Effect of the First Factor VIII Infusions on Immunological Biomarkers in Previously Untreated Patients with Hemophilia A from the HEMFIL Study.

Hemophilia A (HA) is an inherited bleeding disorder which requires continuous replacement with factor (F) VIII concentrate. The main complication of HA is the development of neutralizing alloantibodies which inhibit FVIII activity (inhibitors). The objective of this study was to investigate the effect of the first FVIII infusions on immunological biomarkers in previously untreated patients with HA. Plasma samples were collected at enrollment before any FVIII infusion (T0) and at inhibitor development (INB +/T1) or up to 35 exposure days without inhibitors (INB -/T1). Anti-FVIII antibodies (immunoglobulin M, immunoglobulin G [IgG] 1, IgG3, and IgG4), chemokines (CCL2, CCL5, CXCL8, CXCL9, and CXCL10), and cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, interferon-γ, tumor necrosis factor, and IL-17) were assessed. A total of 71 children with severe HA were included, of whom 28 (39.4%) developed inhibitors. Plasma levels of anti-FVIII IgG4, IL-6, and CXCL8 were higher at INB +/T1 when compared with INB -/T1. This group presented a mixed cytokine profile and higher plasma levels of CXCL9 and CXL10 when compared with INB +/T1. We conclude that exposure to FVIII triggers a proinflammatory response mediated by IL-6 and CXCL8 in patients with HA who developed inhibitors. Regardless of inhibitor status, the immune system of all HA patients is stimulated after infusions of FVIII.

Gene therapy for immune tolerance induction in hemophilia with inhibitors.

The development of inhibitors, i.e. neutralizing alloantibodies against factor (F) VIII or FIX, is the most significant complication of protein replacement therapy for patients with hemophilia, and is associated with both increased mortality and substantial physical, psychosocial and financial morbidity. Current management, including bypassing agents to treat and prevent bleeding, and immune tolerance induction for inhibitor eradication, is suboptimal for many patients. Fortunately, there are several emerging gene therapy approaches aimed at addressing these unmet clinical needs of patients with hemophilia and inhibitors. Herein, we review the mounting evidence from preclinical hemophilia models that the continuous uninterrupted expression of FVIII or FIX delivered as gene therapy can bias the immune system towards tolerance induction, and even promote the eradication of pre-existing inhibitors. We also discuss several gene transfer approaches that directly target immune cells in order to promote immune tolerance. These preclinical findings also shed light on the immunologic mechanisms that underlie tolerance induction.

Quantification of Alloantibody-Mediated Cytotoxicity In Vivo.

BACKGROUND: Preexisting, donor-specific antibodies (DSAs) are culprits of hyperacute rejection. Donor-specific antibodies are also formed de novo, and their role in acute and chronic rejection is increasingly appreciated. However, it is difficult to assess damage inflicted exclusively by DSAs when alloreactive T cell and B cell responses coincide. We reasoned that allosensitization with "costimulation-deficient" cells should induce DSA synthesis but not naive cytotoxic T lymphocyte (CTL) precursors' priming via direct allorecognition. Accordingly, we have developed a novel model to quantify DSA-mediated cytotoxicity in vivo. METHODS: C57BL/6 (H-2b) mice were sensitized with H-2 kidney epithelial cells, and a cytofluorimetric killing assay was tailored to the measurement of allocytotoxicity. We took cell/complement depletion, costimulation blockade, and serum transfer approaches to reveal the mediators of cytotoxicity. "Third-party" controls and a skin allotransplantation model were used to confirm DSAs' specificity for allo-major histocompatibility complex. We validated our experimental approach in other mouse strains primed with different allogeneic cell types, including endothelial cells. To demonstrate the usefulness of our model/method for drug efficacy testing, we examined the effect of CTLA4-Ig and rapamycin on DSA-mediated cytolysis. RESULTS: Allosensitization of MHC-disparate mouse strains with costimulation-deficient cells led to robust cytotoxicity mediated by complement-fixing DSAs and phagocytic cells. This response was independent of CTLs, natural killer or natural killer T cells. It required CD4 T cell help, CD40 signaling and CD28-based costimulation during allosensitization and could be reversed by sustained rapamycin treatment. CONCLUSIONS: The unique model described herein should enable mechanistic studies on sensitization and effector phases of humoral alloreactivity as well as efficacy testing of future immunotherapies to prevent DSA-induced pathology.

