Clinton E. Spencer

Evidence demonstrating poor kidney graft survival when acute rejections are associated with IgG donor-specific lymphocytotoxin.

The current prospective investigation was conducted to determine whether development of IgG donor-specific lymphocytotoxins detected at the onset of acute rejections was predictive of a poor-prognosis acute rejection. Between January 1990 and August 1993, 206 kidney transplants were performed. Cadaver kidney recipients were managed with antilymphocyte globulin as induction therapy and all recipients (i.e., cadaver and living related donor kidneys) received triple immunosuppressive therapy, i.e., CsA, AZA, and prednisone. Rejections were treated with intravenous Solu-Medrol and OKT3. Presence of donor-specific IgG lymphocytotoxin was detected by using dithiothreitol-pretreated sera (obtained at onset of rejection) and frozen donor cells. In addition, percentage of panel reactive antibody was determined on this dithiothreitol-pretreated sera. Of the 82 patients with biopsy-proven acute rejections, 19 were found to have developed donor-specific IgG lymphocytotoxin and a marked increase in panel reactive antibody. One-year graft survival in this group was dismal (16%), despite OKT3 therapy. Over 90% of these patients lost their graft within 2 months of rejection diagnosis. In 63 recipients who had acute rejections without development of IgG anti-HLA antibody, 1-year graft survival was 72%. The majority of these patients lost their grafts from chronic rejection. No anti-HLA activity was found in patients who did not have rejection episodes. Based on this study, evidence indicates that assaying for IgG donor-specific antibody at time of rejection is a valuable tool for selecting a subset of patients with poor-prognosis acute rejections. Identifying this subset will become important as we enter an era of new immunosuppressive agents.

The lack of long-term detrimental effects on liver allografts caused by donor-specific anti-hla antibodies

Recent reports indicate a higher incidence of both acute and chronic liver allograft rejection when, at the time of transplantation, the recipients serum contains donor-specific anti-HLA antibodies. From 9/89 to 5/91, 133 liver allografts were performed at our institution. Thirteen liver recipients had donor-specific IgG anti-HLA antibodies (complement-fixing) at the time of transplantation. In eleven patients, antibodies reacted to donor class I antigens while in 1 patient the donor-specific antibody had class II reactivity. Twelve patients have been followed for a minimum of 12 months (median 18 months, range 28–12 months). No hyperacute rejection was seen in any of the cases and four patients had acute rejections. Thus far only one of the twelve patients has biopsy evidence suggestive of chronic liver injury. The remaining have normal liver enzymes and bilirubin. Three of these twelve patients died (one from a myocardial infarction and the others from sepsis) accounting for a one-year graft survival of 75%. There was no significant statistical difference in the one-year graft survival in those recipients without donor-specific antibodies (i.e., 80.5%). In eight of the twelve patients, pretransplant preformed antibody level (PRA) was >50%. In six of the thirteen patients donor-specific antibody was present at dilutions greater than 1:64. As previously reported, the donor-specific antibody disappeared from the serum posttransplant within hours and did not reappear. In vitro studies demonstrated no factor in portal or hepatic artery blood that could inhibit rabbit complement mediated lysis of anti-HLA antibodies. We conclude that it is not a contraindication to do liver transplants in the presence of donor-specific anti-HLA antibodies.

The use of pronase-digested human leukocytes to improve specificity of the flow cytometric crossmatch

Two-color fluorescence cytometry (FCXM) has recently been introduced to improve the detection of anti-HLA antibodies that react to donor cells, especially in recipients receiving kidney allografts. Although this assay system is highly sensitive, it lacks specificity. Between 70% and 90% of potential kidney recipients with a positive FCXM would have been denied transplant if such an assay had been used alone to detect antidonor antibodies. Lack of specificity is principally due to normal or irrelevant IgG in aggregates or immune complexes binding to Fcλ R receptors on lymphocytes including B cells and a significant subset of T cells. To circumvent this problem, we digested Fcλ R receptors on lymphocytes with pronase. We present data demonstrating that pronase digestion of lymphocytes does not alter HLA antigenicity. In addition, pronased lymphocytes allow one to use either single- or two-color FCXM. With single-color FCXM, one can quantitate antibody reactivity to lymphocytes via a cursor (on the fluorescence histogram) that separates lymphocytes that do not bind to antibodies. We present data demonstrating that this modification renders FCXM highly sensitive and specific. In addition, one can discriminate between IgG and IgM antibodies that react to lymphocytes.

