De novo donor-specific HLA antibodies decrease patient and graft survival in liver transplant recipients.

The role of de novo donor-specific HLA antibodies (DSA) in liver transplantation remains unknown as most of the previous studies have only focused on preformed HLA antibodies. To understand the significance of de novo DSA, we designed a retrospective cohort study of 749 adult liver transplant recipients with pre- and posttransplant serum samples that were analyzed for DSA. We found that 8.1% of patients developed de novo DSA 1 year after transplant; almost all de novo DSAs were against HLA class II antigens, and the majority were against DQ antigens. In multivariable modeling, the use of cyclosporine (as opposed to tacrolimus) and low calcineurin inhibitor levels increased the risk of de novo DSA formation, while a calculated MELD score >15 at transplant and recipient age >60 years old reduced the risk. Multivariable analysis also demonstrated that patients with de novo DSA at 1-year had significantly lower patient and graft survival. In conclusion, we demonstrate that de novo DSA development after liver transplantation is an independent risk factor for patient death and graft loss.

Class II alloantibody and mortality in simultaneous liver-kidney transplantation.

Hyperacute kidney rejection is unusual in crossmatch positive recipients of simultaneous liver-kidney transplants (SLKT). However, recent data suggest that these patients remain at risk for antibody-mediated kidney rejection. To further investigate the risk associated with donor-specific alloantibodies (DSA) in SLKT, we studied 86 consecutive SLKT patients with an available pre-SLKT serum sample. Serum samples were analyzed in a blinded fashion for HLA DSA using single antigen beads (median florescence intensity≥2,000=positive). Post-SLKT samples were analyzed when available (76%). Thirty patients had preformed DSA, and nine developed de novo DSA. Preformed class I DSA did not change the risk of rejection, patient or allograft survival. In contrast, preformed class II DSA was associated with a markedly increased risk of renal antibody mediated rejection (AMR) (p=0.006), liver allograft rejection (p=0.002), patient death (p=0.02), liver allograft loss (p=0.02) and renal allograft loss (p=0.045). Multivariable modeling showed class II DSA (preformed or de novo) to be an independent predictor of patient death (HR=2.2; p=0.043) and liver allograft loss (HR=2.2; p=0.044). These data warrant reconsideration of the approach to DSA in SLKT.

Bortezomib for acute antibody-mediated rejection in liver transplantation.

Antibody-mediated rejection (AMR) is an uncommon, but challenging type of rejection after solid organ transplantation. We review three cases of AMR in ABO-compatible liver transplant recipients. These cases were characterized by severe acute rejection resistant to steroids and antithymocyte globulin, histologic evidence of plasma cell infiltrates, C4d positivity and high serum anti-HLA donor-specific antibodies. All three patients were treated with bortezomib, a proteasome inhibitor effective in depleting plasma cells. After treatment, all patients had improved or normal liver function tests, resolution of C4d deposition and significant decline in their HLA donor-specific antibodies.

High mean fluorescence intensity donor-specific anti-HLA antibodies associated with chronic rejection Postliver transplant.

In contrast to kidney transplantation where donor-specific anti-HLA antibodies (DSA) negatively impact graft survival, correlation of DSA with clinical outcomes in patients after orthotopic liver transplantation (OLT) has not been clearly established. We hypothesized that DSA are present in patients who develop chronic rejection after OLT. Prospectively collected serial serum samples on 39 primary OLT patients with biopsy-proven chronic rejection and 39 comparator patients were blinded and analyzed for DSA using LABScreen(®) single antigen beads test, where a 1000 mean fluorescence value was considered positive. In study patients, the median graft survival was 15 months, 74% received ≥ one retransplant, 20% remain alive and 87% had ≥ one episode of acute rejection. This is in contrast to comparator patients where 69% remain alive, and no patient needed retransplant or experienced rejection. Thirty-six chronic rejection patients (92%) and 24 (61%) comparator patients had DSA (p = 0.003). Chronic rejection versus comparator patients had higher mean fluorescence intensity (MFI) DSA. Although a further study with larger numbers of patients is needed to identify clinically significant thresholds, there is an association of high-MFI DSA with chronic rejection after OLT.

Reducing de novo donor-specific antibody levels during acute rejection diminishes renal allograft loss.

