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Anti-factor VIII (fVIII) alloantibodies, which can develop in patients with hemophilia A, limit the therapeutic options and increase morbidity and mortality of these patients. However, the factors that influence anti-fVIII antibody development remain incompletely understood. Recent studies suggest that Fc gamma receptors (FcγRs) may facilitate recognition and uptake of fVIII by recently developed or pre-existing naturally occurring anti-fVIII antibodies, providing a mechanism whereby the immune system may recognize fVIII following infusion. However, the role of FcγRs in anti-fVIII antibody formation remains unknown. In order to define the influence of FcγRs on the development of anti-fVIII antibodies, fVIII was injected into WT or FcγR knockout recipients, followed by evaluation of anti-fVIII antibodies. Anti-fVIII antibodies were readily observed following fVIII injection into FcγR knockouts, with similar anti-fVIII antibody levels occurring in FcγR knockouts as detected in WT mice injected in parallel. As antibodies can also fix complement, providing a potential mechanism whereby anti-fVIII antibodies may influence anti-fVIII antibody formation independent of FcγRs, fVIII was also injected into complement component 3 (C3) knockout recipients in parallel. Similar to FcγR knockouts, C3 knockout recipients developed a robust response to fVIII, which was likewise similar to that observed in WT recipients. As FcγRs or C3 may compensate for each other in recipients only deficient in FcγRs or C3 alone, we generated mice deficient in both FcγRs and C3 to test for potential antibody effector redundancy in anti-fVIII antibody formation. Infusion of fVIII into FcγRs and C3 (FcγR × C3) double knockouts likewise induced anti-fVIII antibodies. However, unlike individual knockouts, anti-fVIII antibodies in FcγRs × C3 knockouts were initially lower than WT recipients, although anti-fVIII antibodies increased to WT levels following additional fVIII exposure. In contrast, infusion of RBCs expressing distinct alloantigens into FcγRs, C3 or FcγR × C3 knockout recipients either failed to change anti-RBC levels when compared to WT recipients or actually increased antibody responses, depending on the target antigen. Taken together, these results suggest FcγRs and C3 can differentially impact antibody formation following exposure to distinct alloantigens and that FcγRs and C3 work in concert to facilitate early anti-fVIII antibody formation., (Copyright © 2020 Zerra, Arthur, Chonat, Maier, Mener, Shin, Allen, Baldwin, Cox, Verkerke, Jajosky, Tormey, Meeks and Stowell.)

Although factor VIII (FVIII) replacement therapy can be lifesaving for patients with hemophilia A, neutralizing alloantibodies to FVIII, known as inhibitors, develop in a significant number of patients and actively block FVIII activity, making bleeding difficult to control and prevent. Although a variety of downstream immune factors likely regulate inhibitor formation, the identification and subsequent targeting of key initiators in inhibitor development may provide an attractive approach to prevent inhibitor formation before amplification of the FVIII immune response occurs. As the initial steps in FVIII inhibitor development remain incompletely understood, we sought to define early regulators of FVIII inhibitor formation. Our results demonstrate that FVIII localizes in the marginal sinus of the spleen of FVIII-deficient mice shortly after injection, with significant colocalization with marginal zone (MZ) B cells. FVIII not only colocalizes with MZ B cells, but specific removal of MZ B cells also completely prevented inhibitor development following FVIII infusion. Subsequent rechallenge with FVIII following MZ B-cell reconstitution resulted in a primary antibody response, demonstrating that MZ B-cell depletion did not result in FVIII tolerance. Although recipient exposure to the viral-like adjuvant polyinosinic:polycytidylic acid enhanced anti-FVIII antibody formation, MZ B-cell depletion continued to display similar effectiveness in preventing inhibitor formation following FVIII infusion in this inflammatory setting. These data strongly suggest that MZ B cells play a critical role in initiating FVIII inhibitor formation and suggest a potential strategy to prevent anti-FVIII alloantibody formation in patients with hemophilia A., (© 2017 by The American Society of Hematology.)

