Apolipoprotein L1 gene variants in deceased organ donors are associated with renal allograft failure.

Apolipoprotein L1 gene (APOL1) nephropathy variants in African American deceased kidney donors were associated with shorter renal allograft survival in a prior single-center report. APOL1 G1 and G2 variants were genotyped in newly accrued DNA samples from African American deceased donors of kidneys recovered and/or transplanted in Alabama and North Carolina. APOL1 genotypes and allograft outcomes in subsequent transplants from 55 U.S. centers were linked, adjusting for age, sex and race/ethnicity of recipients, HLA match, cold ischemia time, panel reactive antibody levels, and donor type. For 221 transplantations from kidneys recovered in Alabama, there was a statistical trend toward shorter allograft survival in recipients of two-APOL1-nephropathy-variant kidneys (hazard ratio [HR] 2.71; p = 0.06). For all 675 kidneys transplanted from donors at both centers, APOL1 genotype (HR 2.26; p = 0.001) and African American recipient race/ethnicity (HR 1.60; p = 0.03) were associated with allograft failure. Kidneys from African American deceased donors with two APOL1 nephropathy variants reproducibly associate with higher risk for allograft failure after transplantation. These findings warrant consideration of rapidly genotyping deceased African American kidney donors for APOL1 risk variants at organ recovery and incorporation of results into allocation and informed-consent processes.

The association of HLA-DR alleles and T cell activation with allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease caused by the mold Aspergillus fumigatus. We previously reported that the majority of T cell clones (TCC) isolated from three ABPA patients, and specific for a dominant Ag of A. fumigatus, Asp f 1, were IL-4-producing CD4+ Th2 cells capable of responding to Ag in association with the HLA-DR subtypes DRB1*1501, *1503, and *1601 for HLA-DR2, and DRB1*1101, *1104, and *1202 for HLA-DR5. In the present study we extended the previous findings to determine whether the observed restriction with the HLA-DR2/5 subtypes held importance in a larger patient population. Serotyping revealed that 16 of 18 ABPA patients were either HLA-DR2, HLA-DR5, or both. Compared with a normal control population, the frequencies of HLA-DR2 (50 vs 22.3%) and HLA-DR5 (44.4 vs 19.8%) were significantly increased in these ABPA patients. Genotype analyses of an additional 15 patients identified the same HLA-DR subtypes previously shown functional for Asp f 1 Ag presentation. The relative avidities of Asp f 1 peptides for the purified HLA-DR subtypes, DRB1*1501 (functional) and DRB1*1502 (nonfunctional), were examined to determine whether differential binding to the HLA-DR subtypes explains successful Ag presentation. Similar low binding avidities were detected for both HLA-DR subtypes, indicating that the functionality cannot be simply explained by differences in binding affinities. Thus, the limited number and their role in Ag presentation emphasizes the possibility that the six identified HLA-DR subtypes are important in the pathophysiology of ABPA.

Continued mapping of chromosome 2 genes.

This report describes our continued efforts to elucidate the genetic fine structure of the central portion of the mouse chromosome (Chr) 2. Mice from our panel of 28 Chr 2 congenic strains were tested: 1) for the presence of the antigens which stimulate Chr 2-reactive lymphocyte clones in mixed lymphocyte reaction (MLR); 2) for the antigens of histocompatibility (H) genes H-42a and H-45a as determined by allograft rejection; and 3) for their ability to respond to the H-Y antigen in a cell-mediated lysis assay. The results obtained in this study have allowed additional mapping of immunologically involved Chr 2 genes. The gene encoding the antigen which stimulates lymphocyte clone 1C11 can be considered wholly different from other Chr2 H genes on the basis of chromosomal recombination. We have assigned the symbol H-48 to this gene. The following gene order has been established: [H-3, B2m, pa], we, [H-42, H-48,] H-45, IR-H-Y, Hd-1, un, H-13, Aw. The order of the bracketed genes is not known. H-44 maps centromeric to IR-H-Y. The genes encoding the antigens that stimulate lymphocyte clones 2G7, 2C10, 1F6, 1B10, and 1H10 map centromeric to H-45.

The evolutionary origin of the HLA-DR3 haplotype.