The effect of de novo DSA detected at the time of acute cellular rejection (ACR) and the response of DSA levels to rejection therapy on renal allograft survival were analyzed. Kidney transplant patients with acute rejection underwent DSA testing at rejection diagnosis with DSA levels quantified using Luminex single-antigen beads. Fifty-two patients experienced acute rejection with 16 (31%) testing positive for de novo DSA. Median follow-up was 27.0 +/- 17.4 months postacute rejection. Univariate analysis of factors influencing allograft survival demonstrated significance for African American race, DGF, cytotoxic PRA >20% (current) and/or >50% (peak), de novo DSA, C4d and repeat transplantation. Multivariate analysis showed only de novo DSA (6.6-fold increased allograft loss risk, p = 0.017) to be significant. Four-year allograft survival was higher with ACR (without DSA) (100%) than mixed acute rejection (ACR with DSA/C4d) (65%) or antibody-mediated rejection (35%) (p < 0.001). Patients with >50% reduction in DSA within 14 days experienced higher allograft survival (p = 0.039). De novo DSAs detected at rejection are associated with reduced allograft survival, but prompt DSA reduction was associated with improved allograft survival. DSA should be considered a potential new end point for rejection therapy.

Proteasome inhibition reduces donor-specific antibody levels.

BACKGROUND: Current antibody-mediated rejection (AMR) therapies (intravenous immunoglobulin, apheresis, rituximab, polyclonal antibodies) do not target the primary antibody producing B cells, that is, the plasma cell. We report the preliminary results from the first clinical experience with plasma cell targeted therapy with bortezomib. Bortezomib is approved by the US Food and Drug Administration for the treatment of plasma cell tumors (multiple myeloma). METHODS: Kidney transplant patients with mixed acute cellular rejection (ACR) and AMR episodes (by Banff '97 criteria, update 2005) were treated with bortezomib (1.3 mg/m(2) per dose x 4) at standard labeled doses. Patients were monitored by serial donor specific anti-HLA antibody (DSA) determinations [Luminex/Labscreen beads] and quantified by conversion to fluorescence intensity to molecules of equivalent soluble fluorescence (MESF). RESULTS: Five patients were treated with bortezomib. Each patient also had coexisting ACR. In each case, bortezomib treatment led to prompt ACR and AMR rejection reversal. DSA levels decreased significantly in all patients (except 1 patient who had short follow-up). Observed toxicities from bortezomib included a transient grade III thrombocytopenia (1 patient) and mild-to-moderate nausea, vomiting, and/or diarrhea (3/5 patients). Opportunistic infections were not observed. CONCLUSIONS: Bortezomib therapy provides effective reduction in DSA levels with long-term suppression. These preliminary results indicate that proteasome inhibition provides an effective means for reducing HLA antibody levels in transplant recipients.

Analysis of T cell responses in liver allograft recipients. Evidence for deletion of donor-specific cytotoxic T cells in the peripheral circulation.

Analysis of cell-mediated lympholysis in long-term liver allograft recipients indicated that there was a donor-specific unresponsiveness that could not be reversed by the addition of rIL-2 and/or mixed lymphocyte culture supernatant or by nonspecific stimulation of the cultures with PHA. Stimulation of recipient cells with semisyngeneic cells having both donor and third-party HLA antigens failed to reveal the presence of cytotoxic T cells (CTL) specific to the donor, whereas the CTL response to third-party antigens remained normal. Removal of B lymphocytes from the responding cell population did not influence the responses. Furthermore, limiting dilution analysis showed that the liver transplant recipients did not have detectable levels of CTL precursors (CTLp) reactive to the donor antigens, whereas their CTLp to third-party antigens remained normal. Donor-specific CTLp were present before and during the early post-transplant period; these cells were eliminated from the peripheral circulation by 10 mo after transplantation. Taken together, these results indicate that there is a deletion of CTLp specific to donor MHC antigens in the peripheral circulation of long-term liver allograft recipients that may account in part for the success of liver transplantation across MHC barriers.

Phenotype of syngeneic tumor-specific cytotoxic T-lymphocytes and requirements for their in vitro generation from tumor-bearing host and immune spleens.

