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.

Molecular relationship between private and public H-2 antigens as determined by antigen redistribution method.

Molecular relationship of public H-2 antigens 1, 5, 6, 8, 11, 13, 25, and 28 to private antigens controlled by K and D regions was studied using the technique of antibody-induced resistance to complement-mediated cytotoxicity. The results indicate physical association in the cell membrane between H-2 antigens 1 and 23 of H-2-a, 8 and 31 of H-2-d, 11 and 17 of H-2-q, 13 and 30 of H-2-q, 25 and 23 of H-2-k, and 28 and 31 of H-2-g. These results are in agreement with genetic mapping placing the determinants of antigens H-2.8, 11 and 25 in the K region , the determinant of antigen H-2.13 in the D region, and the determinants of antigens H-2.1 and 28 in either the K or the D region. In contrast to genetic mapping placing the determinant for antigen H-2.6 in the D region, we found that in the H-2-b haplotype the antigen is associated with K region antigen H-2.33 and H-2.32, and interpreted this result as evidence for two homologous H-2.5 sites controlled by opposite ends of the H-2 complex. Although the data do not prove that public antigens are carried by the same molecules as private ones, they demonstrate a close physical association in the membrane between the two groups of loci, K and D, coding for the first 33 classical H-2 antigens (with the exception of antigen H-2.7), and thus support the two-locus model. The data also support the duplication model of H-2 by demonstrating two homologous H-2.5 sites associated with K and D molecules.

Histocompatibility antigens controlled by the I region of the murine H-2 complex. I. Mapping of H-2A and H-2C loci.

Skin grafts were reciprocally exchanged in pairs of congenic lines identical in all genes except those located in the central portion of the H-2 complex. Seven such lines were tested: 6R, B10.AQR, A.TL, A.TH, 7R, 9R, and B10.HTT. In all donor-recipient combinations at least some grafts were rejected. In combinations differing at the IA subregion (and other central H-2 regions or subregions), all first-set grafts were rejected within 3 wk after transplantation, and all second-set grafts were rejected within 10 days. In combinations differing at the IC subregion (and other central regions, but not at the IA subregion) between 60 and 100% of first-set grafts were rejected, but some grafts survived for over 100 days. Most of the second-set grafts were rejected within 1 mo after grafting. This behavior of skin grafts indicated the presence of two histocompatibility loci in the I region, a strong one and a weak one. This conclusion was confirmed by genetic mapping which placed the strong locus in the IA subregion and the weak locus in the IC subregion. We designate the former locus H-2A and the latter H-2C. The same strain combinations used for the skin grafting were also used for determination of the capacity of I-region antigens to function as targets in the in vitro cell-mediated lymphocytotoxicity (CML) assay. Spleen cells from mice presensitized in vivo by skin grafting were restimulated in vitro and tested against 51Cr-labeled concanavalin A or lipopolysaccharide blasts. The testing revealed the presence in the I region of two loci coding for CML-target antigens. The two loci comapped with the H-2A and H-2C loci and were most likely identical to them. As in the skin grafting test, in the CML test, the H-2A antigens evoked stronger response than the H-2C antigens. Rejection of skin grafts across the H-2A and H-2C loci was accompanied by the production of Ia antibodies. Direct cytotoxic and absorption tests with Ia antibodies directed against antigens coded for by the IC subregion revealed the presence of IaC antigens on epidermal cells. We suggest that the products of Ia loci might function as transplantation antigens.

Serological distinction of mutants B6.CH(zl) and B6.M505 from strain C57BL-6.

A quantitative serological difference was found between strains Hz1 and M505 carrying mutant H-2 haplotypes ba and bd, respectively, and the original strain B6(H-2(b)). The finding suggests that the mutations occurred in the H-2K(b) gene, and together with data on MLR and CML challenges the current concept of H-2 regions' involvement in immune reactions.

Histocompatibility antigens controlled by the I region of the murine H-2 complex. II. K/D region compatibility is not required for I-region cell-mediated lymphocytotoxicity.

In the cell-mediated lymphocytotoxicity assay, A.TH effector cells sensitized to A.TL lymphocytes lyse not only A.B10.AQR effector cells lyse B10.BR and B10.BYR target cells in addition to B10.AQR cells. These findings indicate that for CML to occur across the IA region barrier, compatibility at K or D regions is not required.

Induction of resistance to antibody-mediated cytotoxicity. H-2, Ia, and Ig antigens are independent entities in the membrane of mouse lymphocytes.

