoclonal and xenoantibodies specific for HLA-DR inhibit primary responses to HLA-D but fail to inhibit secondary proliferative (PLT) responses to allogeneic cells

Monoclonal antibodies (DA-2 and CA-2) and xenoantisera (rabbit anti-human, p23,30) specific for HLA-DR framework determinants were added to primary and secondary mixed lymphocyte cultures. Although such antisera were shown to inhibit primary MLC, primed lymphocytes were much less sensitive to the blocking effects of these antibodies. In the studies shown here, the concentration of antibody required to inhibit primary MLC reactions was 0.1-1.5 micrograms/ml).     

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)     

Population studies of HLA-linked SB antigens and their relative importance in primary MLC typing. Analysis of HLA-D homozygous typing cells and normal heterozygous populations

SB phenotyping was undertaken on 96 HLA-D homozygous typing cells (HTCs) and 129 normal unselected heterozygous donors in the German population, using Interleukin-2-propagated primed lymphocyte typing (PLT) reagents. The results showed that the SB antigens in the normal population behave as a system of alleles at a single locus in Hardy-Weinberg equilibrium (p approximately equal to 0.20). Estimated gene frequencies in the German population appeared to be significantly different (p less than 0.002) from the North American Caucasian population: the principal differences were increased frequencies of the specificities SB1 and SB4, and decreased frequencies of blanks. Of HLA heterozygous donors 41% typed for two distinct SB specificities; 57% typed for one; and 2% were blank. In the HTC group, 20% typed for two specificities; 68% typed for one; and 12% were blank. Thus, a significant proportion of HLA-D homozygous test cells were, nonetheless, heterozygous for HLA-linked SB antigens. Performance of checkerboard mixed leukocyte cultures (MLCs) between 16 SB typed HLA-Dw3 HTCs, however, did not indicate that the observed mutual or one-way responses were influenced in any simple way by SB antigens; neither heterozygosity nor assumed homozygosity for SB antigens appeared to influence the frequency of MLC typing responses of HLA-Dw3-positive donors on these HTCs. These results add further confirmation of the genetic and functional independence of the SB gene product(s) and the HLA-D/DR gene product(s).     

Interactions of allogeneic human mononuclear cells in the two-way mixed leucocyte culture (MLC): influence of cell numbers,subpopulations and cyclosporin

With organ allografts considerable numbers of donor-type mononuclear cells are transferred to the recipient, leading to bilateral immunological interactions between donor and recipient lymphocytes. To study such bilateral immune reactions in detail, human two-way MLC were performed. In this model proliferation kinetics, patterns of activation, and survival of the two populations were analysed, and the relevance of initial cell subset composition, relative cell numbers, and the effect of immunosuppression on this co-culture were evaluated. It could be demonstrated that with an initial 50:50 ratio of two populations of allogeneic cells one population dominated after 21 days of co-culture in 78 out of 80 combinations (97%) tested; the other population decreased markedly after an initially stable phase of 6–7 days. With unequal starting conditions the larger population dominated when resting cells were used, but small populations of preactivated cells or separated CD8⁺ cells could also dominate. Depletion of CD16⁺ natural killer (NK) cells and of CD2^? cells (B cell and monocytes) had no effect on domination. Addition of cyclosporin delayed or blocked the domination process while addition of IL-2 accelerated it. Disappearance of one population was associated with detection of apoptotic cells. The findings indicate that co-cultures of allogeneic mononuclear cells are generally not stable for more than 1 week, but lead to active elimination of one population. CD8⁺ cells and particularly preactivated cells seem to play the most important role in that process, while NK cells are of less importance. Cyclosporin can prolong survival of allogeneic cells in co-culture. These observations suggest that under the conditions of clinical organ transplantation even small amounts of immunocompetent donor cells transferred by the graft may persist for some time and may, thereby, have the chance to exert immunomodulatory functions.     

T cell interactions in active rheumatoid arthritis: insights from the human autologous mixed lymphocyte reaction as a model of T cell activation cascade.

The autologous mixed lymphocyte reaction (AMLR) represents the activation, proliferation and differentiation of T cells in response to signals from autologous non-T cells. Using monoclonal anti-Leu8 antibody to isolate subpopulations of human CD4+ and CD8+ T cells, we have investigated the role of these subpopulations in the T cell activation cascade during the course of AMLR. In normal subjects, CD4+Leu8+ cells are necessary for the initiation of the AMLR response, and sequentially lead to activation and proliferation of both CD4+Leu8- cells and CD8+Leu8+ cells. The activated CD8+Leu8+ cells, in turn, induce CD8+Leu8- cells to generate proliferation of the latter cells. Soluble mediators could be involved in the T cell activation cascade induced by the AMLR. Patients with active rheumatoid arthritis have a profound defect in the AMLR. Further analysis indicates that rheumatoid arthritis CD8+ T cells are markedly defective as responding cells in the AMLR. The impaired AMLR response by CD8+ cells cannot be reconstituted with AMLR-derived supernatants from normal T cells. The data suggest that the defective CD8+ T cell function may contribute to the pathogenesis of the disease.     