Through a glass darkly: seeking clarity in preventing late kidney transplant failure.

A common lament is that long-term kidney transplant outcomes remain the same despite improvements in early graft survival. To be fair, progress has been made-in both our understanding of chronic injury and modestly, graft survival. However, we are still a long way from actually solving this important and difficult problem. In this review, we outline recent data supporting the existence of several causes of renal allograft loss, the incidences of which peak at different time points after transplantation. On the basis of this broadened concept of chronic renal allograft injury, we examine the challenges of clinical trial design in long-term studies, including the use of surrogate end points and biomarkers. Finally, we suggest a path forward that, ultimately, may improve long-term renal allograft survival.

Application of complement component 4d immunohistochemistry to ABO-compatible and ABO-incompatible liver transplantation.

Antibody-mediated rejection (AMR) is difficult to diagnose after ABO-compatible or ABO-identical (ABO-C) liver transplantation. To determine whether complement component 4d (C4d) immunostaining would be useful for diagnosing AMR, we compared the results of C4d immunohistochemistry for allograft biopsy samples with assays for anti-donor antibodies performed at the time of biopsy. One hundred fourteen patients with ABO-C grafts and 29 patients with ABO-incompatible (ABO-I) grafts were included. Linear C4d endothelial staining (identifiable with a 4× objective lens) or staining seen in 50% or more of the portal tracts was considered positive. Five of the 114 patients (4%) with ABO-C grafts and 15 of the 29 patients (52%) with ABO-I grafts showed C4d positivity. In the ABO-C cases, C4d positivity in late biopsy samples (≥30 days after transplantation) was associated with stage 2 or higher fibrosis (METAVIR score; P = 0.01) and with the presence of donor-specific anti-human leukocyte antigen DR antibodies (HLA-DR DSAs) with a mean fluorescence intensity > 5000 according to the Luminex single-antigen bead assay (P = 0.04). Conversely, the presence of HLA-DR DSAs was associated with the presence of stage 2 or higher fibrosis, acute cellular rejection, and C4d positivity. During the 2-year follow-up, neither C4d positivity nor HLA-DR DSAs were related to graft loss. Among ABO-I patients, C4d positivity was not associated with allograft dysfunction or fibrosis. Only 3 of the 15 C4d-positive patients (20%) showed periportal hemorrhagic edema, which could be a histological sign of AMR in ABO-I grafts, and they were the only cases associated with elevations in anti-donor A/B antibody titers. In conclusion, C4d endothelial positivity among ABO-C patients is an uncommon event that could be associated with chronic graft damage with or without clinical AMR. C4d positivity is common among ABO-I patients and may not be associated with allograft dysfunction if alloantibody titers are not elevated.

Antibody-mediated rejection as a contributor to previously unexplained early liver allograft loss.

We analyzed 60 patients with idiopathic early allograft loss (defined as death or retransplantation at <90 days) to determine the relative contribution of preformed donor-specific human leukocyte antigen alloantibodies (DSAs) to this endpoint, and we defined strict criteria for the diagnosis of antibody-mediated rejection (AMR) in liver allografts. The inclusion criteria encompassed the availability of a pretransplant serum sample and both postreperfusion and follow-up tissue specimens for a blinded, retrospective re-review of histology and complement component 4d (C4d) staining. AMR was diagnosed on the basis of the presence of all 4 of the following strict criteria: (1) DSAs in serum, (2) histopathological evidence of diffuse microvascular injury/microvasculitis consistent with antibody-mediated injury, (3) diffuse C4d staining in the portal microvasculature with or without staining in the sinusoids or central veins in at least 1 sample, and (4) the exclusion of other causes of a similar type of injury. Patients thought to be experiencing definite AMR on the basis of routine histopathology alone showed the highest levels of DSA sensitization. Forty percent of patients with pretransplant DSAs with a pattern of bead saturation after serial dilutions developed AMR. Another multiparous female developed what appeared to be a strong recall response, which resulted in combined AMR and acute cellular rejection (ACR) causing graft failure. A contribution of DSAs to allograft failure could not be excluded for 3 additional patients who received marginal grafts. In conclusion, liver allograft recipients with preformed DSAs with a high mean fluorescence intensity despite dilution seem to be at risk for clinically significant allograft injury and possibly for loss from AMR, often in combination with ACR.