Mouse spleen or thymus lymphocytes incubated with monospecific H-2 or Ia alloantisera and then coated with a xenogeneic antimouse Ig serum become specifically resistant to the alloantiserum (and complement) they have been incubated with. This so called "lysostrip method" was used to investigate the molecular interrelationships of antigens in the mouse lymphocyte membrane. The results of this investigation confirm that H-2K and H-2D antigens are carried by two distinct populations of molecules. They provide evidence that the Ia antigens move in the membrane independently of both H2-K and H-2D antigens; and finally they demonstrate absence of any physical linkage between Ig receptors in B cells, on the one hand, and Ia, H-2K, and H-2D molecules on the other hand.

Murine pancreatic beta-cells express H-2K and H-2D but not Ia antigens.

Direct microcytotoxicity testing and absorption analyses were employed to determine whether H-2K, H-2D, and Ia antigens are present on murine islet of Langerhans cells. Products of the H-2K and H-2D loci were found on beta-cells from three different mouse strains, but I-region (Ia) antigens were not detected. The absence of Ia gene products from islet cells may be of importance in explaining the survival of islet allografts across major histocompatibility barriers.

Serological identification of an Ir-region product.

Reciprocal immunization of congenic lines differing in the middle portion of the H-2 complex leads to the production of antibodies which react with an antigen or antigens controlled by the Ir region. The antigen designated Ir-1.1 seems to be present only on a subpopulation of lymphocytes from lymph nodes and spleen. It is absent on bone marrow cells.

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.

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.

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)

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 thymectomy on the competitive potential of C57BL/6 bone marrow graft.

When a mixture of bone marrow cells derived from mice of the CBA-T6T6 and C57BL/6 strains and Lewis rats is injected into lethally irradiated (C57BL/6 X CBA-T6T6)F1 hybrid recipients, a process of cell interaction takes place, which in less than 2 weeks leads to the complete and permanent take over in hematopoietic and lymphoid organs by the C57BL/6 cell population. This is shown by the chromosome marker technique (T6) analysis. Adult thymectomy of F1 hybrid hosts prior to irradiation and bone marrow transplantation does not alter the competitive potential of the C57BL/6 donor cell population. However, neonatal thymectomy of the prospective C57BL/6 donors significantly impairs their "superiority" as donor cells, and CBA-T6T6 dividing cells persist at all times in all tissues analyzed. The results lend support to the concept of thymus-dependent precursor cells residing in "prethymic" hematopoietic tissue.

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.

A new histogenetic method for minor histocompatability antigen typing.

A new method for typing minor H antigens is described. The method is based on the observation that effector T cells, sensitized in vivo to minor H mitigens and then rechallenged in vitro, will lyse labeled target cells in the CML assay, provided that the target, the sensitizing, and the responding cells share the same allele at H-2K or H-2D loci. In this method, two congenic lines differing at a minor H locus are cross-immunized (by skin grafting, injection of lymphoid cells, or, preferably, by a combination of both treatments), rechallenged in vitro, and tested against a panel of strains carrying the same H-2 haplotype as the two lines. Target cells of the panel carrying the same minor H antigens as the strain against which the effector cells were sensitized are lysed; target cells not carrying these antigens are unaffected. The feasibility of the method was demonstrated by testing for H-3 antigens of a panel fo H-2b-bearing strains. The new method is faster and more economical than the classicial F1 method which has been used until recently.

A method for detection of Ia antigens in the absence of appropriate H-2 recombinants.

A method is described which obviates the use of H-2 recombinant strains for production and detection of Ia antibodies. The antibodies are produced by immunization with spleen cells in a strain combination in which the donor differs from the recipient either in the K end of the H-2 complex or in the whole complex. If the right schedule is used, this immunization produces antibodies against both H-2 and Ia antigens. The H-2 antibodies are absorbed out with erythrocytes or T lymphocytes rendering the antiserum specific for Ia antigens. The presence of Ia Ia antibodies is asserted by determining the tissue distribution of the antigens detected with the absorbed antiserum. Another antiserum is then prepared in the same strain combination by immunization with tissue that does not contain Ia but does contain H-2 antigens (e.g., a sarcoma of the proper genotype). The second antiserum contains only H-2 antibodies and the presence of these antibodies is again asserted by determining the tissue distribution of the corresponding antigens. The molecular distinctiveness of the putative Ia and H-2 antigens is then demonstrated by the newly developed technique of antibody-mediated induction of resistance to cytotoxicity (lysis). If the antigens detected with the two antisera move independently in the cell membrane, and if the antigens detected with the first antiserum do indeed have Ia-like properties, it is concluded that the antiserum detects Ia antigens. This method should prove to be useful for detection of Ia antigens in H-2 haplotypes for which no intra-H-2 recombinants are known, and for detection of Ia-like antigens in other mammalian species, particularly in man.