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Background: Chronic allograft rejection manifested as bronchiolitis obliterans syndrome (BOS) is the leading cause of late death after lung transplantation. Although increasing evidence suggests an association between anti-human leukocyte antigens (HLA) antibodies and chronic rejection of kidney or heart allografts, the clinical significance of anti-HLA antibodies in lung recipients is less clear, especially in previously unsensitized recipients. The use of flow cytometry based panel reactive antibody (flow-PRA) provides a highly sensitive means to identify the development of de novo anti-HLA antibodies in lung recipients., Methods: Flow-PRA testing was used to analyze the pre- and posttransplant sera in stable BOS free lung recipients who survived at least 6 months. Patients without prior sensitization as defined by a negative pretransplant flow-PRA were analyzed posttransplant for the presence of anti-HLA antibodies by flow-PRA. A proportional hazards model was used to determine the impact of anti-HLA antibody on BOS risk., Results: Sera from 90 recipients at Duke University with negative pretransplant flow-PRA were tested by flow-PRA at various time points after transplant. Sera from 11% (10/90) of recipients were found to contain anti-HLA antibodies detectable by flow-PRA. Nine patients (90%) developed anti-HLA antibodies specific for donor antigens, and one patient developed anti-HLA class II antibodies, not specific to donor antigens. Among the nine patients with donor antigen specific antibodies, flow-PRA specificity analysis demonstrated eight were specific for class II antigens and one for class I antigens. In a multivariate model that controls for other BOS risk factors, a positive posttransplant flow-PRA was significantly associated with BOS grades 1,2, or 3 (hazard ratios [HR] 3.19; 95% confidence interval [CI]: 1.41-7.12, P=0.005) and BOS grade 2 or 3 (HR 4.08; 95% CI: 1.66-10.04, P=0.002). Four patients with de novo anti-HLA antibodies died during follow-up; all four had BOS. Among BOS patients, the presence of anti-HLA antibodies was associated with a significantly worse survival (P =0.05, log-rank test)., Conclusions: Although uncommon, previously unsensitized lung transplant recipients can develop anti-HLA antibodies to donor class II antigens. The development of de novo anti-HLA antibodies significantly increases the risk for BOS, independent of other posttransplant events. Furthermore, de novo anti-HLA antibodies identify BOS patients with significantly worse survival. Additional studies are needed to determine if class II-directed anti-HLA antibodies contribute mechanistically to the chronic rejection process in lung recipients.

Seven murine anti-H-2 and three nonimmune mouse sera were tested for cytotoxicity toward B and T lymphocytes from a panel of human donors. One group of sera, including two anti-H-2.33 sera, exhibited cytotoxicity directed exclusively toward human B but not T cells from all donors. Absorption studies on human lymphoblastoid cell lines (LCLs) of B or T cell origin corroborated these findings. Some nonimmune sera showed similar characteristics, indicating that the observed reactions were not attributable to cross-reactivities between mouse H-2K or D specificities and human antigens coded by the HLA-A, B, or C locus. Another set of mouse sera (anti-H-2.28b and anti-H-2.31) was highly cytotoxic to both B and T cells of some donors but not of others, suggesting that activity in these sera may arise from cross-reactions between mouse and human specifities. A third set of anti-H-2 as well as normal mouse sera showed only background cytotoxicity when tested on human cells. The ability of the B cell cytotoxic mouse sera to block the human mixed lymphocyte culture reaction (MLR) was compared to that of appropriate human alloantisera with exclusive B cell activity or a rabbit serum raised against human B cells. None of the mouse sera resulted in a significant reduction in the human MLR, whereas the human alloantisera as well as the rabbit antiserum caused a significant amount of blocking at several dilutions beyond their highest cytolytic titer.

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.

HL-A genotyping was accomplished in 30 families (8 black, 21 white, and 1 American Indian) based on serological results obtained by our ususal lymphocytotoxicity assays. Each 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-identical 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 gave 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.