The role of non-complement-activating alloantibodies in humoral graft rejection is unclear. We hypothesized that the non-complement-activating alloantibodies synergistically activate complement in combination with complement-activating antibodies. B10.A hearts were transplanted into immunoglobulin knock out (Ig-KO) mice reconstituted with monoclonal antibodies to MHC class I antigens. In allografts of unreconstituted Ig-KO recipients, no C4d was detected. Similarly, reconstitution with IgG1 or low dose IgG2b alloantibodies did not induce C4d deposition. However, mice administered with a low dose of IgG2b combined with IgG1 had heavy linear deposits of C4d on vascular endothelium. C4d deposits correlated with decreased graft survival. To replicate this synergy in vitro, mononuclear cells from B10.A mice were incubated with antibodies to MHC class I antigens followed by incubation in normal mouse serum. Flow cytometry revealed that both IgG2a and IgG2b synergized with IgG1 to deposit C4d. This synergy was significantly decreased in mouse serum deficient in mannose binding lectin (MBL) and in serum deficient in C1q. Reconstitution of MBL-A/C knock out (MBL-KO) serum with C1q-knock out (C1q-KO) serum reestablished the synergistic activity. This suggests a novel role for non-complement-activating alloantibodies and MBL in humoral rejection.

Antibody-mediated rejection of human cardiac transplants is correlated with C4d deposits and macrophage infiltrates in capillaries of endomyocardial biopsies. We produced an antibody to rat C4d to study C4d deposition and clearance in Lewis rats that were sensitized with a blood transfusion from DA rats 7, 14 or 21 days before cardiac transplantation. Cyclosporin A (CsA) immunosuppression was initiated after transplantation at a dose that inhibited graft rejection, antibody production and C4d deposition in unsensitized recipients. Blood transfusion elicited high levels of circulating IgG alloantibodies, predominantly of the complement-activating IgG2b subclass, that peaked 14 days after transplantation. At this time, macrophages accumulated in capillaries, and C4d deposits were diffuse and intense on arteries, capillaries and veins. Grafts that survived 90 days in sensitized recipients still had deposits of C4d that were associated with increased interstitial fibrosis and vasculopathy in arteries. Clearance of C4d was determined by retransplanting DA cardiac allografts from Lewis recipients back to DA recipients. C4d deposits were decreased to minimal levels within 5 days after retransplantation. Thus, C4d deposition is not limited to the capillaries, but extends throughout the arterial tree, and despite formation of a covalent bond, C4d is cleared within days.

Background: Alloantibody is an intrinsic component of the immune response to organ transplants. Although alloantibodies have been correlated with decreased graft survival, the mechanisms of alloantibody-mediated injury remain largely undefined in vivo. In the present study, we have established a model of alloantibody-mediated graft injury using B10.A (H-2a) hearts transplanted to wild type (WT) or immunoglobulin knock out (IgKO) C57BL-Igh-6 (H-2b) mice., Methods: Alloantibodies were measured in the circulation and graft by flow cytometry and in immunofluorescence staining, respectively. Intragraft cytokine mRNA expression was evaluated using a competitive template reverse transcriptase polymerase chain reaction (RT-PCR) technique. P-selectin and von Willebrand factor expression were localized by immunoperoxidase staining. The capacity of alloantibodies to restore acute cardiac allograft rejection was tested by passive transfer of monoclonal antibodies (mAbs) against donor major histocompatibility complex (MHC) class I antigens to IgKO recipients., Results: B10.A cardiac allografts are rejected acutely by WT C57BL/6 recipients, but over 50% of the cardiac allografts survived more than 50 days after transplantation in IgKO mice. Competitive template RT-PCR on the cardiac transplants demonstrated similar levels of IL-1-alpha, IL-12 (p40), TNF-alpha, IL-2, IFN-gamma, IL-4, and IL-10 mRNA in WT and IgKO recipients 8-10 days after transplantation, indicating that macrophage- and T-cell-dependent immune responses were intact in IgKO recipients. The rejection of B10.A hearts in WT recipients was characterized by interstitial and perivascular cellular infiltration; IgG, IgM, and complement (C3) deposition; vascular cell injury and intravascular platelet aggregation; and release of von Willebrand factor and P-selectin. In IgKO recipients the lower degree of vascular injury in the absence of alloantibody responses was reflected by the lack of release of von Willebrand factor and P-selectin, which remained confined to cytoplasmic storage granules of endothelial cells and platelets. Acute rejection of cardiac allografts was restored to IgKO recipients by passive transfer of proinflammatory IgG2b mAbs against donor MHC; recipients injected with isotype-matched control mAbs did not reject. In contrast, passive transfer of IgG1 mAbs against donor MHC failed to restore acute rejection of cardiac allografts to IgKO recipients. Passive transfer of IgG2b, but not IgG1 mAbs was associated with endothelial cell activation and plate. let aggregation together with the release of preformed von Willebrand factor and P-selectin from storage granules., Conclusions: Acute rejection of cardiac allografts can be reconstituted in IgKO recipients by passive transfer of IgG2b, but not IgG1 antibody. This model allows the mechanism of alloantibody-mediate graft injury to be dissected in vivo.

