Solid-Phase C1q/C3d Fixing Readouts Correlate with High Median Fluorescence Intensity (MFI) De Novo Donor-Specific HLA Antibodies and C4d⁺ Antibody-Mediated Rejection in Kidney Transplant Recipients.

BACKGROUND Solid-phase assays to investigate the complement-activating capacity of HLA antibodies have been utilized to optimize organ allocation and improve transplant outcomes. The clinical utility of C1q/C3d-binding characteristics of de novo donor-specific anti-HLA antibodies (dnDSA) associated with C4d-positive antibody-mediated rejection (C4d⁺ AMR) in kidney transplants (KTx) has not been defined. MATERIAL AND METHODS Sera from 120 KTx recipients that had dnDSA concurrent with protocol/cause biopsy (median 3.8 years after transplantation) were screened for C1q and C3d-binding dnDSA. The difference in the incidence of C4d⁺ AMR between recipients with and without C1q/C3d-binding dnDSA was assessed. RESULTS Over 86% of dnDSAs were class II antibodies. The immunodominant dnDSAs characterized by the highest median fluorescence intensity (MFI) in most recipients were HLA-DQ antibodies (67%). Most recipients (62%, n=74) had either C1q⁺ (56%), C3d⁺ (48%), or both C1q⁺C3d⁺ (41.2%) dnDSA, while the remaining 38% were negative for both C1q and C3d. Of those with C1q⁺/C3d⁺ dnDSA, 87% had high-MFI IgG (MFI=14144±5363 and 13932±5278, respectively), while 65% of C1q⁻C3d⁻ dnDSA had low-MFI IgG (MFI=5970±3347). The incidence of C4d+ AMR was significantly higher in recipients with C1q⁺ (66%), C3d+ (74%), and C1q⁺C3d⁺ (72%) dnDSA than in those with C1q⁻C3d⁻ dnDSA (30%) recipients. Recipients with C3d⁺/C1q⁺ dnDSA had higher C4d⁺ scores on biopsy. CONCLUSIONS C1q⁺/C3d⁺ dnDSA were associated with C4d⁺ AMR and high-IgG MFI. Our data call into question the predictive utility of C1q/C3d-binding assays in identifying KTx recipients at risk of allograft failure. In conclusion, IgG MFI is sufficient for clinical management, and the C1q/C3d-assays with added cost do not provide any additional information.

HLA-specific B cells: I. A method for their detection, quantification, and isolation using HLA tetramers.

BACKGROUND: The development of highly sensitive and specific assays for detecting and characterizing HLA-specific antibodies has contributed to an appreciation of the extensive involvement of those antibodies in graft injury and dysfunction. However, understanding the regulatory processes of the humoral response to transplantation and the mechanisms underlying therapeutic agents and protocols for preventing and treating sensitization requires a way to study HLA-specific B cells. METHODS: Lymphocyte preparations enriched for B cells were stained with one or more of three different HLA tetramers. Tetramer-positive (tet+) B cells were enumerated and evaluated for an association of their frequencies with known sensitization. In some cases, tet+ B cells were isolated and placed in culture with supplements known to activate B cells in a nonspecific fashion. RESULTS: For all tetramers used, the frequencies of tet+ B cells were significantly higher (4.1%-5.5%) among sensitized patients than among nonsensitized patients (1.6%-3.2%, P<0.001). Binding of the tetramers occurred by the surface immunoglobulin antigen receptor with little or no binding to antibody captured in the Fc receptor. Cultured tet+ B cells produced antibodies specific for epitopes of the tetramer antigen. There appeared to be a certain amount of crossreactivity in the binding of tetramers. The frequency of CD27+ cells among tet+ B cells was higher, on average (34.4%-38.8%) than among all B cells (26.2%) whereas the frequencies of CD38 were comparable in the two groups. CONCLUSIONS: Staining with HLA tetramers provides a means for identifying, quantifying, and isolating HLA-specific B cells.

HLA-specific B cells: II. Application to transplantation.

BACKGROUND: Differences in the antibody response to allogeneic transplantation exist between groups defined by race or gender. These differences may reflect differences in immune competency and/or exposure to alloantigens. We have investigated the frequencies and phenotypes of HLA-specific B cells to address those possibilities. METHODS: HLA-specific B cells were identified by staining with HLA tetramers (tet) as described previously and the distribution of CD27 and CD38 among those cells were measured in groups defined by various parameters. Possible correlation between frequencies of HLA-specific B cells and production of HLA-specific antibody after transplantation was also investigated. RESULTS: We found no correlation between the frequencies of CD27+tet+ (33%-44% vs. 34%-36%) or CD38+tet+ (57%-65% vs. 59%-66%) B cells and a previous mismatch for the HLA antigen of the tetramer. However, there was an increase in CD38+tet+ B cells among patients making antibody to the tetramer antigen (67%-72% vs. 53%-56%). Blacks had lower frequencies of CD27+ B cells than did whites (11.8% vs. 28.9%, P=0.003), but had greater increases of these cells among tet+ cells than did whites. There was a higher frequency of tet+ B cells among patients who developed "new" antibody to the HLA antigen (3.9%-8.6%) of the tetramer after transplantation than among those who did not (1.1%-3.7%). CONCLUSIONS: The phenotype of HLA-specific B cells reflects current or historic sensitization to HLA and may reflect inherent differences between groups defined by race and/or gender. The frequencies of HLA-specific B cells may predict patients at risk for production of donor-specific antibody after transplantation.