Cells required for the in vitro generation of syngeneic cytotoxic T-lymphocytes (CTL) against the P815 mastocytoma in the DBA/2 mouse strain were investigated. For both immune and tumor-bearing host spleen cells, CTL effector cells were eliminated by treatment with anti-Thy1.2, anti-Lyt1.1, or anti-Lyt2.1 and C', but were resistant to anti-L3T4 (GK1.5). Thus, CTL effectors (and their precursors) were Lyt1+2+, L3T4-. However, P815-specific CTL could not be generated in the absence of L3T4+ cells, whose function could be replaced with exogenous interleukin-2 (IL-2). When monoclonal antibodies against L3T4 were added to mixed leukocyte tumor cultures, CTL generation was markedly inhibited. Depletion of accessory cells also led to a marked reduction in CTL generation, which could be restored to control levels by adding adherent cells from normal spleens or with exogenous IL-2, but not with IL-1. Thus, accessory cells are apparently required to present the tumor antigens of this Ia-negative tumor to T-helper cells.

Inhibition of tumor-specific cytotoxic T-lymphocyte responses by transforming growth factor beta 1.

Transforming growth factor beta (TGF-beta) is a potent immunosuppressive cytokine that is produced by neoplastic and normal cells. It has not been demonstrated directly, however, that TGF-beta can inhibit antigen-specific T-cell responses to tumor cells in vitro. We show here that generation of antitumor cytotoxic T-lymphocyte (CTL) activity in mixed-lymphocyte tumor cultures of splenocytes from DBA/2 mice immunized with the syngeneic P815 mastocytoma + Corynebacterium parvum was consistently and profoundly inhibited when 0.675 to 10 ng/ml of TGF-beta were added on Day 0 of culture. TGF-beta added on Day 1 or later had little or no effect. In contrast to the results with P815 immune mice, mixed-lymphocyte tumor cultures established with splenocytes from P815 tumor-bearing hosts showed variable degrees of inhibition by TGF-beta, depending on the stage of the ongoing in vivo immune response. Addition of recombinant murine tumor necrosis factor alpha (1,000 or 10,000 units/ml) partially reversed inhibition of CTL responses by TGF-beta, while recombinant interleukin 2 nearly completely reversed the suppression. These data indicate that one level at which TGF-beta may act to inhibit mixed-lymphocyte tumor cultures is that of cytokine production. To determine whether TGF-beta also has any direct effect on CTL, P815-specific CTL clones derived from tumor-bearing host mice were utilized. We found that proliferation of rested CTL clones in response to tumor cells + interleukin 2 was inhibited by 5 ng/ml of TGF-beta, while the interleukin 2-dependent reactivation of cytolytic activity was not affected by TGF-beta. In contrast to rested CTL, when TGF-beta was added to cultures of previously activated CTL, proliferation was not inhibited. These data demonstrate that TGF-beta has profound inhibitory effects on the in vitro generation of effector CTL from tumor-specific murine splenocytes, and this inhibition may be an indirect result of suppressed cytokine production as well as a direct antiproliferative effect on CTL.

Histocompatibility testing predicts acute rejection risk in early corticosteroid withdrawal regimens.

UNLABELLED: Histocompatibility testing has been shown to predict acute rejection risk in steroid-based immunosuppression. However, little evidence exists of its ability to predict acute rejection risk in corticosteroid-free patients, with no evidence in early corticosteroid withdrawal (CSWD) under modern immunosuppression. The purpose of this study was to evaluate the ability of histocompatibility testing to identify patients at high risk for acute rejection after early CSWD. METHODS: One hundred eighty-one patients were entered into six IRB-approved early CSWD regimens. Histocompatibility testing included serologic PRA, flow cytometric PRA testing by Class I and Class II MHC beads, and B cell crossmatching with pronase treatment. All rejection episodes were biopsy proven, and grading was assigned using Banff criteria. Influence of individual tests was examined using Chi square univariate and multivariate logistic regression analysis. RESULTS: Median follow-up was 23.5 months (range 7-48 months). Of 181 patients, 16% were repeat transplant recipients, 36% received deceased donor renal transplants, 48% received living related donor renal transplants, and 16% received living unrelated transplants. Overall patient survival was 97%, and death-censored graft survival was 96.5%. Acute rejection rates in the entire follow-up period were 17.7%. 12.4% in primary transplant recipients and 37% in repeat transplant recipients. Multivariate analysis revealed that HLA AB and DR locus mismatching were associated with increased acute rejection risk. Similarly, serologic PRA analysis predicted acute rejection risk; however, flow cytometry crossmatching did not predict acute rejection risk. The greatest single influence on acute rejection risk appeared to be a flow cytometric B cell crossmatch (7.94-fold increased risk). In conclusion, histocompatibility testing can identify patients at high risk for acute rejection following early CSWD. HLA matching, serologic PRA testing, and flow cytometry-based B cell crossmatching can all be used to predict acute rejection risk.