The human HLA-DR3 haplotype consists of two functional genes (DRB1*03 and DRB3*01) and one pseudogene (DRB2), arranged in the order DRB1...DRB2...DRB3 on the chromosome. To shed light on the origin of the haplotype, we sequenced 1480 nucleotides of the HLA-DRB2 gene and long stretches of two other genes, Gogo-DRB2 from a gorilla, "Sylvia" and Patr-DRB2 from a chimpanzee, "Hugo". All three sequences (HLA-DRB2, Gogo-DRB2, Patr-DRB2) are pseudogenes. The HLA-DRB2 and Gogo-DRB2 pseudogenes lack exon 2 and contain a twenty-nucleotide deletion in exon 3, which destroys the correct translational reading frame and obliterates the highly conserved cysteine residue at position 173. The Patr-DRB2 pseudogene lacks exons 1 and 2; it does not contain the twenty-nucleotide deletion, but does contain a characteristic duplication of that part of exon 6 which codes for the last four amino acid residues of the cytoplasmic region. When the nucleotide sequences of these three genes are compared to those of all other known DRB genes, the HLA-DRB2 is seen as most closely related to Gogo-DRB2, indicating orthologous relationship between the two sequences. The Patr-DRB2 gene is more distantly related to these two DRB2 genes and whether it is orthologous to them is uncertain. The three genes are in turn most closely related to HLA-DRBVI (the pseudogene of the DR2 haplotype) and Patr-DRB6 (another pseudogene of the Hugo haplotype), followed by HLA-DRB4 (the functional but nonpolymorphic gene of the DR4 haplotype). These relationships suggest that these six genes evolved from a common ancestor which existed before the separation of the human, gorilla, and chimpanzee lineages. The DRB2 and DRB6 have apparently been pseudogenes for at least six million years (myr). In the human and the gorilla haplotype, the DRB2 pseudogene is flanked on each side by what appear to be related genes. Apparently, the DR3 haplotype has existed in its present form for more than six myr.

Production of marked prolongation of islet xenograft survival (rat to mouse) by local release of mouse and rat antilymphocyte sera at transplant site.

Polymer rods impregnated with lyophilized particles of mouse (M) or rat (R) antilymphocyte serum (ALS) were placed adjacent to rat islet xenografts transplanted beneath the kidney capsule of diabetic mice. Insertion of rods containing only MALS or RALS had no effect on the survival time of the rat islet xenografts. In contrast, the insertion of both MALS and RALS rods with the graft produced a marked prolongation of islet xenograft survival (mean survival time greater than 55.5 +/- 10.9 days) compared with controls (14.7 +/- 2.5 days). One recipient was still normoglycemic at 100 days, and removal of the graft returned the animal to a diabetic state. The islet graft had a normal degree of beta-granulation, and a slight fibrotic reaction was present around the rods. The effect of the rods in prolonging survival of the xenografts resulted from a local slow release of MALS and RALS, because implantation of the MALS and RALS rods in the right kidney and the islets in the left kidney had no effect on prolonging islet xenograft survival. These findings indicate that local immunosuppression produced marked prolongation of rat islet xenograft survival in mice. This raises the possibility of using polymer rods for the local slow release of monoclonal antibodies to lymphokines and other agents for prevention of rejection of islet allografts and xenografts and to determine the effect of lymphokines in vivo on islet function.

Induction of Ia antigen expression on murine islet parenchymal cells does not diminish islet allograft survival.

Aberrant Ia antigen expression has been implicated in the pathogenesis of type 1 diabetes. Ia antigen expression was induced on isolated B10.BR murine islet parenchymal cells by culturing them for 5 days with lymphokine supernatants containing interferon-gamma (IFN-gamma) or with recombinant murine IFN-gamma + recombinant tumor necrosis factor. Ia positivity was confirmed by indirect immunofluorescence. Islets cultured for 5 days without cytokines were Ia-negative. Purified B10.BR islets allografted into the portal veins of C57BL/6J mice do not reject, which allowed the authors to determine whether aberrant expression of Ia on parenchymal cells has a deleterious effect on allograft survival. Ia-positive or Ia-negative islets were transplanted via the portal vein into diabetic C57BL/6J mice. All mice remained normoglycemic until they were killed at 30 to 60 days. Well-granulated islet allografts were identified histologically in all mice. The experiment was repeated using Balb/cJ mice as donors. Purified Balb/cJ islets are rapidly rejected by C57BL/6J mice. Induction of Ia expression on Balb/cJ islets significantly improved allograft survival. These findings indicate that Ia-positive islet cells do not induce rejection in these allograft models but may actually have a protective effect.

Tumor necrosis factor enhances interferon-induced Ia antigen expression on murine islet parenchymal cells.

