D. B. Amos

Genetic Mapping of Ir Locus in Man: Linkage to Second Locus of HL-A

Fifty-seven members of a family that spanned three generations were studied for antigen E and ragweed skin sensitivity and HL-A antigens. There was significant association between the haplotype HL-A 2-12 and antigen E skin hypersensitivity (F = .22 to .26) in this family. The map order is first locus of HL-A, second locus of HL-A, and IrE. These determinants are considered to be part of the linkage group HL-1.

An Analysis of an Antigenic System in the Mouse (the H-2 System)

The H-2 complex of mice plays a major part in transplantation immunity. Genetical studies by Snell and his colleagues have revealed nine different combinations. Serological investigations have demonstrated five basic antigens, C, D, E, F and K. The distribution so far as it is known is given in the table. We postulate two alleles of E, and at least three of D. No allele of K has been definitely established as yet. The extreme polymorphism of the system is largely contributed by D and possibly by K as well. Difficulty in demonstrating allelomorphs may be attributable to the following factors: (1) technical difficulties in the demonstration of some partial antibodies in mice, (2) low concentration of certain antigens on the erythrocytes, (3) the small population so far studied serologically. Mutations have been shown to arise not infrequently in some strains studied in America and involve the simultaneous transformations of three antigens, we have found that two of the antigens known to have been involved are separable by crossing-over.

Serological analysis of a recombination in the H-2 region of the mouse.

The H-2 phenotype determined by H-2o, a new allele derived from a recombination between H-2d and H-2k, has been analyzed for 21 of the 25 defined H-2 specificities by direct hemagglutination, by absorptions and by immunizations. The recombinant allele determines specificities 1(A), 3(C), 5(E), 8(H), 29(C′), 31 (Ed) and 32(Dk). The results of this crossover support the previous recombination maps of the H-2 region, extend them to include additional specificities, and newly define a separable genetic determinant for factor 5(E). One anomalous immunization result raises the possibility either of unequal crossing over or of alteration of one antigenic specificity as a result of recombination.

Human renal transplantation with donor selection by leukocyte typing.

Advances are being made in controlling or reducing rejection of renal allografts. The authors find leukocyte typing helpful in selecting compatible donors.

Cross reactions of HL-A antibodies. I. Characterization by absorption and elution.

A human alloantiserum produced by multiple immunizations of subject BC with lymphocytes from donor MF was analyzed by quantitative absorption and elution techniques. The antiserum demonstrated three distinct patterns of reactivity with lymphocytes from a donor cell panel. Of these three classes of reactivity, only one class showed absolute correlation with an identifiable HL-A specificity, HL-A3. By absorption and elution criteria the serum appeared “monospecific”; complete absorption was attained by cells not carrying HL-A3 and eluates from these cells had the same specificity as the unabsorbed serum. Three additional HL-A antisera were used for comparison and gave similar results.

Renal Transplantation between HL-A Identical Donor-Recipient Pairs: FUNCTIONAL AND MORPHOLOGICAL EVALUATION

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.

Factors which have a significant effect on the survival of human skin grafts.