Cord blood-derived plasma factor (CBPF) potentiates the low cytokinetic and immunokinetic profile of bone marrow cells in pesticide victims suffering from Acquired Aplastic Anaemia (AAA): an in vitro correlate

Cases of Acquired Aplastic Anaemia (AAA) in patients with a long history of pesticide exposure from agricultural fields have been investigated in our laboratory using an immunological approach. These patients showed moderate to severe degrees of bone marrow aplasia as a result of 9-12 years protracted exposure to pesticides which were mainly comprised of organophosphorous and organochloride compounds. The bone marrow aspirate culture was found to be severely deficient both in terms of differentiation and proliferation, and cell mediated immune function (CMI). We attempted ex vivo manipulation of the bone marrow population of patients in two different protocols: in one, stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-colony stimulating factor (G-CSF) were administered and, in the second set, cord blood-derived plasma factors (CBPF) were supplemented to evaluate the effects, if any. Simultaneously, two control groups including one for healthy normal control (N) and the second, for non-pesticide induced aplastic anaemia group of patients (NPAA) was also investigated for all the above parameters. Active colony formation and improved cellular immune activity (CMI) was observed more frequently in the CBPF treated group rather than that in the cytokine treated group. Surprisingly, administration of cytokines in the first set and CBPF in the second set triggered CD34 + cell generation as revealed through flow cytometric analysis (FACS). The effect was more pronounced in the second set. Investigations carried out with NPAA showed relatively insignificant effects with both cytokine and CBPF set up. The investigations indicated that AAA as induced by pesticides could be therapeutically manipulated by exogenous cytokines and growth factors and, more efficiently, by CBPF by way of immunopotentiation through microenvironmental supplementation.     

Analysis of human class II antigens by cloned cytolytic T cell reagents: a study using HLA loss mutant lymphoblastoid cell lines and monoclonal antibodies detecting the HLA-DP product(s)

We have utilized cloned T cell reagents and ionizing radiation-induced mutants of an HLA heterozygous lymphoblastoid cell line (LCL) to investigate the determinants detected by the cell-mediated lympholysis (CML) assay. Cells of an LCL clone, 721.501, an HLA haplotype loss mutant expressing the HLA-A2-Cw1-Bw51-DR1-Dw1-DQw1-DPw2-GLO haplotype were used as sensitizing cells for responder cells in vitro. "Cloned" reagents were generated by single-cell deposition of cells of a bulk reagent primed against 721.501 cells. Those clones were screened for cytolytic activity against HLA loss mutant targets (derived from LCL 721) of four different categories; HLA-A2 loss only, A2-Cw1-Bw51 loss, A2-Cw1-Bw51-DR1-DQw1 loss, and the entire HLA haplotype loss. Of 196 clones tested, 36 were cytolytic, including three anti-A2, five anti-Bw51/Cw1, 12 anti-DR1/DQw1, 13 anti-DP region associated with DPw2, and three of undetermined specificity, based on cytolytic patterns against the HLA loss mutant targets. Of 25 anti-HLA class II lytic clones, 23 (92%) fitted the characteristics of helper cell-independent cytolytic T cells (HITc), whereas only two of eight (25%) anti-class I clones were HITc. The 13 anti-DP region clones were divided into three subgroups defined by blocking by anti-FA and not Tü39 monoclonal antibodies (MoAb), by Tü39 and not anti-FA, and by both MoAbs.     

Analysis of intraepithelial lymphocytes from major histocompatibility complex (MHC)-deficient mice: No evidence for a role of MHC class II antigens in the positive selection of Vδ4+γδ T cells

Three-color flow cytometric analysis was carried out with intraepithelial lymphocytes from mice deficient in expression of major histocompatibility complex (MHC) antigens. These experiments were done to address the possible role of MHC class II molecules in the positive selection of Vδ4⁺ γδ T cells. By analyzing mice deficient MHC class II antigens alone or in combination with MHC class I antigens, no evidence was found for positive selection of Vδ4⁺ cells among CD8a⁺ or CD4^?CD8^? subpopulations of γδ T cell receptor-positive cells. Because V54⁺, CD8a⁺ cells were reported to be positively selected on I-E^k and hybrid I-E^k/b molecules, class II-deficient animals were crossed with I-E^k transgenic mice and progeny examined for Vδ4 expression. Again, no evidence for positive selection was found. Interestingly, in MHC class I-deficient animals, the total number of γδ T cells was about twofold higher than in control and MHC class II-deficient mice and the proportion of V8δ-expressing cells was correspondingly decreased. Taken together, these results cast doubt on a major role for conventional MHC antigens in shaping the γδ T cell repertoire of intraepithelial lymphocytes.