Islets from male B10.BR mice (H-2k) were isolated by the collagenase technique, hand-picked with a Pasteur pipette, and incubated in tissue culture media supplemented with recombinant murine interferon-gamma (rIFN-gamma), recombinant murine tumor necrosis factor (rTNF), or both. IAk-molecules could be identified on the surface of islets incubated for 5 days with a combination of rIFN-gamma (1, 10, or 100 ng/ml) and rTNF (10 or 50 U/ml) by indirect immunofluorescence. Optimal concentrations of rIFN-gamma and rTNF, 10 ng/ml and 50 U/ml, respectively, were used in all subsequent experiments. Weak Ia-positivity could be identified on the surface of islets cultured with both cytokines for as little as 48 hours; however, the staining appeared most intense after 5 days of culture. Intensely Ia-positive islets were then carefully washed and cultured in media without either cytokine; Ia positivity could be identified on the surface of these islets for up to 1 week. Dispersed islet cells were cultured with rIFN-gamma alone (10 ng/ml), rTNF alone (50 U/ml), or both cytokines for 5 or 10 days. After either 5 or 10 days of culture with both cytokines, intense immunofluorescent staining for Ia could be identified on the surface of greater than 80-90% of the viable islet cells. Culture with IFN alone for 10 days resulted in 15-20% Ia positivity; culture with TNF alone did not cause Ia expression.

Studies on recombination within the mouse H-2 complex: IV. Characterization of new recombinant haplotypes H-2t7, H-2t8, H-2as2, and H-2as3.

Four new intra-H-2 recombinants were characterized serologically and functionally. In two of these recombinants, B10.ASR1 and B10.ASR7, crossing over occurred between the A alpha and E alpha subregions, very probably in E beta since most intra-I region recombinants thus far investigated at the DNA level appear to involve recombination within the E beta gene. In the other two recombinants, B10.ASR2 and B10.ARS8, crossing over occurred between the S and D subregions. B10.ASR2 and B10.ASR8, crossing over occurred between the S and D subregions. B10.ASR7, which is serologically indistinguishable from B10.BASR1 and B10.S(8R), slightly stimulates and strongly responds to both of these strains in MLR. The probable location of the B10.S(8R) stimulatory product is E beta. The H-2 composition of B10.ASR1 is closest to that of B10.S(9R) and B10.HTT; therefore, precise definition of the cross-over point at the DNA level will be of particular interest.

Effect of low-temperature culture and site of transplantation on hamster islet xenograft survival (hamster to mouse).

Isolated hamster islets were transplanted either into the liver via the portal vein or into the renal subcapsular space of diabetic C57BL/6J mice. The mean survival time (MST) of hamster islets cultured overnight at 37 degrees C was 8.5 +/- 0.6 days when transplanted into the liver as compared to an MST of greater than 21.7 +/- 4.9 days with 1 recipient still normoglycemic at 60 days when the islets were placed in the renal subcapsular space. Low-temperature culture (24 degrees C) of the hamster islets for 7 days produced a further significant prolongation of xenograft survival when the islets were placed beneath the renal capsule (MST greater than 43.3 +/- 4.7 days) with 2 recipients normoglycemic at 60 days. A single injection of anti-T-lymphocyte serum in conjunction with low-temperature culture did not produce a further increase in MST; however, 3 recipients were normoglycemic at 60 days. Removal of the kidney bearing successful xenografts at 60 days resulted in a rapid return to the diabetic state. It was interesting that the xenografts maintained normoglycemia in the mice at a level equivalent to the normal hamster (66.2 +/- 4.7 mg/dl) instead of the nonfasting level found in normal C57BL/6J mice (128.4 +/- 6.4 mg/dl). The findings indicate that low-temperature culture of the donor islets in conjunction with using the renal capsule as the site of transplantation produced a marked prolongation of hamster islet xenograft survival. Slow rejection of the xenografts did occur in this site, and histologic studies indicated that this rejection may be antibody mediated.

Interferon-mediated induction of Ia antigen expression on isolated murine whole islets and dispersed islet cells.

Islets from male B10.BR mice (H-2k) were isolated by the collagenase technique, handpicked with a Pasteur pipette, and incubated (either intact or after dispersion with Dispase) for 0, 3, 5, 7, 10, or 14 days in tissue culture medium supplemented with either lymphokine supernatants or recombinant murine interferon-gamma. Islets and single cells were examined for IAk molecules by use of indirect immunofluorescence. Ia-positive islet cells were identified on the surface of islets incubated with 5-10% lymphokine for greater than 4 days or with 10, 100, or 1000 ng/ml interferon for greater than 6 days. Islets incubated in unsupplemented medium were Ia negative. Incubation with 5% lymphokine induced Ia expression on 10-40% dispersed islet cells cultured for greater than 9 days. Dual immunofluorescent staining for Ia and insulin revealed that Ia-positive cells included both beta- and non-beta-cells.