SUMMARY Survival of 436 ABO-compatible skin grafts exchanged in 97 Caucasian families was prolonged if donor and recipient were genotypically, as compared with phenotypically, HLA identical. Among skin grafts between haploidentical family members, a mismatch at the A locus was equivalent to a mismatch at the B locus. Skin grafted from child to mother survived longer than did skin grafted between other family members, other variables being equivalent. A highly significant positive correlation was found between the age of recipient and skin graft survival. In addition, a significant interaction was found between the relationship of donor and recipient and degree of antigen match. Genetic factors contribute significantly to the survival of grafted tissues and organs in experimental animals and in man. In particular, prolonged graft survival is associated with compatibility for a genetic complex, usually referred to as the major histocompatibility system. The major histocompatibility complex, called HLA in man and H-2 in mouse, includes a number of distinguishable genes, each of which may contribute by one mechanism or another to graft survival or rejection. By exchanging grafts between congenic strains of mice, it has been possible to measure directly the effect of incompatibility for segments of the H-2 complex on graft survival (13). Such direct measurements cannot be made in man since a human equivalent of congenic strains does not exist. Two individuals, whether related or unrelated, may differ for several genes which influence graft survival in addition to the genetic factor(s) in question. Therefore, in man the effect of single genes or chromosome segments must be measured indirectly and evaluated by statistical methods. Attempts have been made in the past to measure the relative strength of incompatibilities. The earliest attempts were through the third man test, in which a recipient (the third man) was sensitized by receiving leukocytes (10) or a skin graft (19) from a given subject and was then tested by receiving grafts from a donor panel. Donors whose skin was rejected as second-set or white grafts were considered to share antigens, absent from the third man, with the original donor. Although it was possible to show antigenic variation in this way, no information was obtained about specific antigens. The next step was to type for the then identified HLA-related specificities and match in such a way that the sensitizing donor possessed a specificity absent from the third man. Test grafts were then placed on the recipient from two or more donors possessing and two or more lacking the specificity in question (controls). Sensitization was by injection of leukocytes or platelets, or by skin graft. In almost every case skin from the antigen-positive donors was rejected in an accelerated fashion, and control grafts were usually rejected as first-set grafts 3 or 4 days later. Unfortunately, the sophistication of antigen definition was low and the validity of some of the data (e.g., with platelet immunization) seems rather doubtful. In any event the tests did not show that some specificities were consistently more immunogenic than others. Skin grafts between “matched” and “mismatched” donor-recipient pairs did not show any consistent differences (30); although from a consideration of variations in length of survival of skin grafts and of the survival and function of kidney grafts, there are great differences between pairs matched for one HLA haplotype. The relevance of the HLA system to transplant survival, however, is beyond question. Grafts from HLA-identical siblings are consistently better accepted than are grafts from siblings, parents, or others, clearly mismatched for one or more haplotype. In this study, we have attempted to evaluate the relative importance of matching for HLA antigens per se and the effects of other variables on the survival of skin grafted to nonimmunosuppressed normal, healthy family members. Our results suggest that matching for HLA antigens themselves, or consequently, for factors in significant linkage disequilibrium with them, significantly prolongs skin graft survival. Other significant factors are the familial relationship of graft donor and recipient and age of recipient. Our data demonstrate the importance of multivariate analysis in assessing the significance of individual variables on skin graft survival.

HL-A TYPING, MIXED LEUKOCYTE REACTIVITY, AND SKIN GRAFT SURVIVAL IN A FAMILY WITH A RECOMBINANT AT THE HL-1 CHROMOSOMAL REGION (MAJOR TRANSPLANTATION REGION)1

SUMMARY The members of family Sch were studied by three different parameters: HL‐A typing, mixed leukocyte reaction (MLR), and skin grafting. There were two HL‐A‐identical, mixed leukocyte reaction‐identical siblings (MLR‐S); two HL‐A‐identical, MLR‐S‐different siblings; and one sibling who was haploidentical to the two sets of HL‐A identicals. We have named the HL‐A‐MLR‐S complex the HL‐1 haplotype. The father (HL‐A1‐8/9‐12) and two siblings (1‐8/2‐12) show mutual stimulation in MLR despite identity at the second locus. Similarly, the sibling who is homozygous for HL‐A12 showed mutual stimulation in MLR with the parents and all siblings. These results are further proof that the MLR results from a product of a gene linked to the second locus and not the second locus itself. Skin graft rejection times within this family indicate that the gene products responsible for rejection of skin grafts were probably inherited in association with HL‐A and independent of MLR‐S.

B-Lymphocyte Alloantigens

Seventy B‐cell alloantisera were tested by microcytotoxicity against a panel of B‐cell‐enriched and T‐cell‐enriched lymphocytes. Six groups were discerned and have tentatively been designated DIg 1–7 (for Duke immunoglobulin‐positive cell groups). These alloantisera were used to type an HLA‐A/B recombinant family. Two groups were observed to segregate in this family, DIg 7 with the HLA‐B locus and DIg 2 with the HLA‐A locus.

THE ANTIGLOBULIN MICROCYTOTOXICITY ASSAY IN HL-A GENOTYPED FAMILIES

HL-A genotyping was accomplished in 30 families (8 black, 21 white, and 1 American Indian) based on serological results obtained by our ususal lymphoeytotoxicity assays. Bach family was further tested by an antiglobulin microcytotoxicity method. Segregation patterns obtained by the latter method, compared with those of the former, showed few discrepancies. HL-A-identieal siblings were serologically similar by the antiglobulin assay with frequency of discordant reactions at the same level as that of our regular two-stage lymphocytotoxicity assay. The antiglobulin method was shown to be highly reproducible with 1.3% discordance between duplicate tests. Many sera employed in routine cytotoxicity testing give positive reactions with all family members in the antiglobulin assay (39% all positive by antiglobulin versus 16% by the regular cytotoxicity test), so that this method has limited usefulness for routine HL-A typing. The antiglobulin assay may have particular value, however, for identifying HL-A haplotypes in families, especially non-Caucasian, which give infrequent positive reactions in the usual lymphocytotoxicity assays.