B-cell regulation and its application to transplantation.

There has been increasing interest in the role played by B cells and their associated antibody in the immune response to an allograft, driven by the need to undertake antibody-incompatible transplantation and evidence suggesting that B cells play a role in acute T-cell-mediated rejection and in acute and chronic antibody-mediated rejection. This review focuses on the molecular events, both activating and inhibitory, which control B-cell activation, and considers how this information might inform therapeutic strategies. Potential targets include the BAFF (B-cell-activating factor belonging to the tumour necrosis factor family) and CD40-CD40L pathways and inhibitory molecules, such as CD22 and FcγRIIB. B cells can also play an immunomodulatory role via interleukin (IL)10 production and may contribute to transplant tolerance. The expansion of allograft-specific IL10-producing B cells may be an additional therapeutic goal. Thus, the treatment paradigm required in transplantation has shifted from that of simple B-cell depletion, to that of a more subtle, differential manipulation of different B-cell subsets.

Down-regulating humoral immune responses: implications for organ transplantation.

Alloantibody can be a major barrier to successful organ transplantation; however, therapy to control antibody production or to alter its impact on the allograft remains limited. The goal of this review is to examine the regulatory steps that are involved in the generation of alloreactive B cells, with a specific emphasis on how known mechanisms relate to clinical situations in transplant recipients. Thus, we will examine the process of activation of mature, naïve B cells and how this relates to de novo antibody production. The role of long-lived plasma cells in persistent antibody production and the factors regulating their longevity will be explored. The regulation of memory B cells and their possible roles in alloimmunity also will be assessed. Finally, we will review current therapeutic approaches aimed at controlling alloantibody and assess their efficacy. By examining the pathways to antibody production mechanistically, we hope to identify important gaps in our current knowledge and gain insight into possible new therapeutic approaches to overcoming antibody in transplant patients.

A new anti-c-Met antibody selected by a mechanism-based dual-screening method: therapeutic potential in cancer.

c-Met, the high affinity receptor for hepatocyte growth factor (HGF), is one of the most frequently activated tyrosine kinases in many human cancers and a target for cancer therapy. However, inhibitory targeting of c-Met with antibodies has proven difficult, because most antibodies have intrinsic agonist activity. Therefore, the strategy for reducing the agonism is critical for successful development of cancer therapies based on anti-c-Met antibodies. Here we developed a mechanism-based assay method for rapid screening of anti-c-Met antibodies, involving the determination of Akt phosphorylation and c-Met degradation for agonism and efficacy, respectively. Using the method, we identified an antibody, F46, that binds to human c-Met with high affinity (Kd = 2.56 nM) and specificity, and induces the degradation of c-Met in multiple cancer cells (including MKN45, a gastric cancer cell line) with minimal activation of c-Met signaling. F46 induced c-Met internalization in both HGF-dependent and HGF-independent cells, suggesting that the degradation of c-Met results from antibody-mediated receptor internalization. Furthermore, F46 competed with HGF for binding to c-Met, resulting in the inhibition of both HGF-mediated invasion and angiogenesis. Consistently, F46 inhibited the proliferation of MKN45 cells, in which c-Met is constitutively activated in an HGF-independent manner. Xenograft analysis revealed that F46 markedly inhibits the growth of subcutaneously implanted gastric and lung tumors. These results indicate that F46, identified by a novel mechanism-based assay, induces c-Met degradation with minimal agonism, implicating a potential role of F46 in therapy of human cancers.

The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-Cß antibody.

The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-Cß antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 × 10(-5)M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-Cß antibody, its binding affinity for p/MHC increased by 5-fold (2.2 × 10(-6)M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-Cß antibody, which is probably due to the stabilization of the Vα/Vß region of the TCR. These findings provide new insights into the binding of sTCRs to p/MHCs and will hopefully be instrumental in establishing functional sTCR as a diagnostic and therapeutic tool for cancer.

Epitope mapping of antibodies directed against the human neutrophil alloantigen 3a.

