HLA alloantibodies and the mechanism of the antiglobulin-augmented lymphocytotoxicity procedure.

HLA Class I alloantigens express multiple epitopes which can be defined serologically using human HLA alloantibodies (aAb). We have shown that the vast majority of HLA antisera exhibit the CYNAP phenomenon (complement-dependent cytotoxicity (CDC) negative, adsorption positive) which can be identified by conversion to direct CDC positive reactivity with the addition of an antihuman immunoglobulin (Ig) light chain (AHG) reagent. In this study, the immunochemical mechanisms responsible for the CYNAP phenomena and how AHG overrides CYNAP have been further characterized using affinity-purified HLA aAb, class-specific anti-IgH reagents and human C1q binding assays quantified by flow cytometry. We have found that CYNAP reactions are not the result of low affinity aAb or generally caused by non-complement fixing HLA aAb. Our experiments illustrate that only anti-human IgL AHG reagents can consistently augment CDC and override CYNAP; anti-IgH have not effective. Two noncompeting HLA aAb of different epitopic specificity or one aAb in conjunction with the AHG-augmenting reagent results in striking synergy with a 200 to 400% increase in binding of C1q. We conclude from these and other experiments detailed in this article that an IgM aAb or either two adjacent, noncompeting IgG HLA aAb bound to spatially distinct epitopes on a single HLA molecule or a monospecific IgG HLA aAb in concert with the AHG binding to this HLA aAb, is required for efficient (bivalent) C1q binding and initiation of C-mediated lympholysis. In contrast, the CYNAP phenomenon usually occurs because monospecific HLA aAb directed against a single epitope cannot effect high affinity, bivalent interaction with Clq and activate complement that would ultimately lead to cytolysis.

The outcome of unrelated donor bone marrow transplantation in patients with hematologic malignancies using tacrolimus (FK506) and low dose methotrexate for graft-versus-host disease prophylaxis.

Initial studies of FK506 combined with methotrexate (MTX) in patients receiving unrelated donor BMT have demonstrated a possible-decrease in the incidence of severe GVHD but high rates of severe stomatitis and nephrotoxicity. With this background, we undertook a pilot study evaluating FK506 in combination with a lower than usual dose of MTX in an attempt to improve the tolerability of this immunoprophylaxis regimen. Between July 1993 and October 1994, 26 consecutive adults receiving unrelated donor BMT at Emory University Hospital were enrolled on this study. All patients received FK506 intravenously at an initial dose of 0.03 mg/kg/day beginning day -1 and continuing until oral FK506 was tolerated. Patients also received MTX intravenously at 5 mg/m2 on days 1, 3, 6, and 11. The preparative regimen administered to all but one patient included cyclophosphamide at 200 mg/kg over 4 days followed by total body irradiation (TBI) at 1400 cGy in twice daily fractions over 4 days. The median age of patients was 31 years (range: 19 to 52). Sixteen donor/recipient pairs were matched for HLA-A, -B, and -DR by serology and molecular typing. Ten paris were minor mismatches at either class I or class II. Twenty-two of 26 patients (85%) completed four doses of MTX on schedule. Nephrotoxicity was the most common adverse event associated with the administration of FK506: 88% of patients experienced a doubling of their serum creatinine. One patient died of central nervous system hemorrhage prior to engraftment. Twenty-four of the remaining 25 patients (96%) engrafted. Fourteen of 24 patients (50%) evaluable developed grades 2-4 acute GVHD. The rate of severe (grades 3-4) acute GVHD was 25%. Chronic GVHD developed in 11 of 20 (55%) evaluable patients. At a median follow-up of 461 days, 14 patients (54%) are alive. All are relapse-free with a median Karnofsky performance status of 90% (range: 70-100%). The cumulative probability of 2-year disease-free survival is 50% (95% confidence interval [CI]: 0.33 to 0.77); for low risk patients 67% (95% CI: 0.47 to 0.95) and for high risk patients 23% (95% CI: 0.049 to 1.00). These preliminary data indicate that FK506-based immunosuppression following unrelated donor BMT is effective in preventing severe acute GVHD and warrants comparison to CSA-based regimens.

Epitope specificity of HLA class I alloantibodies: II. Stability of cross-reactive group antibody patterns over extended time periods.