The effect of cyclosporin-A, low-temperature culture, and anti-Ia antibodies on prevention of rejection of rat islet allografts.

The effect of cyclosporin-A, low-temperature culture, and anti-Ia antibodies on prevention of rejection of rat islet allografts was determined. Wistar-Furth islets were isolated by the collagenase technique and transplanted via the portal vein into diabetic Lewis recipients. Cyclosporin-A (30 mg/kg) injected at 0, 1, and 2 days after transplantation produced a significant prolongation of survival of the islet allografts (MST greater than 35.7 +/- 7.0 days) when hand-picked donor islets were used, whereas only a modest prolongation of survival (14.0 +/- 1.6 days) was obtained using donor islets removed directly from Ficoll gradients. This difference in survival was apparently due to the large number of lymphoid, antigen-presenting cells that were present in the islet fraction removed directly from the Ficoll gradients. Treatment of donor, hand-picked islets with a mixture of cross-reactive anti-Ia antibodies and complement without cyclosporin-A therapy did not prolong the survival of islet allografts (MST, 6.5 +/- 0.4 days versus 7.0 +/- 0.5 days in controls). In contrast, treatment of the donor islets with the mixture of anti-Ia antibodies and complement in conjunction with the 3-day course of cyclosporin-A therapy produced an 83% survival of the islet allografts at 60 days after transplantation. In vitro culture of hand-picked donor islets at 24 degrees C for 7 days and the 3-day course of cyclosporin-A therapy produced a 100% survival of the allografts at 60 days after transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of rejection of murine islet allografts by pretreatment with anti-dendritic cell antibody.

Previously we have demonstrated that islets of Langerhans treated with donor-specific anti-Ia serum and complement survive when transplanted across the major histocompatibility complex of the mouse. In this study, using immunofluorescence, we demonstrate two morphologically distinct populations of Ia-positive cells scattered within the Ia-negative islet tissue. A large irregularly shaped Ia-positive subset of cells were identified as dendritic cells by using the 33D1 antibody specific for a mouse dendritic cell antigen. The other small, round Ia-positive subset was 33D1 negative. Islets pretreated with anti-dendritic cell antibody and complement prior to transplantation survived in their histoincompatible recipients for greater than 200 days. Rejection of stable islet allografts promptly occurred when transplant recipients were challenged with 1 X 10(5) donor dendritic cells 60 days after transplantation. These results demonstrate an important in vivo role for donor dendritic cells in the stimulation of allograft rejection.

Prevention of allograft rejection by immunization with donor blood depleted of Ia-bearing cells.

Strain-specific unresponsiveness was induced in adult mice by immunizing them with donor blood treated with antiserum to Ia (I region-associated antigens) prior to the transplantation of islets of Langerhans. This regimen alone produced greater than 100-day survival of islet allografts transplanted across a major histocompatibility barrier.

Demonstration of active tolerance in maintenance of established islet of Langerhans allografts.

Streptozotocin-induced diabetes in mice can be reversed by transplantation of islets of Langerhans from histoincompatible mice if the islets are treated with anti-Ia-serum and complement before transplantation. Here we show that anti-Ia-treated islets most likely induce tolerance in the recipient animals. Daily injections of recipient-specific anti-I-J-serum (beginning 80 or more days after transplantation) and small numbers of donor splenocytes caused the prompt rejection of the islets in half of the animals; neither anti-I-J-serum nor donor splenocytes alone were effective. It is likely that rejection of established allografts after this treatment is the result of abolition of the activity of allograft-specific suppressor cells.

Differences in expression of MHC products between several H-2-restricted CTL clones.

Five cytotoxic T lymphocyte (CTL) clones, all specific for influenza virus A/JAP/57 and all H-2Kd-restricted, were typed for expression of MHC products. All express H-2K and H-2D encoded antigens, but they differ in their expression of I region products. One clone, 14-2, is positive with antisera directed against serologically detectable products apparently related to three different I regions, namely I-A, I-J, and I-E. Two clones, 11-1 and 14-1, lack I-E-associated antigens but express I-A and I-J products, and two other clones, 14-7 and 14-13, lack detectable I region products. These observations show that although discrete CTL clones may be homogeneous in their function, they differ in their expression of I region products.