MLC Tests Research Articles

Possible mechanisms by which alloantisera inhibit in the MLC test.

MLC inhibition studies were performed with two human alloantisera: one specific for HLA-B7, the other for HLA-DRw7. The stimulator cell inhibitory effects of these sera were tested in primary and secondary MLC tests. Both sera inhibited in the primary MLC, whereas only the anti-DRw7 serum was capable of blocking the secondary MLC test. The difference in inhibiting properties of these sera was further analyzed in primary MLC tests using selected MLC combinations, Fc receptor negative cell populations and pepsin digests of the anti-B7 serum. Anti-DRw7 antibodies could inhibit by masking the stimulatory DR antigens. The inhibition of the anit-B7 antiserum was dependent on Fc, which suggested that anti HLA-B antibodies inhibited by some other mechanism. This inhibition could have been caused by antibody dependent cellular lympholysis of the stimulator cells or by the induction of suppressor cell activity.

Homozygous cell typing for the rabbit RLA-D antigens in animals from commercial breeders

We have previously reported that several of theRLA haplotypes of our rabbits have anRLA-D allele in common, i. e., they fail to cross stimulate in the MLC test. To investigate the possibility that these haplotypes originated in unrelated animals, a panel of homozygous rabbits was selected to partially characterize noninbred rabbits from five commercial sources for their alleles at theRLA-D locus. In the 47 rabbits tested, 4 homozygous animals and 12 heterozygous animals were detected with the four alleles chosen for typing. At least two independent pairs of rabbits shared identicalRLA-D genotypes. Our results indicate that theRLA-D locus is not extremely polymorphic and that rabbits cannot be assumed to be completely mismatched for theRLA complex simply because they are from different breeds or from independent suppliers.

GENETIC ANALYSIS OF THE MIXED LYMPHOCYTE REACTION IN H‐1 SEROLOGICALLY IDENTICAL INBRED RATS

SUMMARYH‐1d and H‐11 serologically identical combinations of inbred rat strains were studied, in which there was a strong mixed lymphocyte reaction (MLR). The H‐1d identical combination was formed with the Maudsley reactive (MR) and BD IX strains, in which there was bidirectional stimulation. The LAD differences in the combination BD IX and MR were shown in experiments using back‐crosses derived from two different sources, BD IX x LEW and MR x LEW.The H‐11 identical combination was the F344 (Fisher) and LEW (Lewis) strains; here the MLR was unidirectional—LEW stimulating F344. Two other H‐11 serologically identical strains, AS and HCS, also stimulated F344 lymphocytes. Genetic segregation analyses using back‐crosses showed linkage with H‐1 in both instances. The LADs of AS, HCS and LEW appeared to be the same.These experiments show that the rat LADs so far detectable using these strains are all encoded by genes linked to the major histocompatibility system of the rat, despite serological identity. In addition to disassociation of the major histocompatibility system antigens detected by serological means and those defined by MLC testing, there was also disassociation of transplantation antigens as shown in the BD IX x MR model.

F1-hybrid anti-parental-strain reactivity. II. Proliferation of C3H X CBA hybrid lymphocytes in the spleens of irradiated CBA mice.

Experiments were conducted to explore whether C3H X CBA lymphocytes can react by proliferation when they meet parental CBA spleen cells. MLC tests could not detect such a reaction, but infusion of C3H X CBA lymph node cells into irradiated CBA hosts resulted in rapid cell proliferation in the host's spleen. Such a cell proliferation was also observed after infusion of T-cell-enriched lymph node cell preparations or thymic cells from C3H X CBA donors. Upon transfer to new irradiated mice these proliferating cells continued to proliferate in CBA mice but not in C3H X CBA mice. Further evidence that the injected T-cells were the proliferating emerged from experiments where AKR X CBA lymphocytes were found to proliferate in spleens of irradiated CBA mice and that most of these cells posessed the theta antigen determined by AKR. Proliferation of F1-hybrid lymphocytes in the spleens of its irradiated parental strains was found not to be a general phenomenon and is probably restricted to some Mls-antigen-compatible strain combinations. The possibility that the C3H X CBA hybrid lymphocytes are stimulated to proliferations by CBA lymphocytes reactive against the C3H-determined Mls antigen is discussed.