Global cardiovascular risk under early corticosteroid cessation decreases progressively in the first year following renal transplantation.

UNLABELLED: A primary reason to eliminate corticosteroids from immunosuppressive regimens in solid organ transplant recipients is improved cardiovascular risk profiles. Although a number of studies have documented that corticosteroid withdrawal (CSWD) regimens reduce hypertension, hyperlipidemia, diabetes, and weight gain, global assessments of cardiovascular risk under CSWD have not been reported. The purpose of this study was to document cardiovascular risk under CSWD using a global risk assessment by Framingham risk assessment. METHODS: Framingham global cardiovascular risk assessments were performed at baseline and 3, 6, and 12 months posttransplant on patients enrolled in prospective, IRB-approved early (<7 days of corticosteroids) CSWD trials. Framingham score was based on age, sex, presence of diabetes, HDL and total cholesterol, and systolic blood pressure. All patients were nonsmokers. Left ventricular hypertrophy assessment by EKG criteria was not available at all time points and therefore were not included. RESULTS: One hundred eighty-three patients were included in the analysis. Fourteen percent of patients had evidence of coronary heart disease (prior MI, CABG, PTCA, or significant cardiovascular disease as evidenced by angiography) prior to transplant. Complete information was available for 160 patients at baseline, 132 at 1, 3, and 6 months, and 93 at 12 months posttransplant. Mean 10-year risk (expressed as percent) for developing coronary heart disease decreased over time: 8.03 at baseline, 8.31 at 3 months, 7.40 at 6 months, and 7.20 at 12 months, indicating that global cardiovascular risk fell at 1 year posttransplant by about 10% in renal transplant recipients undergoing early CSWD. CONCLUSIONS: Estimation of cardiovascular risk by Framingham risk factor assessment allows incorporation of several cardiovascular risk factors into a single estimate, thereby accounting for differential effects of each individual factor on global cardiovascular risk. This experience indicates that global cardiovascular risk decreases by approximately 10% at 1 year posttransplant in renal transplant recipients who undergo early corticosteroid withdrawal (CSWD).

African American renal transplant recipients benefit from early corticosteroid withdrawal under modern immunosuppression.

African Americans have historically been considered high-risk renal transplant recipients due to increased rejection rates and reduced long-term graft survival. Modern immunosuppression has reduced rejections and improved graft survival in African Americans and may allow successful corticosteroid withdrawal. Outcomes in 56 African Americans were compared to 56 non-African Americans enrolled in early withdrawal protocols. Results are reported as African American versus non-African American. Acute rejection at 1 year was 23% and 18% (P = NS), while patient and graft survival was 96% versus 98% and 91% versus 91% (P = NS), respectively. In conclusion, early withdrawal in African Americans is associated with acceptable rejection rates and excellent patient and graft survival, indicating that the risks and benefits of early withdrawal are similar between African Americans and non-African Americans. Additional followup is needed to determine long-term renal function, graft survival, and cardiovascular risk in African Americans with early steroid withdrawal.

Can an immunologically, nonreactive potential allograft recipient undergo transplantation without a donor-specific crossmatch?

Performance of the pretransplant crossmatch requires 4 or more hours . Delays in the crossmatch might alter operating room availability and thereby increase donor organ cold ischemia time that might then result in increased risk of delayed graft function. To avoid these problems, recipients could be identified who would be expected to display negative donor crossmatches and who could be transplanted with a concurrent or retrospective rather than a pretransplant crossmatch. We, therefore, evaluated the percent reactive antibodies and donor IgG-antihuman globulin (AHG) crossmatch results of 1165 sera from 220 potential allograft recipients. Twenty-five (11%) of 220 recipients consistently displayed a 0% PRA and, with only one exception, their sera (n= 156) tested IgG-AHG crossmatch-negative against potential cadaveric donors (a 0.6% IgG-AHG positive crossmatch risk). These data suggest that the timing of the pretransplant serum crossmatch could be altered for a highly selected group of immunologically nonreactive recipients.