The stability of HLA alloantibodies was studied in 128 antibody-positive, potential kidney transplant recipients over an average period of 3 years. Antibody detection was performed using an anti-human globulin-complement-dependent cytotoxicity technique. In this study, the specificity of antibodies was categorized as against either private epitopes or cross-reactive group (CREG) epitope clusters. Definable antibodies were found in 94% of patients, and 89.5% of the definable antibodies had specificity for CREG clusters. Patterns of antibody reactivity were stable in most of the patients evaluated, even though the percentage of panel-reactive antibody (PRA) often demonstrated considerable fluctuations. Of the 220 definable private-specific or CREG cluster-specific antibodies identified in the patients, nearly 80% persisted throughout the observation period. The fluctuations in % PRA were common, but usually were not due to the acquisition of new HLA antibodies. Most fluctuations were attributable to variable detection of specificities within the same CREG cluster, possibly due to technique variation or changes in antibody avidity or titer or in cell panel composition. This study demonstrates that patterns of antibody specificity are remarkably stable in this patient population, even though PRA values fluctuated. This study further suggests that HLA antibody specificity analysis is a more useful clinical parameter of lymphocytotoxicity testing than simple reporting of % PRA when identifying potential donors for individual patients.

Low incidence of red cell and HLA antibody formation by bone marrow transplant patients.

BACKGROUND: Bone marrow transplant (BMT) patients, although immunosuppressed, are at risk for the development of red cell (RBC) and HLA antibodies, and they often are given filtered blood in an effort to prevent the latter complication. This study attempts to determine the rate of formation and the specificity of both RBC and HLA alloantibodies in this patient population. STUDY DESIGN AND METHODS: BMT patients (148 received autologous marrow; 45 received allogeneic marrow) from an 18-month period, including patients with leukemia (57 patients), lymphoma (54), breast cancer (68), myeloma (8), myelodysplastic syndrome (5), and aplastic anemia (1), were studied to determine the rate of alloantibody formation to RBC and HLA antigens. A total of 2,410 RBC antibody screens were performed. The patients received 3,921 packed RBCs and 5,915 single-donor platelet units; all were irradiated and administered via white cell-reduction filters. RESULTS: Seven (3.6%) of 193 patients had RBC antibodies upon hospital admission. Four (2.1%) of 193 developed RBC antibodies during the course of BMT: 3 patients had one RBC antibody and 1 patient had two RBC antibodies. RBC antibodies included anti-E (n = 2), anti-M (n = 1), anti-Jkb (n = 1), and anti-Lu14 (n = 1). Thus, 98 percent of patients (189/193) did not develop new (182/186) or additional (7/7) RBC antibodies during BMT. BMT patients were also screened weekly for HLA antibody formation (60-cell panel). Upon admission, 170 (85%) patients were negative. Of these, 8 (4.7%) developed persistent HLA antibodies (mean panel-reactive antibody score, 33 +/- 29%) and 9 (5.3%) were variably positive. Thus, in our setting and population, RBC antibody formation was 0.1 percent per unit transfused, and the HLA alloimmunization rate was 5 to 10 percent. CONCLUSION: As RBC antibody screens are done every Monday, Wednesday, and Friday on this BMT service and as RBC antibody formation is low in these patients, screening for unexpected antibodies might be possible on a more infrequent basis. Also, the rate of HLA alloimmunization in this population receiving filtered blood components is low.

Epitope specificity of HLA class I alloantibodies. I. Frequency analysis of antibodies to private versus public specificities in potential transplant recipients.

Sera obtained sequentially from 419 patients awaiting solid organ transplantation were screened and analyzed for HLA class I epitope specificity. Antibodies detected in each serum were defined as "private" if reactivity could only be demonstrated against a single specificity within one of the eight major CREGs, or as "public" if reactivity in a serum could be demonstrated against two or more specificities within a single CREG. A total of 139 sera contained % PRA > 0, in which 147 specific antibodies were identified. Of the 103 positive sera, 93 (90%) contained antipublic antibodies, with or without additional antiprivate antibodies, whereas just 10 (10%) sera contained only apparent antiprivate antibodies. The success rate in defining antibody specificities was low at PRA values of 1%-20% due to weak reactivity and high false-positive rates. Specificity analysis with high test sensitivity and specificity was achieved with PRA values between 40% and 80%. At PRA values > 80%, test sensitivity remained high but specificity declined. We conclude that most anti-HLA antibodies are directed against high frequency public epitope clusters (CREGs), and highly sensitized patients develop antibodies in a fairly predictable fashion, a feature that significantly improved the success rate of specificity analysis. Since high frequency antipublic antibodies are common sequelae of CREG mismatches, further definition of HLA class I public epitopes eventually may be important in donor-recipient matching.