Allosensitization is a fundamental problem that limits the effectiveness of blood transfusions. Patients who receive multiple transfusions of blood or blood components frequently develop alloantibodies against donor alloantigens. Allosensitized patients are refractory to further transfusion and difficult to transplant successfully. CTLA4Ig fusion protein, which blocks the CD28-B7 costimulatory pathway in T-lymphocyte activation, was tested for its capacity to inhibit allosensitization to blood transfusions. Groups of LEW (RT1') rats were transfused with ACI blood (RT1a) together with L6 (a human immunoglobulin G1 [IgG1] antibody as isotype control) or CTLA4Ig in different doses (0.004, 0.02, 0.1, and 0.5 mg). Rats were retransfused with ACI blood after 28 and 84 days without any additional CTLA4Ig therapy. Weekly sera samples were tested for alloantibody against donor leukocytes using flow cytometry. CTLA4Ig caused a dose-dependent decrease in the IgM alloantibody response against donor major histocompatibility complex (MHC) class I antigens. In addition, 0.02-, 0.1-, and 0.50-mg doses of CTLA4Ig totally inhibited the IgG responses to the first transfusion, and this immunosuppressive effect persisted for the second and third transfusions. To study the capacity of CTLA4Ig to prevent a secondary immune response, three groups of LEW rats were transfused with ACI blood with no accompanying treatment. Animals were retransfused 28 days later with ACI blood together with L6 control antibody or 0.5 or 2.5 mg CTLA4Ig. CTLA4Ig, but not L6, prevented an increase in IgG alloantibody response despite repeated transfusions. The effects of CTLA4Ig treatment on helper T-lymphocyte proliferation was tested by limiting dilution analysis (LDA). Peripheral blood cells taken 30 days after blood transfusion and CTLA4Ig treatment contained significantly decreased donor-specific T-lymphocyte precursors compared with L6-treated rats. These data support the idea that blocking the B7/CD28 signal of T-lymphocyte activation by CTLA4Ig treatment at the time of transfusion may be an important therapeutic tool to inhibit alloantibody responses to blood transfusions.

The accelerated (24 h) rejection of (LEWxBN)F1 cardiac allografts (Tx) in LEW rats sensitized with BN skin grafts, is abrogated with CD4 mAb (BWH-4) administration between skin (day -7) and heart (day 0) transplantation (Tx survival ca. 11 days, p < 0.0001). This study analyzed the effects of CD4-targeted therapy upon host IgG and IgM alloantibody (allo-Ab) within the serum by two-color flow cytometry, and within the Tx, by immunohistology. These data were correlated with mRNA and protein production profiles of Th1 (IL-2, IFN-gamma) vs Th2 (IL-4) specific cytokines (polymerase chain reaction and/or immunohistology). Skin grafts elicited a strong systemic IgM allo-Ab response, which peaked at the time of cardiac Tx rejection at 24 h. It was associated with extensive deposits of IgM on Tx endothelium. Treatment with BWH-4 mAb diminished circulating IgM allo-Ab levels, and only low levels of IgM could be detected at the Tx site. Conversely, the low circulating IgG allo-Ab levels during rejection at 24 h in untreated recipients were accompanied by a strong labeling for intra-Tx IgG. BWH-4 mAb therapy did not prevent totally the switch of the IgM to IgG, but the IgG allo-Ab response was earlier, less intense and more transient than in untreated recipients. Accelerated rejection triggered sequential lymphokine mRNA expression in cardiac Tx, with the peak of transcription for IL-2 (6-12 h) preceding that for IL-4 (24 h). Interestingly, although CD4 targeted therapy virtually ablated the induction of IL-2 mRNA, it preserved transcription of the IL-4 gene. BWH-4 mAb therapy decreased otherwise abundant intra-Tx IL-2 and IFN-gamma, but allowed a vigorous elaboration of IL-4, confirming the translation of mRNA to the protein in vivo. Thus, CD4 mAb-mediated abrogation of accelerated cardiac Tx injury correlates with suppression of Th1 responses (depressed IL-2 and IFN-gamma production), but sparing of the Th2 function (enhanced IL-4 elaboration). Indeed, CD4 mAb-induced allo-Ab depression and immunosuppressive effects may reflect selective targeting of proinflammatory Th1-like cells and the multifaceted effects of IL-4 produced by unopposed Th2-like cells.