The impact of elevated serum IgA and race on primary recipient renal allograft survival.

This study correlated overall serum IgA levels in pretransplant (preTx) sera with graft survival. IgA levels, determined by nephelometry, were normally distributed, with a mean level of 255 +/- 139 mg/dl and a median of 234 mg/dl in 631 adult primary kidney allograft recipients and a mean level of 213 +/- 123 mg/dl with a median of 196 mg/dl for 100 retransplant recipients. Improved 3-year survival was associated with a high preTx IgA serum level in primary recipients (Kaplan-Meier analysis, P = 0.01), but not in retransplant patients. After stratifying by race, IgA correlated with graft survival in Caucasian, Hispanic, and "other" (Middle Eastern, Indian subcontinent, and Asian) primary recipients (P < or = 0.04), but not in African Americans. Higher survival rates were not associated with IgA in primary recipients stratified for rejection episodes, blood transfusions, or HLA-DR mismatches. Graft survival was improved in patients with > 2 HLA-AB mismatches and serum IgA above the median. PreTx IgA level and IgA alpha-HLA activity were significantly associated in preTx sera of primary renal allograft recipients (chi 2 = 7.145, P = 0.01), although only 9% (12/133) of sera tested displayed IgA anti-HLA class I reactivity. Thus, enhanced graft survival mediated by elevated serum IgA levels may due in part to competition for allograft HLA class I binding with deleterious Ig subclasses or immune effector cells. Elevated serum IgA may also reflect an altered immunoregulatory state. The results suggest that, depending on the racial group, preTx serum IgA levels are a prognostic indicator of graft survival in primary renal allograft recipients.

Flow cytometry-detected IgG is not a contraindication to renal transplantation: IgM may be beneficial to outcome.

BACKGROUND: At our transplant center, primary recipients of either a haplo-identical (haplo-ID) living related (LRD) or a cadaveric (CAD) donor renal allograft are transplanted after a negative donor-specific IgG anti-human globulin (AHG) cross-match (XM). Testing included the historically highest panel-reactive antibody and the immediate (0-7 days) pretransplant sera. A positive donor specific IgM-AHG XM has not been a contraindication to transplant. Reports suggest that donor-specific flow cytometry cross-matches (FCXM) may be more clinically informative than the AHG-XM. METHODS: We therefore evaluated the impact of a positive FCXM (IgG or IgM) on the rejection frequency (0-12 months after transplant) and 1-year graft survival for cyclosporine-prednisone-treated primary (haplo-ID and CAD) renal allograft recipients. All transplants were performed after a negative donor-specific IgG AHG-XM regardless of the IgM-AHG XM status. RESULTS: Rejection frequencies (26% vs. 31%, P = NS) and 1-year graft survivals (92% vs. 89%, P = NS) were comparable for haplo-ID LRD FCXM-negative and IgG-FCXM-positive recipients. However, IgM-FCXM-positive LRD recipients experienced significantly fewer rejections (13% vs. 26% P<0.02) and an improved 1-year graft survival (100% vs. 92%, P<0.02) than FCXM-negative LRD recipients. Similar results were observed for primary CAD recipients. Rejection frequencies (40% vs. 44%, P = NS) and 1-year graft survivals (83% vs. 81%, P = NS) were comparable for primary CAD FCXM-negative and IgG-FCXM-positive recipients. Again, IgM-FCXM-positive primary CAD recipients experienced significantly fewer rejections (22% vs. 40%, P<0.02) and improved 1-year graft survivals (89% vs. 83%, P<0.05) than FCXM-negative recipients. CONCLUSION: These data suggest that, after a negative donor-specific IgG-AHG XM, an IgG-positive FCXM is not a contraindication to transplantation. The presence of IgM may be beneficial in reducing the occurrence of rejection episodes and improving graft survivals.

The role of flow cytometry-detected IgG and IgM anti-donor antibodies in cardiac allograft recipients.