BACKGROUND: Severe transfusion-related acute lung injury is often caused by antibodies directed against the human neutrophil alloantigen (HNA)-3a. HNA-3a results from an amino acid exchange (Arg154Gln) in the first extracellular loop of the choline transporter-like protein 2 (CTL2). The characteristics of the binding domain(s) of HNA-3a antibodies are unknown. STUDY DESIGN AND METHODS: For epitope mapping, a library of 23 different HNA-3a (R(154)) and three HNA-3b (Q(154)) peptides covering different parts of the first extracellular loop of CTL2 (aa(55-231)) was synthesized in Escherichia coli and tested by Western blot with two HNA-3a alloantibody-containing plasma samples and by enzyme immunoassay (EIA) with different HNA-3a- (n = 21) and HNA-3b- (n = 1) positive plasma samples. RESULTS: Despite promising Western blot results using highly reactive plasma samples, we found widely varying reactivities of different HNA-3a plasmas in the EIA, with only 11 of 21 HNA-3a antibodies binding to any of the tested HNA-3a peptides and with no peptide recognized by more than nine of the 21 antibodies. The HNA-3b plasma did not react with R(154) peptides in the EIA nor with R(154) or Q(154) peptides in Western blot experiments. Plasma reactivity profiles with the peptides did not correlate with those observed using granulocyte agglutination and granulocyte immunofluorescence tests. CONCLUSION: Binding of HNA-3a alloantibodies depends on the conformation of the intact CTL2 protein and their binding sites may differ substantially. Peptide-based assays for detection of HNA-3a antibodies bear the risk to be insensitive and require systematic validation with a large panel of antibodies.

Peptide inhibitors of xenoreactive antibodies mimic the interaction profile of the native carbohydrate antigens.

Carbohydrate-antibody interactions mediate many cellular processes and immune responses. Carbohydrates expressed on the surface of cells serve as recognition elements for particular cell types, for example, in the ABO(H) blood group system. Antibodies that recognize host-incompatible ABO(H) system antigens exist in the bloodstream of all individuals (except AB individuals), preventing blood transfusion and organ transplantation between incompatible donors and recipients. A similar barrier exists for cross-species transplantation (xenotransplantation), in particular for pig-to-human transplantation. All humans express antibodies against the major carbohydrate xenoantigen, Galalpha (1,3)Gal (alphaGal), preventing successful xenotransplantation. Although antibody binding sites are precisely organized so as to selectively bind a specific antigen, many antibodies recognize molecules other than their native antigen. A range of peptides have been identified that can mimic carbohydrates and inhibit anti-alphaGal antibodies. However, the structural basis of how the peptides achieved this was not known. Previously, we developed an in silico method which we used to investigate carbohydrate recognition by a panel of anti-alphaGal antibodies. The method involves molecular docking of carbohydrates to antibodies and uses the docked carbohydrate poses to generate maps of th antibody binding sites in terms of prevalent hydrogen bonding and van der Waals interactions. We have applied this method to investigate peptide recognition by the anti-alphaGal antibodies. It was found that the site maps of the peptides and the carbohydrates were similar, indicating that the peptides interact with the same residues as those involved in carbohydrate recognition. This study demonstrates the potential for "design by mapping" of anti-carbohydrate antibody inhibitors.

Suppressing memory T cell activation induces islet allograft tolerance in alloantigen-primed mice.

Memory T cells are known to play a key role in prevention of allograft tolerance in alloantigen-primed mice. Here, we used an adoptively transferred memory T cell model and an alloantigen-primed model to evaluate the abilities of different combinations of monoclonal antibodies (mAb) to block key signaling pathways involved in activation of effector and memory T cells. In the adoptively transferred model, the use of anti-CD134L mAb effectively prevented activation of CD4(+) memory T cells and significantly prolonged islet survival, similar to the action of anti-CD122 mAb to CD8(+) memory T cells. In the alloantigen-primed model, use of anti-CD134L and anti-CD122 mAbs in addition to co-stimulatory blockade with anti-CD154 and anti-LFA-1 prolonged secondary allograft survival and significantly reduced the proportion of memory T cells; meanwhile, this combination therapy increased the proportion of regulatory T cells (Tregs) in the spleen, inhibited lymphocyte infiltration in the graft, and suppressed alloresponse of recipient splenic T cells. However, we also detected high levels of alloantibodies in the serum which caused high levels of damage to the allogeneic spleen cells. Our results suggest that combination of four mAbs can significantly suppress the function of memory T cells and prolong allograft survival in alloantigen primed animals.