Can anti HLA-A and -B antibodies inhibit the MLC test?

Eleven lymphocytotoxic pregnancy sera were tested for inhibition in the MLC test. Responder inhibition and stimulator inhibition were analyzed separately by using cells from the serum producer as stimulator and responder respectively. Only three sera showed inhibitionof responder function, whereas all sera showed inhibition of stimulator function. The specificity of the latter inhibition was at least in part attributable to antibodies directed to specificities other than B-cell specificities (presumed to be coded for in the HLA-D region). This was deduced from the fact that platelet eluates devoid of anti B-cell antibodies effectively inhibited in MLC.

Histiocytosis X: IV—Immunologic response assessed by lymphocyte transformation tests

Eight children with histiocytosis X were investigated with reference to immunologic reactivity in vitro (PHA, PWM, Con-A and MLC testing). No significant impairment of immune function was detected, but a subnormal (statistically insignificant) response in the MLC test may suggest some abnormality of the T lymphocytes reacting against allogeneic cells. In a patient who relapsed a marked, but insignificant, increase in lymphocyte reactivity in vitro was observed.

Linkage between C2 deficiency and theHLA-A10,B18,Dw2/BfS haplotype in a French family

A linkage between C2 deficiency and the HLA-A10,B18/BfS antigens has been found in a French family from the Strasbourg area. The propositus, suffering from a chronic glomerulonephritis, is homozygous forHLA-A10,B18/BfS and totally C2-deficient. The parents and the brother are heterozygous for C2 deficiency and share theHLA-A10,B18/BfS haplotype. MLC tests and HLA-D typing revealed that the homozygous C2-deficient patient is also homozygous at theHLA-D locus for the w2 specificity. Evidence was obtained for a heterogeneity of the HLA-Dw2 specificity. This observation confirms the remarkable association between C2 deficiency and theHLA-A10,B18,Dw2 haplotype.

B-cell antibodies, Ia-like determinants, and their relation to MLC determinants in man.

The HLA supergene is located in the 6th chromosome. Its position to the centromere and the position of a number of polymorphic isoenzymes has been elucidated. The HLA supergene codes not only for determinants present on all nucleated cells, but also for determinants present on B cells and absent from T cells and platelets. These determinants can be recognized by serology, and evidence is presented that some of them are coded for by a hither to unrecognized locus Ag, which is very closely linked to the MLC determinants of the D locus can be recognized with the help of the MLC test using unprimed cells, homozygous for the MLC determinants, so-called typing cells primed against one MLC determinant in the PLT test. So far, 8 MLC determinants have been recognized. Significant disease-association studies in different racial groups appear to be especially informative. They already indicate that the association found so far must rest on different mechanisms. Whether some of them could be caused by partial deficiency for one or more of the complement factors remains to be proven.

Lymphocyte stimulation by soluble subcellular fractions.

Nuclear material can produce inhibition or stimulation of healty leucocytes under different experimental conditions, Reactivity could not be produced in cultures using intact nuclei and allogeneic lymphocytes. The effect of nuclear and cytoplasm fractions was compared with that of whole cells on intact healthy lymphocytes. The HLA activity in the individual fractions was assessed. Stimulation was produced by certain nuclear and cytoplasmic fractions and these were closely related to the peaks of HLA activity. The response to these fractions showed less activity than that achieved in conventional one way MLC tests.

LD antigens associated with HL-A8 and myasthenia gravis.