BACKGROUND: At our transplant center, cardiac allograft recipients undergo transplantation following a negative IgG anti-human globulin (AHG) crossmatch (XM). Flow cytometry crossmatching (FCXM) is a more sensitive XM procedure than the AHG XM procedure, yet there is limited information regarding the clinical relevance of FCXM to cardiac allograft outcome. METHODS: FCXM was performed retrospectively using the pretransplant sera from 140 recipients of primary cardiac allografts who underwent transplantation after AHG-IgG-NEG XM. The FCXM results were correlated to posttransplant rejection and patient survival. RESULTS: All of the patients were auto-XM-NEG. Twenty-two of 140 patients (16%) displayed IgG(+) FCXM and had a significantly poorer 1-year survival rate than did 57 of the FCXM-NEG recipients (68% vs. 86%, P<0.02). Moreover, 50% of the IgG(+) FCXM recipients experienced early rejections (< or =14 days postoperatively) compared with only 16% for the FCXM-NEG recipients (P<0.01). The survival rate of 92% for IgM(+) FCXM recipients (n=37) was significantly improved compared with the 86% survival rate for FCXM-NEG control recipients (P<0.05), suggesting a protective role for IgM. Consistent with this interpretation is that the 1-year survival rate of 79% for the IgG, IgM FCXM(+) recipients (n=24) was significantly better than the 68% survival rate for the IgG(+) FCXM recipients (P<0.02). CONCLUSIONS: These data suggest that IgG(+) FCXM identifies a subset of AHG-IgG-NEG XM cardiac allograft recipients who are at risk for early rejections and poor survival. In contrast, the presence of IgM may be beneficial to survival.

Comparison between enzyme-linked immunosorbent assay and cytotoxic cross-match procedures for detecting IgG anti-donor antibodies.

BACKGROUND: Disadvantages inherent to complement-dependent cytotoxicity cross-match (CDC XM) methods are the requirements for complement and viable target cells, detection of antibodies (Abs) against non-HLA antigens, and subjective scoring. Cross-Stat (SangStat Medical Corp., Menlo Park, CA), a recently developed enzyme-linked immunosorbent assay XM procedure for the detection of IgG anti-donor HLA Abs, is theoretically devoid of these flaws. METHODS: We compared results of Cross-Stat and our standard anti-human globulin (AHG)-enhanced CDC XM procedure on 524 sera from 230 transplant candidates, which were evaluated against 51 cadaveric donors. RESULTS: There was a significant correlation between AHG-CDC IgG XM and Cross-Stat results (P<0.001). For false negative sera, repeat AHG-CDC IgG XMs were still positive after platelet absorption, indicating that the Abs present were either non-HLA Abs or anti-HLA class II. Flow cytometry testing of false positive sera usually (42/62) substantiated Cross-Stat results, indicating that the discrepancy with AHG-CDC IgG XM is caused by greater sensitivity of Cross-Stat. Relative to the AHG-CDC XM, the sensitivity of Cross-Stat was 100%, the specificity was 93%, the positive predictive value was 73%, and the negative predictive value was 100%. A technical shortcoming of the Cross-Stat assay is that the frequency of indeterminate samples in the assays was 15%. Among 49 Cross-Stat negative vs. 13 Cross-Stat positive primary cadaveric renal allograft recipients (all AHG-CDC IgG-XM negative), there was no statistical difference in overall graft survival. CONCLUSION: Given the important theoretical advantages of enzyme-linked immunosorbent assay-based XM methods over the CDC XM, however, further testing of the clinical relevance of the Cross-Stat is warranted.

Correlation of ELISA-detected IgG and IgA anti-HLA antibodies in pretransplant sera with renal allograft rejection.