Donor-specific blood transfusion (DSBT) in animals and humans can either promote subsequent renal graft survival or lead to sensitization and graft rejection. Using a rat renal allograft model, we have examined whether the effects of DSBT on renal allograft outcome and IgG alloantibody responses are linked to the host MHC. In F1 rats produced by mating PVG (RT1c), a low IgG alloantibody responder to transfused ACI (RT1a) blood, with 3 different high-IgG responders [W/F (RT1u), LOU (RT1u), and LEW (RT1l)], high IgG alloantibody production was found to be inherited as a dominant trait and associated with acute rejection of ACI renal allografts. DSBT given to offspring of (PVG x W/F)F1 rats backcrossed to W/F with either RT1u/c (u/c) or RT1u/u) (u/u) phenotype induced high-IgG-alloantibody responses that were associated with acute renal allograft rejection. Likewise, offspring of (PVG x W/F)F1 rats backcrossed to PVG expressing the u/c phenotype had high IgG responses to ACI DSBT associated with acute renal allograft rejection. In contrast, DSBT given to backcrossed recipients expressing the RT1c/c (c/c) phenotype elicited a transient IgM response that switched to a very low IgG response and was associated with renal allograft acceptance. Analysis of IgG isotypes demonstrated that DSBT prevented production of IgG1 and IgG2a, and to a lesser extent IgG2b and IgG2c alloantibodies in c/c but not u/c renal allograft recipients. The differences in the level and isotype of IgG alloantibody responses found in sera of DSBT-pretreated backcross rats of u/c and c/c phenotypes were also present in allograft eluates and splenocyte cultures. Likewise, DSBT-pretreated renal allograft recipients of the c/c phenotype produced lower levels of alloantibodies directed to class I RT1.Aa antigens compared with their u/c counterparts; in contrast, no difference was found in alloantibody responses to class II RT1.Ba antigens. These findings demonstrate that the variable ability of DSBT to enhance renal allograft survival correlates with the inhibition of antidonor class I alloantibody responses of all IgG subclasses by a mechanism that is linked to host MHC.

