Abstracts 123

1985. We found marked differences in matching requirements for male and female recipients. Overall male recipients benefitted little from HLA matching while female recipients showed a 10% increase in 1-yr graft survival with matching for DR locus antigens. Male recipients who produced no panel reactive lympho- cytotoxic antibodies (PRA) as a result of pretransplant transfusions had 1-yr graft survival ranging between 77 and 80% whether they were matched for HLA- ABDR antigens or not. In males with PRA, graft survival decreased by 5% at 1 yr for each mismatched HLA-AB antigen. Females who had PRA as a result of pregnancies, transfusions, or unknown causes showed a very marked beneficial effect of combined matching for B and DR locus antigens. The production of HLA reactive antibodies as a result of immunologic stimulation appears to be a crude marker for responsiveness and recipients who had such antibodies had significantly better graft outcomes if they received well-matched grafts. Respon- sive males improved with HLA-AB matching and responsive females with BDR matching. Matching for A and B locus antigens was, to some extent dictated by cross-matching. The percentage of well-matched transplants was higher in recip- ients with >20% PRA than in those with <20% PRA. Twenty percent of re- cipients with >75% PRA had no mismatches at A or B. One hundred two patients who received very well-matched grafts (zero BDR mismatches) had excellent 87% graft survival if they had no PRA and 100% if they had more than 20% PRA at the time of transplant. Emphasis on appropriate HLA matching in those recipients who will benefit the most has the potential to raise the already high graft survival rates in cyclosporine treated recipients.

ABSTR A C T SESSION II: MOLECULAR GENETICS

DEFECTIVE EXPRESSION OF THE DR BETA-2 CHAIN IN HLA-Bw57,DR7,DQw3 HOMO- ZYGOUS INDIVIDUALS IS ASSOCIATED WITH A POLYMORPHIC RESTRICTION FRAGMENT MAPPING 3' TO THE EXON ENCODING THE FIRST EXTERNAL DOMAIN OF A DR BETA GENE. Vivien R. Sutton and Robert W. Knowles; MemorialSloan-Kettering Cancer Center, New York, N Y

Individuals who are homozygous for the HLA-Bw57,DR7,DQw3 extended hap- lotype have been found to lack the expression of one of two DR beta chains normally expressed on DR7( + ) individuals. These chains can be distinguished biochemically by their two-dimensional gel patterns. The DR beta-1 chain, which carries the DR7 allotypic determinant, continues to be expressed while the DR beta-2 chain, normally carrying two distinct supertypic determinants, is com- pletely absent. In order to compare the genomic structure of the HLA-DR beta genes of individuals inheriting the DR7,DQw3 haplotype with those inheriting normal DR7,DQw2 haplotypes, Southern blot analysis was performed using DNA isolated from B-cell lines from homozygous individuals, digested with various restriction enzymes and probed with a DR beta gene cDNA probe. A Hind IlI restriction fragment length polymorphism was found which correlated with lack of expression of the DR beta-2 gene. An 8.5 kb fragment was detected only in the DR7,DQw2 individual whereas a larger fragment of 9.4 kb was present only in the DRT,DQw3 individuals. Failure to detect these fragments with a DQ beta cDNA probe indicated that the observed polymorphism does not involve DQ genomic sequences. Probes which consisted of either 5' (leader sequence and exon for the first external domain) or 3' (exons for the second external domain, cytoplasmic, and transmembrane regions, and the 3' untranslated region) se- quences were used to determine that the polymorphic fragment is located 3' to

124 Abstracts

the exon encoding the first external domain in the DR beta-2 gene. To further analyze the relationship of the observed restriction fragment length polymor- phism and expression of the DR beta-2 gene, the 8.5 kb and the 9.4 kb Hind III fragments have been cloned from size selected genomic libraries.

EFFECT OF METHYLAT1ON ON THE EXPRESSION OF HLA CLASS I1 ANTIGEN. Linda O'Neill and Janet S. Lee; Memorial Sloan Kettering Cancer Center. New York, N Y

Methylation of selected cytosine residues in DNA is thought to play a role in the regulation of gene expression in higher eukaryotes. We have attempted to determine whether methylation of class II MHC genes might affect their expres- sion. We have used a series of melanoma cell lines all derived from patient DX and compared characteristics from the melanoma lines with those of a B lym- phoblastoid cell line derived from the same patient. Each of the cell lines displays different class II expression profiles as visualized on Northern blots. DX3 ex- presses class II constitutively at levels at least equivalent to B-cell expression. DX2 does not normally express class II, but can be induced to do so with gamma interferon. In contrast, DX4 is constitutively negative and noninducible for expression of class II RNA. For this study, high molecular weight DNA from each cell line was isolated and digested with either MspI or HpaII. Both enzymes recognize the sequence CCGG, but HpaII does not digest at this site when the second cytosine residue is methylated. Radiolabeled probes made from DR alpha cDNA sequences were used to hybridize Southern blots of the digests. All MspI digested samples showed a 3.0-kb band. However, Hpa II digests showed an additional band of 3.8 kb in DX2 and DX3, the two cell lines which are inducible and constitutive, respectively, for class II expression. Interestingly, DXB showed a larger band of approximately 4.5 kb, indicating that methylation in this cell occurs at more than one site, or at a different site than those in the melanomas. In our analyses, the presence of methylation at or near the gene, appears to be correlated with expression of class II antigens. This contrasts with the negative regulatory role for methylation proposed for other systems. Studies are currently underway to determine the location of the methylated sites around the DR alpha gene in these cell lines, and to analyze the role of methylation at other class II subloci.

DIFFERENTIAL EXPRESSION OF HLA CLASS II ANTIGENS IN MYELOMONOCYTIC LEUKEMIA CELL LINES. Hamid Band, Joseph Lustgarten, Vladimir Corredor, and Edmond J. Yunis; Division of lmmunogenetics, Dana Farber Cancer Institute, and Department of Pathology. Harvard Medical School, Boston, MA

The class II HLA antigens are heterodimeric glycoproteins important in antigen presentation and cell-cell interactions during hematopoietic cell differentiation. At least three classes of these antigens, namely DP, DQ, and DR, have been recognized both at the protein and DNA level. These antigens appear to be differentially expressed on cells of the myeloid lineage. In the present study our aim was to characterize the expression of HLA class II antigens on myelo- monocytic leukemia cell lines representing different stages of differentiation. Human leukemic cell lines KG-1 (myeloblast), HL-60 (myelomonocytic), and U- 937 (monocytic) were studied by: (a) immunofluorescence with monoclonal an- tibodies. (ST2.45A-anti class II; L243-anti DR; B7/21 and PL3-anti-DP; and Leul0-anti DQ), (b) Northern-blot analysis using cDNA probes. By immunoflu-