The present study compared the occurrence of rejection episodes during the first twelve posttransplant (Tx) months and the 1-, 2-, and 3-year graft survivals among recipients stratified by the percent panel reactive antibody (% PRA) of pre-Tx sera as detected using either an antihuman globulin determined PRA (AHG-% PRA) or an ELISA methodology detecting IgG reactive against soluble HLA class I antigens (% PRA-STAT). There was a significant correlation between AHG-PRA greater than or equal to 10% and a PRA-STAT greater than or equal to 10% (P<0.001). However, among 200 sera displaying an AHG-PRA greater than or equal to 10% (mean 57 +/- 2l%), only 69% (138/200) displayed a PRA-STAT greater than or equal to 10%. With further study the discrepant finding, of 62 sera that were AHG-PRA greater than or equal to 10% but PRA-STAT <10%, was due to the presence of IgM and/or IgG non-MHC reactivity. In contrast, among 293 sera displaying an AHG-PRA < 100% (mean 3 +/- 2%), 15% (43/293) displayed a PRA-STAT greater than or equal to 10%. There was no correlation between AHG-% PRA and rejection episodes occurring during the first twelve post Tx months. In contrast, however, there was a highly significant correlation between PRA-STAT greater than or equal to 10% and the occurrence of rejection episodes during the first twelve post-Tx months (P < 0.001). Patients with PRA-STAT greater than of equal to 10% experienced a 70% rejection frequency compared with the 35% rejection frequency for patients with PRA-STAT sera < 10% (P<0.001). A significant correlation was observed between the presence of IgG-1 and rejection (P<0.01) but not IgG-subclasses 2, 3, or 4. Of particular interest was the observation in 11 patients that the presence of ELISA-detected IgA anti-HLA class I antigen (ELISA-IgA PRA greater than or equal to 10%) was associated with a significantly reduced rejection risk compared with sera where only PRA-STAT greater than or equal to 10% was present (27% vs. 70% incidence of rejection episodes, P<0.01). Finally, patients displaying pretransplant PRA-STAT results < 10% experienced significantly improved l-, 2-, and 3- year graft survivals of 85% vs. 74%, 82% vs. 70% and 81% vs. 67%, respectively (P<0.01 for each time point), compared with patients displaying PRA-STAT results greater than or equal to 10%. These data suggest that the use of the ELISA methodology to detect IgG reactivity against soluble HLA class I antigens (PRA-STAT) may allow for the determination of a more clinically informative % PRA than the AHG-% PRA. Moreover, the presence of ELISA-detected IgA anti-HLA may act to inhibit rejection mechanisms associated with ELISA-detected IgG anti-HLA greater than or equal to 10%.

Indirect allorecognition of HLA class I peptides by CD4+ cytolytic T lymphocytes.

T-cell responses to alloantigens can occur either by "direct" recognition of donor MHC molecules, or "indirect" recognition of MHC peptides in association with self-MHC. To evaluate human T cells mediating indirect allorecognition, a CD4+ TCL and clones specific for HLA-A1 or HLA-B8 (residues 60-84) were generated from normal PBLs (A2,29 B62,- DR1,4 DQ3). Most clones were A1 specific (16 out of 17 tested), HLA-DR4 restricted (8 out of 8), and lysed targets pulsed with A1 peptide (16 out of 16). An amino acid substitution at position 86 of the DR4 beta chain (G -> V) abrogated the capacity of CD4+ CTLs to lyse target cells. Chloroquine treatment of A1-pulsed targets reduced their susceptibility to lysis, indicating a requirement for peptide processing. The TCL and clones were stimulated to proliferate by cells bearing intact HLA-A1 when autologous APCs were present, indicating that the epitope contained within the A1 60-84 peptide being recognized is produced when APCs process native HLA-A1. Furthermore, the clones and TCL did not recognize HLA-A1 on target cells carrying this allele plus self-HLA-DR4. These studies suggest a much wider role for CD4+ T cells in allograft immunity.

Cytolytic effector mechanisms of human CD4+ cytotoxic T lymphocytes.

To elucidate mechanisms by which human CD4+ cells mediated cytolytic activity, we studied the expression of cytolytic proteins and the effects of inhibitors and mAbs on T-cell clones. Of seven cytolytic CD4+ clones, three were specific for the HLA-DR17, while four recognized DR18. Anti-HLA-DR mAb and anti-CD4 mAb blocked lysis. In addition, N alpha-p-tosyl-L-lysine chloromethylketone (TLCK), a serine esterase inhibitor, as well as cytochalasin B and monensin, antagonists of secretory pathways, inhibited CD4+ CTLs, whereas the absence of extracellular Ca+2 or the presence of Ca+2 channel blockers partially inhibited cytotoxicity. CD4+ CTLs induced apoptosis of target cell nuclei and membrane damage simultaneously. The CD4+ clones synthesized perforin and granzyme B and expressed the granule-associated protein TIA-1. Our studies indicate that two distinct mechanisms may contribute to cytolysis by CD4+ clones: (1) a Ca+2-dependent mechanism associated with the cytotoxic granules and (2) a Ca+2-insensitive mechanism.