Renal allograft rejection in rats and humans is a potent inducer of alloantibody to donor major histocompatibility complex antigens. Alloantibody in such presensitized recipients can cause hyperacute rejection of subsequent renal allografts. In order to characterize alloantibody production in rats presensitized by renal graft rejection, ACI (RT1a) kidneys were transplanted into untreated fully allogeneic PVG (RT1c) recipients and allowed to reject while one native kidney remained in situ for host survival. Serum samples collected at weekly intervals were analyzed by flow cytometry for IgM and IgG antibody binding to ACI lymphoid target cells. The specificity of alloantibody responses was assessed by (1) differential binding to congenic rat strain target cells expressing only donor class I (PVG.R1) versus both donor class I and II (PVG.1A) antigens, (2) differential binding to unseparated donor lymphoid target cells versus lymphoid target cells depleted of class II MHC antigen-expressing cells, and (3) specific blocking of monoclonal antibodies to donor class I (R2/10P, R2/15S) or class II (F17.23.2) epitopes. Alloantibody responses to both donor class I and II MHC antigens were detected. The initial IgM response to donor class I MHC antigens peaked at the time of rejection, followed by a steady decline to relatively low levels by 4 weeks posttransplantation. The IgM response to donor class II MHC antigens was found to be cyclical with apparent peaks at day 7 and 5-6 weeks. The IgG response to donor class I and class II MHC antigens reached maximum by 5-6 weeks before slowly decreasing. IgM and IgG alloantibody specific for class I and class II MHC antigens could be detected through 19 weeks posttransplantation. The effects on circulating alloantibody of two manipulations, posttransplantation donor specific blood transfusion and allograft removal, were examined in this model. The alloantibody responses to class I MHC antigens were not affected by giving DSBT weekly beginning at day 14 after transplantation. However, posttransplantation DSBT eliminated the second peak of IgM alloantibody to class II MHC antigens seen approximately 5-6 weeks posttransplantation and also decreased circulating IgG specific for class II antigens. Transplantectomy at day 5-7 days after transplantation had no apparent effect on circulating IgM or IgG alloantibody through 7 weeks posttransplantation. These data indicate that in a fully allogeneic rat renal allograft model alloantibody responses are elicited to both class I and II MHC donor antigens, but that the kinetics and regulation of the responses to class I differ from those to class II alloantigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Donor-specific blood transfusion prolongs the survival of fully allogeneic ACI (RT1a) renal allografts in PVG (RT1c) recipients from 7-10 days to greater than 100 days. We have observed significant differences in the alloantibody (Ab1) responses to ACI renal allografts in control and DSBT-treated PVG recipients: DSBT is associated with decreased IgG and IgM alloantibody circulating in serum, deposited in the allograft, and produced in culture by splenocytes. In the present studies the effects of DSBT on alloantibody production and renal allograft survival were extended to examine other recipient strains: F344 (RT1lv1), BN (RT1n), W/F (RT1u) and LEW (RT1l). Animals of each recipient strain were injected i.v. with 0.5 ml of ACI blood alone or followed by a renal allograft. Studies on the kinetics of IgM and IgG alloantibody responses were performed by flow cytometry on lymphocytes from donor ACI, PVG, and PVG.R1 (RT1.Aa class I MHC antigen on PVG background) rats. In F344 and PVG rats, DSBT from ACI rats elicited a transient IgM response that peaked at day 7 and was not followed by a switch to IgG. In control PBS transfused F344 recipients, an ACI renal allograft stimulated both IgM and IgG alloantibody production. DSBT pretreatment significantly decreased circulating IgG alloantibody following ACI renal transplantation and prolonged graft survival in F344 recipients. In DSBT-treated F344 recipients that rejected ACI renal allografts acutely, small amounts of IgG (5-12 mode channel shift) were detected in sera harvested 7 days after transplantation, whereas almost no IgG was detected in the sera from DSBT treated F344 rats that accepted their renal allografts indefinitely. In contrast, DSBT alone from ACI to BN, W/F, or LEW strains elicited a transient IgM response that peaked at day 7 and was followed by a strong IgG response that peaked on days 10-14 and remained high through day 21. DSBT failed to prolong ACI renal allograft survival in any of these strains (survival less than 11 days in control and DSBT rats). The alloantibody response to DSBT in all five recipient strains examined was directed primarily to RT1.Aa class I MHC antigens, as determined by binding studies on lymphocytes from ACI, PVG and PVG.R1 rats and alloantibody blocking studies using biotinylated rat monoclonal antibodies to distinct epitopes of the RT1.Aa antigen. The relative magnitude of blocking of R2/10P and R2/15S binding by sera from BN, W/F, and LEW rats was: control allograft recipients greater than DSBT pretreated allograft recipients greater than DSBT alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Untreated PVG (RT1c) rats reject ACI (RT1a) renal grafts in 6-8 days. Autologous (PVG) blood transfusions (ABT) do not alter ACI allograft rejection, but donor-specific blood transfusions (DSBT) 7 or 11 days prior to transplantation usually results in indefinite graft survival. We have reported previously that DSBT is associated with the development of antiidiotypic antibody and reduced circulating cytotoxic alloantibodies in this model. To further define the effects of DSBT on the alloantibody responses to renal allografts, we examined PVG rats that received DSBT or ABT prior to ACI renal transplantation. Antibody production by cells in the spleen was investigated by tissue culture techniques; circulating antibody titers were measured by antibody binding to target ACI lymphoblasts with flow cytometry; and antibodies bound to the ACI allograft were recovered by hypertonic acid elution and quantitated by flow cytometry and ELISA. Seven days after DSBT alone, circulating IgM alloantibodies to ACI reached peak titers. After renal allografting, serum IgM alloantibody titers decreased in DSBT-pretreated rats and little IgG could be detected. In contrast, renal allografts in ABT-pretreated rats elicited high titers of IgM and moderate titers of IgG in the circulation by 5-7 days posttransplantation. Spleens harvested one week posttransplantation from ABT-pretreated rats produced high titers (16-32) of IgM and IgG antibodies to ACI antigens, but no such antibody production was detected in spleens cultured from DSBT-pretreated rats. In addition, 4-32-fold more IgM and IgG was eluted from kidneys removed 6-7 days after grafting to ABT-treated rats than from allografts in DSBT-treated rats. IgG2a was the predominant subclass of IgG that bound to target ACI cells. No IgA was detected in graft eluates from any rat. Polyacrylamide gel electrophoresis demonstrated that the eluates contained predominantly IgM and IgG without significant contamination by other serum proteins. These data suggest that DSBT decreases the levels of IgM and IgG normally produced in the spleen and deposited in the graft following renal transplantation. Because IgM fixes complement and IgG (especially IgG2a) triggers ADCC, the reduced deposition of IgM and IgG in the graft may be of particular importance in DSBT enhancement.