The finding that the SD antigen HL-A8 is associated with myasthenia gravis has raised the question as to whether or not there is also an association between some specific LD antigen and myasthenia gravis and/or HL-A8. MLC technique was used to study LD antigens in 33 myasthenic patients. The cells of a myasthenic patient who was homozygous for both HL-A and LD products were used in MLC tests as stimulators with HL-A8 bearing cells from 24 myasthenics, their 17 relatives and 16 controls and to non-HL-A8 cells from nine myasthenics and 16 controls. At least three different LD genes were found to be associated with HL-A8. One of them, called LDm' was present in 17 (63 %) of the 27 HL-A8-bearing haplotypes of myasthenics, and in nine (47 %) of the 19 HL-A8-bearing haplotypes of controls. In our study LDm was not found in the 84 haplotypes devoid of HL-A8. LDm is strongly associated with HL-A8 and through this also with myasthenia. Calculated from phenotype frequencies for HL-A8 and its association to LDm' the LDm frequencies are 9 % in the control population, 30 % in myasthenics and 48 % in young females with onset of myasthenia below 35 years. LDm had no indendent correlation with any of the clinical parameters of myasthenia gravis, even though is was found more often in HL-A8+ females with the onset of myasthenia before 35 years than in the whole myasthenic group. LDm appears to be similar to LD8a antigen.

Identification of five lymphocyte activating determinants in man.

Eleven putative LD-1 locus homozygous cell donors were found by MLC tests in non-consanguineous families. Lymphocytes from each of them were used as stimulating typing cells in MLC tests together with responding cells from the other homozygous cell donors and from 45 random unrelated individuals. Four of the stimulating typing cells were found to identify a determinant in non-random association with HL-A7; LD-7a, three other cells typed for a determinant non-randomly associated with HL-A8; LD-8a, and one cell seems to identify LD-W5 in non-random association with W5. Two possible additional LD-1 determinants, LD-oh and LD-om, were less well defined with two of the cells. The combined gene frequency of the five possible LD-1 determinants is approximately 0.45 among Norwegians.

An estimation of the recombination fraction between the MLC locus and the FOUR locus.

Thirty-nine families with 167 children were typed for HL-A and studied in the MLC test. Two maternal recombinations between the FOUR and the MLC locus in two different families were found. An estimate of the recombination fraction between the FOUR and the MLC locus was calculated.

MLC TYPING IN HL‐A IDENTICAL NON‐SIBLING PAIRS IN AN INBRED POPULATION

SUMMARYMixed Lymphocyte Culture (MLC) tests were performed between HL‐A identical non‐sibling members of an inbred population. Three pairs were known to be related, and the descent of chromosomes is followed by both HL‐A serology and MLC tests. The implication of a high frequency of negative MLC tests between HL‐A identical non‐sibling pairs in this population as compared with other populations is discussed.

Mixed Lymphocyte Culture Technique: Standardization of a Test‐System with 105 Responding and 105 Stimulating Lymphocytes per 1 ml

An MLC test system is described utilizing 105 responding and 105 stimulating lymphocytes in a total of 1 ml of culture medium. Some of the variables of the test have been evaluated and the reproducibility of the test within a given day and between days investigated according to a Workshop protocol. This system was first applied to three groups: HL‐A identical siblings, one haplotype different family members and two haplotype different individuals. Within each group, the log converted stimulation ratios are normally distributed, allowing the use of simple statistical analysis in repeated testings. In addition, a group of weakly positive MLC reactions can be detected. This group includes the LD identical but SD different sibling combinations. Within the same group we find some unrelated HL‐A sero‐identical combinations and the so‐called LD typing response, used to identify LD determinants by means of LD and SD homozygous stimulating test cells.It is concluded that this MLC test system allows identification of identical combinations, weakly positive combinations and one and two haplotype responses even within unrelated combinations. This test system may have advantages over the microtiter test system because the large culture volume gives a low concentration of blastogenic factors in typing responses.

Severe rejection of an HL-A identical sibling renal transplant. Results of MLC tests.

Severe rejection of an HL-A identical sibling renal transplant. Results of MLC tests.