Specific and durable elimination of antibody to donor HLA antigens in renal-transplant patients.

BACKGROUND: Donor-specific antibody (DSA) is the major barrier to success of kidney transplants. Attempts to deal with this problem have used plasmapheresis to remove antibodies or high-dose pooled immunoglobulin (IVIg) to down-regulate DSA. However, elimination of antibodies by these methods has been limited in duration or scope. METHODS: We have confirmed the presence of immunoglobulin (Ig)G antibody to one or more donor HLA antigens in 49 patients treated with alternate-day, single-volume plasmapheresis followed by low-dose cytomegalovirus (CMV) hyperimmune globulin (CMV-Ig) combined with quadruple immunosuppression. We examined the effect of the treatment protocol on antibodies to donor HLA, third-party HLA, and nominal antigens. RESULTS: At the end of treatment, 63% of patients had lost antibody to donor HLA, whereas only 27% had lost antibody to third-party HLA (P<0.001). More strikingly, loss of antibody to donor and third-party HLA antigens occurred in 89% and 19%, respectively, of patients followed for 2 or more months after end of treatment (P<0.0001). No elimination of antiviral antibodies tested was seen. With one exception, elimination of DSA appeared to be independent of antibody titer or specificity, the number of different antibody specificities, or whether or not the target antigen was a repeat mismatch. The effect appears to be long lasting, with no return of DSA observed in patients followed for an average of 13 months. CONCLUSIONS: Plasmapheresis and low-dose CMV-Ig combined with traditional immunosuppression is effective in producing a specific and durable elimination of antibody to donor HLA.

Characterization of an antibody specific for HLA-A36 and not reactive with HLA-A1.

Humoral sensitization to HLA often results in antibodies to public determinants shared among two or more antigens. Although monoclonal antibodies to A36 have been produced, there are no reports of polyclonal antibodies that react with A36 but not A1. We report here sera from a heart transplant recipient that reacted with A36 but not A1 in tests with both phenotype and single antigen panels on the Luminex platform. Flow cytometric crossmatch tests yielded positive results with an A36 bearing phenotype but not with a phenotype containing A1. A36 reactivity in solid phase assays was abrogated by absorption with cells bearing A36, but not with A1-positive cells. The frequency of B cells in this patient specific for A1 was comparable to that for individuals not sensitized to A1. These data indicate that reactivity was to an epitope present on A36 but absent from A1.

Immunogenetics and immunology in transplantation.

Advances in immunogenetics and histocompatibility have facilitated the clinical transplantation of solid organs and tissues. Improved definition of HLA antigens, alleles, and haplotypes has clarified the diversity of the HLA system among different racial/ethnic populations. Knowledge of allele and haplotypes frequencies derived from these studies can be applied to the estimates of transplant compatibility and the likelihood finding suitable donors. Humoral sensitization that has been a major barrier to transplantation is being successfully treated with desensitization and paired donor exchanges. Sensitive solid phase immunoassays for HLA-specific antibodies permit analyses that guide both desensitization and donor exchanges through antibody monitoring, virtual crossmatches, and detection of cryptic sensitization. Ongoing studies are investigating the mechanisms underlying the down-regulation of donor HLA-specific antibodies.

Reconstitution of peripheral allospecific CD19+ B-cell subsets after B-lymphocyte depletion therapy in renal transplant patients.

BACKGROUND: Desensitization protocols, which frequently use lymphocyte depleting agents have increased access to successful transplantation for sensitized candidates. Here, we report on the reconstitution of human leukocyte antigen (HLA)-specific B lymphocytes in renal transplant patients after treatment with B-lymphocyte depletion. METHODS: Sixteen renal transplant candidates were included in the study. Eleven patients were treated with anti-CD20 monoclonal antibody (Ab), four of whom also underwent splenectomy perioperatively. Five patients who did not undergo B-cell depletion were studied as controls. Blood samples were obtained before any treatment and transplant, and at later time points up to 44 months posttransplant. HLA-specific B-cell subpopulations were identified by staining with fluorochrome-labeled HLA tetramers and anti-CD19, CD27, and CD38 monoclonal Abs. RESULTS: Total circulating B lymphocytes repopulated within 12 months post-B-cell depletion. The majority of the recovering cells had the phenotype of transitional CD38 B cells and the percentages of mature, memory CD27 B cells remained significantly depressed. There was a sustained reduction in the proportion of HLA-specific CD27 memory B cells, whereas the HLA-specific CD38 B-cell population returned to near pretreatment levels by 12 months. The presence of mismatched HLA antigens seemed to affect the reconstitution kinetics. The delay in reconstitution of HLA-specific CD27 memory B cells was greater for donor-specific compared with third party. CONCLUSIONS: A delay in functional maturity of repopulating HLA-specific B cells, and in particular those specific for donor HLA, after B-lymphocyte depletion treatment in renal transplant recipients may contribute to the efficacy of desensitization protocols.