Pretransplant donor-specific blood transfusion (DSBT) has been shown to enhance renal allograft survival in man and indefinitely prolong renal transplants among various MHC-disparate rat strains. Using PVG (RT1c) recipients and ACI (RT1a) donor-strain rats, DSBT alone was found to elicit complement-dependent cytotoxic IgM antibody (Ab) to donor class I (RT1.Aa) alloantigens that peaked at 7 days. An enzyme-linked immunosorbent assay was developed to measure host Ab against allospecific (idiotypic) determinants on the anti-RT1.Aa monoclonal Ab R2/10P, R2/15S, and YR1/100. Following DSBT alone, antiidiotypic Ab were detected in the circulation within 7-11 days after transfusion. Transplantation of a donor strain kidney in the presence of antiidiotypic Ab at day 7 or 11 post-DSBT resulted in enhanced graft survival and a rapid decline in circulating alloantibody, such that by days 4-6 posttransplantation little IgM or IgG alloantibody was detected. In contrast, all 6 PVG rats that were transplanted 4 days after DSBT (prior to development of detectable antiidiotypic Ab) rejected their grafts within 30 days, and 4 of 6 showed elevated alloantibody titers within 3 days posttransplantation. Control PVG rats receiving autologous blood transfusion (ABT) alone developed no alloantibody response but developed high titers of donor-specific alloantibody by 6 days posttransplantation, at the time of irreversible rejection. ABT alone did not elicit antiidiotypic Ab and ABT pretreated graft recipients developed antiidiotypic Ab only after the onset of rejection at day 4. In both DSBT and ABT groups, the antiidiotypic Ab were primarily IgM, IgG1, and IgG2c. These findings indicate that DSBT induces production of cytotoxic alloantibodies followed by an antiidiotypic Ab response at days 7-11, during which time transplanted renal allografts are not rejected and there is a reduction in circulating alloantibody. In contrast, renal allografts placed in DSBT-treated rats prior to antiidiotypic Ab development (less than or equal to 4 days) or in ABT-treated rats that do not develop any antiidiotypic Ab, elicit a rapid rise in alloantibody and are rejected.

Pre- and post transplant sera from 51 cases of bone marrow transplants performed for severe aplastic anemia were tested on monocytes (M) and corresponding B cells (B) from a panel of unrelated donors. One-third of the sera were cytotoxic for B and M either from different or from the same individuals, while 45% reacted only to M and appeared to recognize non-HLA M-associated antigens. No significant reaction to endothelial cells was obtained from these sera. The subsequent clinical course was not associated with any reaction pattern of pre-transplant sera. There was a significant relationship between rejection and the development of M antibodies after grafting, but since these were also found in many other patients without rejection, their occurrence has no predictive value for an individual patient.

Clearly the pioneering studies on hyperacute rejection, which lead to the concept of antibody-mediated injury to transplants, still present challenges to the clinician today. It is obvious that tests for antibodies before and after transplantation must be further refined to more reliably distinguish among destructive, protective and innocuous antibodies. The current crossmatch tests detect antibodies to donor lymphocytes, but lymphocytes may not always represent adequately the antigenic composition of the tissue that will be transplanted. Moreover the distribution of antigens in transplanted tissues must be considered in relation to the functional anatomy of the organs in order to understand the ultimate effects of antibodies on specific transplants. Thus some antibodies that produce a positive crossmatch may be safely ignored because they exert either a beneficial or no effect on the organ that is to be transplanted, while other antibodies that remain undetected by crossmatches on lymphocytes may damage some types of transplants. Moreover, the balance between protective and destructive antibodies is not dynamic; with time potentially dangerous antibody responses may be inhibited by a variety of autoregulatory mechanisms, some of which may be mediated by antibodies. Regulatory as well as cytotoxic antibody production may be actively stimulated by pretransplant blood transfusions. These antibody-mediated immune responses probably are magnified in the clinical setting by our current therapeutic immunosuppressive drugs which have more impact on cell-mediated immunity than on antibody-mediated mechanisms. New immunosuppressive interventions devised to inhibit undesired antibody-mediated immune responses might be of benefit to those patients who fail to respond to current rejection therapy as well as to patients who have pre-existing antibodies and are waiting for transplants.