Characterization of human heart-infiltrating cells after transplantation. V. Suppression of donor-specific allogeneic responses by cloned T-cell lines isolated from heart biopsy specimens of patients after transplantation.

In vitro culture of heart biopsy specimens from patients after transplantation in media containing recombinant human interleukin-2 led to the exudation of host mononuclear-cell infiltrates. Cloned T-cell lines were prepared from such infiltrates and studied for donor-specific mixed lymphocyte reaction and cytotoxic T-lymphocyte activity. Although most T-cell clones (greater than 50%) showed donor-specific reactivity, a small but distinct frequency (2% to 10%) of the cloned T-cell lines did not proliferate against donor or third-party stimulator cells. Of interest was our finding that addition of these non-donor-reactive cloned T-cell lines to autologous peripheral blood mononuclear cells markedly suppressed their donor-specific, but not third-party major histocompatibility complex, unrelated proliferative response and prevented the generation of donor, but not third-party, major histocompatibility complex unrelated cytotoxic T-lymphocyte function. The suppression was not secondary to lysis of donor stimulator cells, lysis of autologous donor-specific CD4+ lymphoblasts, or by selective consumption of interleukin-2. The suppression was mediated at the initiation of sensitization (precursor cell level). These suppressor cells expressed CD3, CD8, CD45RO, and the alpha, beta T-cell receptor, but not CD4 or CD56. These cloned T-cell lines will provide unique reagents to study the molecular basis by which these cells exert their regulatory function.

Characterization of human heart-infiltrating cells after transplantation. VI. Differences in the cytokines produced by individual CD4+ cloned T-cell lines with apparently identical antiidiotype-like reactivity.

Studies of cultures and cloned T-cell lines from mononuclear cell infiltrates in cardiac biopsy specimens have provided a unique resource to study the cellular basis of human organ allograft rejection. Our laboratory has previously shown that biopsy specimens placed on autologous donor MHC-class-II-specific cloned T-cell lines from previous cardiac biopsies led to the isolation of cloned T-cell lines, which appeared to be functionally "antiidiotypic" in nature. Detailed functional analysis of such CD4+ individual antiidiotype-reactive cloned T-cell lines revealed that although some augmented the proliferative response of autologous idiotype-bearing cloned T-cell lines against donor stimulator cells, others markedly suppressed the proliferative response; thus, although each of these antiidiotype-like reactive cloned T-cell lines appears to specifically react with the same idiotype-bearing donor MHC-class-II-specific cloned T-cell line, they were functionally heterogeneous. Analysis of cytokines secreted by these individual clones showed that the antiidiotype-reactive cloned T-cell lines that suppressed the response of idiotype-bearing cells appear to secrete predominantly interferon gamma, whereas those antiidiotype-reactive cloned T-cell lines that augmented the response do not secrete interferon gamma but secrete interleukin-2, -4, and -6. These preliminary data suggest that differences in the predominant cytokines secreted by these individual antiidiotype-reactive cloned T-cell lines may account for their functional differences.

Anti-idiotypic-like antibodies detected following alloimmunization. Their characterization and relevance to allograft acceptance.

Anti-idiotypic-like antibodies (Ab2) develop during the course of HLA alloimmunization. Several reports indicate that their presence in alloimmunized patients is associated with superior allograft survival, and their appearance correlates inversely with specific HLA antibodies. These features suggest that the induced auto-Ab2 may interact with regulatory idiotypes of HLA-specific antibodies, and may be part of an early immune regulatory mechanism that facilitates the induction of donor-specific immunosuppression. The observations have great potential clinical implications for pretransplant manipulations of the immune response to facilitate donor-specific immunosuppression, and to predict the fate of allografts in previously alloimmunized recipients. Further development and general application of Ab2 testing, however, require better documentation that regulatory immune networks between HLA antibodies and T cells exist, and that improved methods of Ab2 detection that could be used routinely and duplicated in the clinical laboratory, can be developed.

Conclusions: the maintenance of allograft acceptance.

This article summarizes all of the findings of the authors in this issue who examined various laboratory procedures used to implement and monitor transplantation treatment strategies. Histocompatibility matching, crossmatching, monitoring immunosuppression and rejection, and immunologic monitoring of allograft rejection and acceptance are reviewed.

Topographic map of the HLA-A2 CREG epitopes using human alloantibody probes.