Naturally Occurring IgM Anti-Leukocyte Autoantibodies (IgM-ALA) Inhibit T Cell Activation and Chemotaxis

The physiological relevance of naturally occurring IgM-ALA remains to be elucidated. These autoantibodies are present from birth and increase in diverse inflammatory states that are both infectious and noninfectious. Clinical observations showing significantly less acute allograft rejections in recipients having high IgM-ALA levels, led us to investigate whether IgM-ALA could have a functional role in attenuating T cell mediated inflammatory responses. In pursuit of this hypothesis, we did studies using IgM purified from the serum of normal individuals, patients with end stage renal disease, and HIV-1 infection. All preparations of IgM immunoprecipitated certain receptors e.g., CD3, CD4, CCR5, and CXCR4 from whole cell lysates but failed to immunoprecipitate IL-2R and HLA Ags. In physiological doses IgM down-regulated CD4, CD2 and CD86 but not CD8 and CD28, inhibited T cell proliferation, decreased production of certain proinflammatory cytokines e.g., TNF-α, IL-13 and IL-2, but not IFN- γ, IL-1β, GM-CSF, IL-6 and IL-8 and inhibited leukocyte chemotaxis. These inhibitory effects were more pronounced when using IgM from patients with high levels of IgM-ALA and these inhibitory effects were significantly reduced after using IgM preabsorbed with leukocytes. IgM-ALA binding to leukocytes was found to be highly specific, as <10% of IgM secreting B cell clones had IgM-ALA specificity with some clones having specificity for either T cells or monocytes. These findings support the concept that IgM-ALA provides an innate mechanism to regulate T cell mediated inflammatory responses.

Hyperacute renal allograft rejection from anti‐HLA class 1 antibody to B cells ‐antibody detection by two color FCXM was possible only after using pronase‐digested donor lymphocytes

Abstract We present a report of a transplant recipient who lost her renal allograft from hyperacute rejection. This was secondary to a weak IgG anti‐HLA class I antibody that was only reactive to donor B lymphocytes. This antibody was not detected in her pretransplant serum by the conventional complement‐dependent cytotoxicity assays using donor blood lymphocytes. Pretransplant sera were analyzed retrospectively by two‐color flow cytometric crossmatching (FCXM). It was difficult to determine if the recipients serum contained an IgG antibody specific for HLA on donor B cells since IgG from control AB sera and pretransplant sera bound equally well to CD19 B cells. However, when donor lymphocytes were pretreated with pronase to digest the membrane receptor for Fc domain of IgG (FcyR) on non‐T‐cells, control IgG in AB serum did not bind to B cells and, hence, it was easy to detect binding of IgG (in pretransplant sera) to HLA on B cells. This case underscores the importance of identifying weak anti‐HLA class I antibodies reactive only to B cells. Moreover, it shows that the currently used two‐color FCXM lacks the specificity to detect such antibodies.

HLA matching for simultaneous pancreas-kidney transplantation in the United States: a multivariable analysis of the UNOS data.

BACKGROUND As the incidence of diabetic nephropathy increases, especially in minority populations, more simultaneous pancreas-kidney (SPK) transplants are being performed both in the United States and worldwide. The role of matching on SPK outcomes and organ allocation remains controversial. The purpose of this analysis was to determine the influence of HLA matching using currently employed criteria on 5-year SPK graft survival. METHODS We performed an analysis of all 3,316 SPK transplants performed in the United States reported to the United Network for Organ Sharing (UNOS) between December 31, 1988 and December 31, 1994. Kaplan-Meier unadjusted 1- and 5-year graft survival with log rank comparisons and Cox multivariable regression models that adjusted for 12 confounding variables were used to analyze the influence of HLA matching on outcomes. RESULTS Despite low-grade HLA or DR matching or high levels of common reactive groups (CREG) mismatching, 1- and 5-year allograft survival rates were 90% and 78% for kidney, and 85% and 75% for pancreas transplantation. CONCLUSIONS SPK transplantation is associated with excellent outcomes independent of the level of HLA matching. These data support the hypothesis that SPK transplants need not be allocated based on matching criteria, thus minimizing organ ischemia time and promoting a more racially equitable allocation for SPKs in the US today.