K. K. Mittal

Serotyping for homotransplantation. 18. Refinement of microdroplet lymphocyte cytotoxicity test.

The microdroplet lymphocyte cytotoxicity test was examined thoroughly in an effort to increase the reproducibility of the test. The discrepancy rate in a large series of tests was reduced from 5.16% at the start of this study to the present 0.95% by introducing certain modifications in the technique. Variables connected with the isolation of lymphocytes, handling of antisera, quality of antisera, amount of complement, incubation temperature, duration of incubation, fixing of reactions, and reading of reactions were studied. The method which has resulted appears to be reproducible, simple, and readily usable on a large scale.

Serotyping for homotransplantation. XVII. Preliminary studies of HL-A subunits and alleles.

The relationship between 7 antigenic groups of lymphocytes was studied in 36 families with 144 children and a population of 2208 people. Gene frequencies of the individual antigens were deduced from the phenotype of the subjects in the population and the parents in the families as well as the genotype of the latter. In families these antigenic determinants were inherited en bloc, confirming the concept of Dausset, van Rood, Ceppellini, Amos and others that these antigens belong to a single complex locus, the HL-A locus. Evidence is presented to suggest that there are two mutational sites in the HL-A locus. One site corresponds to antigens 1 and 2 (LA series of Payne-Bodmer). The second site is principally defined by the antithetic groups 5 and 6 (roughly 4d and 4c). Two other groups, 3 and 7 (approximately 4a and 4b of van Rood), which behave allelic but are more difficult to define sharply, appear to constitute a partially independent site next to the 5–6 site. The mutational site next to 5 appears to be group 7, whereas the site next to antigen 6 could be either 3 or 7. Thus the HL-A locus seems to have two sites, of which one is composed of two partially independent subunits.

SEROTYPING FOR HOMOTRANSPLANTATION: XXXI. A 45-Minute Microcytotoxicity Test1

SUMMARY A rapid method for microcytotoxicity testing is presented, requiring 45 min for a complete test. The time required to make lymphocyte suspensions has been reduced to 10 min by eliminating red cells before differentially removing granulocytes. Incubation time has been reduced to 15‐20 min by enzymatic (ficin) treatment of lymphocytes.

Cross-reactivity between human and murine lymphocyte antigens: III. Reactivity of H-2 allo- and xenoantisera with human lymphoid cells

H-2 alloantisera and antimouse lymphocyte xenoantisera react with 14%–100% of human lymphocytes from a panel of at least 80 unrelated people. Population and family studies did not reveal HL-A specificity of such lymphocytotoxic antibodies but indicated that the antibodies are directed against polymorphic antigenic determinants inherited in association with HL-A antigens. H-2 allo- and xenoantisera absorbed with human lymphoid cells and a panel of platelets bearing all the known HL-A specificities were still cytolytic when tested against murine lymphocytes, suggesting that only a small proportion of the heterogeneous population of H-2 antibodies react with human lymphocytes. On the other hand, HL-A alloantisera could be absorbed by lymphocytes from certain murine strains. These results suggest that the crossreactivity between human and murine lymphocytes is caused by antigens common to several HL-A (or H-2) types or by antigens linked to HL-A but not identical with them.

A quantitative study of cross reactivity in the HL-A system with human cultured lymphoid cells and soluble HL-A antigens.

SUMMARY Cross reactivity in the HL-A system has been evaluated quantitatively by utilizing human cultured lymphoid cells and soluble HL-A antigens in cross absorption and cross inhibition assays. Most of the cross reactions among different HL-A specificities determined with platelets or peripheral lymphocytes were confirmed in this study. Some cultured lymphoid cells or soluble HL-A antigens possessing a cross reacting specificity cross absorbed a certain anti-HL-A antiserum, whereas others with the same specificity did not. The number of cells required to absorb cytotoxic HL-A alloantisera directed against cross reacting specificities is significantly greater than that necessary to absorb alloantisera directed against HL-A determinants present on the cell surface. This observation suggests that relatively small numbers of available antigenic determinants react with the cross reacting antibodies. The differential absorbing capacity of different cultured cell lines for the same cross reacting HL-A alloantibodies suggests a variability in the cell surface expression of cross reacting HL-A determinants.

Serologic specificity and crossreactivity of soluble HL-A alloantigens.

The serologic activity of soluble human histocompatibility (HL-A) antigens extracted from four human cultured lymphoid cell lines by the 3M KCI method has been evaluated by their ability to inhibit specifically the cytotoxicity of 38 operationally monospecific alloantisera recognizing 18 HL-A specificities. Several HL-A alloantisera directed against the same HL-A specificity, although used at functionally comparable levels of activity, varied in their susceptibility to inhibition by the same soluble HL-A alloantigen preparation. Similarly, lymphocytes from a number of subjects, when used as target cells for the same alloantiserum, detected quantitatively different activities of a given soluble HL-A alloantigen preparations in the inhibition test. Soluble HL-A alloantigens can block the cytotoxicity of antisera directed against HL-A specificities either crossreacting with or present on the cultured lymphoid cells used as source for extraction of soluble HL-A alloantigens. Crossreactivity was exhibited only...

Detection of B cell antibodies in renal transplant recipients.

SUMMARY A retrospective study for the presence of lymphocytotoxic antibodies was performed on sera collected from 119 kidney graft recipients. Sera that had been collected on days 12 to 19 post-transplant were tested for cytotoxic reactions against a panel of human peripheral blood lymphocytes from 60 unrelated donors and 37 to 47 cultured human lymphoid cell lines (LCL). Forty-nine sera were negative against peripheral blood lymphocytes but contained cytotoxic antibody against cells on the LCL panel. Several sera were tested on E rosette-purified peripheral blood lymphocyte B cells and T cells from five donors whose LCL had also been tested. LCL appeared to be more sensitive to cytotoxic reactions than their B cell counterparts and may identify additional specificities which may not be related to the B cell alloantigenic system. Mixed lymphocyte culture blocking experiments were carried out against all combinations of these five cells. Some sera showed reactions of identity for B cells and LCL, and blocked the appropriate stimulator cells in mixed lymphocyte culture. Two sera that were positive for LCL but negative for B cell blocked only responder cells in the mixed lymphocyte culture.

Serotyping for Homotransplantation XXVIII Sib‐Pair Assay for NonHL‐A Antigens

Summary. Negative results were obtained in an investigation of 118 selected lymphocytotoxic antisera for activity against antigens determined by a locus or loci independent of the HL‐A locus. The sera studied were those not readily identifiable in terms of either generally recognized or tentative HL‐A antigenic specificities, and accordingly were considered appropriate for study to search for nonHL‐A antibodies. It is concluded that antisera active in lymphocytotoxicity tests against nonHL‐A antigens are quite rare.