The topographic architecture of the epitopes expressed on the HLA-A2 glycoprotein using murine monoclonal antibody (mAb) probes indicates at least two sterically distinct domains. Previously, we have demonstrated using human HLA alloantibodies (aAb) that multiple determinants are expressed on each HLA antigen: the highly polymorphic private epitopes and the public determinants that are shared within a family of crossreactive groups (CREG). Our objectives now focus on probing the antigenic structure of the HLA-A2-28-9-B17 CREG using highly specific aAb in conjunction with mAb that have previously been used for structural studies. Both mAb-mediated blockage of complement-dependent cytotoxic aAb and reciprocal antibody (Ab) binding inhibition assays with quantitation by fluorescence flow cytometry have been utilized. We have found that xenogeneic mAb directed against A2-69, A2-B17, and A2-28 crossblock aAb of the same serologic specificity, and vice versa, indicating that the epitopes they respectively recognize are at least in close steric proximity. However, additional HLA-A2, A28, and B17 aAb of private specificity and A2-28-9 aAb of public specificity, for which there are no known mAb counterparts, paint an additional complexity not previously known. We conclude that at least four different alloepitopes can be expressed by each serologically defined HLA antigen. Based on the primary sequence data, we have assigned the location and the amino acid substitutions which most likely account for these discrete epitopes. The unique private determinants are located on the alpha 1 domain together with the interlocus A2-B17 epitope while the public epitopes A2-69, A2-28-9 and A2-28 are located on the alpha 2 domain.

Epitope map of the HLA-B7 CREG using affinity-purified human alloantibody probes.

Monoclonal antibodies (mAb) recognizing the B7 CREG have been used to construct an epitopic map of HLA-B7. Similar studies with human HLA alloantisera have been lacking due to the polyclonal nature of the alloantibodies (aAb). Detergent-solubilized HLA Class I antigens were purified and coupled to activated CH-Sepharose 4B. Sequential affinity isolation of aAb populations using a series of HLA antigen columns enabled us to produce a battery of aAb eluates against both the private B7, B13, B27, and B omega 60 determinants and the public B7-42, B7-60, B7-60-61, B7-27-13-60, B7-42-22-27, B7-8-42-60-41, and B omega 6 epitopes. The topographic relationship of the B7 family of determinants recognized by the Ab probes was derived using crosscompetition Ab blocking assays with quantitation by indirect immunofluorescence and FACS analysis. We have found that aAb and mAb of similar specificity crossblock; Ab of different specificity give complex patterns including both overlapping blocking between the alpha domains and Ab-induced conformational change of the molecule. From these investigations, we conclude that HLA Class I alloantigens bear both multiple, topographically distinct public epitopes and separate private determinants that can be distinguished using human aAb probes. At least four discrete epitopes are expressed by each molecule of the HLA-B7 CREG and can be ascribed to unique aa substitutions on the hydrophilic beta loops of the distal heavy chain domains and also on several exposed areas of the alpha helices. These findings are extremely similar to those of the HLA-A2 CREG and suggest that possibly all Class I molecules possess a comparable, complex degree of serologic polymorphism.

Characterization of human cardiac infiltrating cells posttransplantation: II. CD4+ cloned T-cell lines with "anti-idiotype"-like reactivity.

CD4+ T cells were cultured from posttransplant cardiac biopsies placed on irradiated feeder cells of autologous cloned donor major histocompatibility complex class II-specific T-cell lines cultured and grown from previous biopsies. Fourteen of the CD4+ T-cell cultures were expanded and cloned using the same feeder cells. Two of the 14 cloned T-cell lines were examined in detail for their ability to proliferate in vitro. Clones 7E4 and 8G2 proliferated (as determined by primed lymphocyte testing) only when cocultured with a series of distinct autologous cloned T-cell lines from previous biopsies that were specific for donor-specific HLA-DR3 and HLA-DR4, respectively. In addition, when HLA-DR-specific T-cell lines were established using recipient peripheral blood mononuclear cells and a series of HLA-DR-expressing homozygous typing cells, clone 7E4 only responded to the series of distinct HLA-DR3-specific autologous cloned T-cell lines but not to autologous HLA-DR2 and -DR4, and clone 8G2 responded to 3 of 8 distinct autologous HLA-DR4-specific T-cell lines, but not HLA-DR2-specific T-cell lines. These data demonstrate that cardiac biopsies contain CD4+ T cells of recipient origin which show anti-idiotype-like reactivity against T-cell receptors specific for donor-specific major histocompatibility complex class II molecules.

The development and specificity of antiidiotypic antibodies in renal transplant recipients receiving single-donor blood transfusions.

Multiple pretransplant sera obtained from alloimmunized renal transplant recipients were tested for the presence of antiidiotypic-like antibodies (AB2) that inhibit donor-specific HLA antibodies in the microlymphocytotoxicity assay. Fourteen patients received repetitive single-donor blood transfusions (SDT). In this patient group, sera were collected prior to each blood transfusion and prior to transplantation. Three additional patients were studied in whom prior donor-specific HLA antibodies had been lost over a period of 6 months preceding transplantation. Donor-specific AB2-like antibodies were found in the sera of 13/14 SDT patients who did not develop HLA antibodies, and in the 3 patients who had lost donor-specific HLA antibodies. All patients had received prior random blood transfusions in the year preceding the study. Five (38%) of the SDT patients had detectable donor-specific AB2 prior to the initiation of single-donor blood transfusion, presumably related to previous blood transfusions. In the remaining six SDT patients in whom complete serum sets were available, AB2 always appeared after the first blood transfusion. The specificity of HLA antibodies inhibited by AB2 was studied, and antibodies against HLA-A, -B, -C, -DR, and DQw were all identified. Thus, there was no predilection for patients to develop AB2 against locus-specific HLA gene products. This study also confirms the apparent polymorphism of putative crossreactive idiotypes. Approximately 25% of donor-specific HLA antibodies were not inhibited by relevant AB2. This study confirms and extends previous observations that alloimmunization is associated in many patients with the development of antiidiotypic-like antibodies that are capable of inhibiting the binding and cytotoxicity of HLA alloantibodies.

Association of antiidiotypic antibody with successful second transplant of a kidney sharing HLA antigens with the previous hyperacutely rejected first kidney.

The evolution of HLA antibodies and autoantiidiotypic antibodies (AB2) were studied during an 18-month period in a patient who hyperacutely rejected an HLA-A2-positive kidney, but tolerated a second HLA-A2-positive kidney one year later. Following rejection of the first kidney, the patient's serum contained an HLA-A2 antibody that reacted with 100% of HLA-A2-positive panel cells. After several months, the HLA-A2 antibody activity was precipitously lost over a one-month period and could no longer be identified by sensitive lymphocytotoxicity procedures. Approximately one year later, the patient received a second HLA-A2-positive kidney that has survived for a 2-year period and was not associated with significant rejection episodes during the early posttransplantation period. Prior to and episodically following the second transplant, the patient's sera contained antiidiotypic-like antibodies that specifically inhibited HLA alloantibodies directed against HLA-A2. AB2, with specificity for a putative idiotype on HLA-A2 alloantibodies, existed concurrently with other HLA alloantibodies in the patient's serum that had not been lost over the course of several months. This case study demonstrates a temporal association between the loss of a specific HLA antibody and the development of an AB2 with inhibitory specificity for the antibody. The study also confirms that anamnestic responses to donor-specific antigens do not always occur in previously alloimmunized patients rechallenged with the same HLA antigens.

Nonenzymic glycation of human immunoglobulins does not impair their immunoreactivity.

Diabetic patients have an increased proportion of their immunoglobulins nonenzymically glycated. To investigate the possibility that this may contribute to increased susceptibility to infection, we compared the immunoreactivity of glycated and nonglycated human immunoglobulins against rubella and hepatitis; streptococcal exoenzyme and infectious mononucleosis; human lymphocytotoxic antigens (HLA); and Varicella zoster in terms of antigen-antibody binding, cell agglutination, cytotoxicity, and complement-fixation properties, respectively. We found no evidence to support the supposition that glycated immunoglobulins are functionally impaired.

Concomitant immunosuppression and donor-specific transfusions prior to renal transplantation.

The induction of immunologic unresponsiveness to improve renal allograft survival was attempted in 151 patients by the pretransplant administration of donor-specific whole blood or buffy coat in conjunction with continuous Aza immunosuppression. All donor-recipient combinations were at least one-haplotype disparate and 21 were two-haplotype disparate. Presensitization was present in ten patients and attempts at desensitization were uniformly unsuccessful. Of the 151 nonpresensitized patients, transient sensitization occurred in 3% and permanent sensitization in 7%. Of 140 nonsensitized patients, 135 underwent renal transplantation from the specific blood donor and 56% have never experienced a rejection episode. The allograft survival rate at two years (93%) and seven years (87%) is significantly better (p less than .01) than our historical experience with one-haplotype living-related transplants at two years (68%) and seven years (59%). The low rate of sensitization (7%) has permitted almost all patients to undergo eventual renal transplantation from the specific blood donor. This and the low rate of early rejection (2%) argues for a modification of the immunologic response, perhaps by clonal deletion, rather than a selecting out process as the mechanism for improved allograft survival.