Successful renal transplantation with desensitization in highly sensitized patients: a single center experience.

Intravenous immunoglobulin (IVIG) and/or plasmapheresis (PP) are effective in preventing antibody-mediated rejection (AMR) of kidney allografts, but AMR is still a problem. This study reports our experience in living donor renal transplantation in highly sensitized patients. Ten patients with positive crossmatch tests or high levels of panel-reactive antibody (PRA) were included. Eight patients were desensitized with pretransplant PP and low dose IVIG, and two were additionally treated with rituximab. Allograft function, number of acute rejection (AR) episodes, protocol biopsy findings, and the presence of donor-specific antibody (DSA) were evaluated. With PP/IVIG, six out of eight patients showed good graft function without AR episodes. Protocol biopsies revealed no evidence of tissue injury or C4d deposits. Of two patients with AR, one was successfully treated with PP/IVIG, but the other lost graft function due to de novo production of DSA. Thereafter, rituximab was added to PP/IVIG in two cases. Rituximab gradually decreased PRA levels and the percentage of peripheral CD20+ cells. DSA was undetectable and protocol biopsy showed no C4d deposits. The graft function was stable and there were no AR episodes. Conclusively, desensitization using PP/IVIG with or without rituximab increases the likelihood of successful living donor renal transplantation in sensitized recipients.

Evidence of heterogeneity in the antibody response against the platelet antigen 3a; recognition of an 11-mer peptide carrying the HPA-3a polymorphic determinant.

BACKGROUND: The immune processes involved in the development of alloantibodies against the human platelet antigens in alloimmune disorders remain unclear. Antibody recognition of the platelet antigens on their respective platelet glycoproteins has been shown to be dependent on glycoprotein conformation. Furthermore, the post-translational modification of glycoproteins adds complexity to the alloantigenic determinants. METHODS: Nine anti-HPA-3a sera along with several control sera were tested for reactivity to an 11-mer peptide straddling the HPA-3a/b polymorphism. Sera found to specifically recognize the 3a peptide were further assessed by platelet pre-exposure and immunoblotting. RESULTS: Three of the nine antisera were found to specifically recognize an 11-mer synthetic 3a peptide by ELISA. Further analysis of all anti-HPA-3a sera by Western blot showed that only those reactive to the 3a peptide were able to bind both reduced and non-reduced GPIIb. CONCLUSION: The results presented in this study provide the first known evidence for the identification of an antibody population capable of recognizing a linear and non-glycosylated form of the HPA-3a epitope.

Anti-human leukocyte antigen antibodies, vascular C4d deposition and increased soluble c4d in broncho-alveolar lavage of lung allografts.

BACKGROUND: The hallmark of humoral rejection is the presence of subendothelial C4d in the allograft. A simultaneous determination of vascular C4d with soluble C4d in broncho-alveolar lavage fluid (BAL) and circulating anti-human leukocyte antigen (HLA) antibodies (HLA-Ab) has not been reported in lung transplantation. METHODS: Forty-two consecutive lung-transplant patients were included in this cross-sectional study. The presence and specificity of HLA-Ab was determined at the same frequency with transbronchial biopsies. Soluble C4d levels were measured by enzyme-linked immunosorbent assay in all 42 patients. In a subgroup of 32 patients with available timely matched paraffin-embedded tissue sections, the vascular C4d deposition was also assessed. RESULTS: The presence of HLA-Ab in 16 patients was associated with biopsy-proven acute rejection (10/16 vs. 3/16, P<0.01) and increased immunosuppression (13/16 vs. 4/16, P<0.005). Pulmonary function was also decreased in patients with HLA-Ab (mean forced expiratory volume in 1 second=49%) when compared with the control group (mean forced expiratory volume in 1 second=66%, P<0.05). Nine patients exhibited specific vascular C4d deposition and in eight of nine (89%) cases HLA-Ab were detected, versus 8 of 23 (35%) in C4d-negative patients (P<0.05). Soluble C4d in BAL was highly (>0.5 microg/mL) elevated in patients with HLA-Ab and vascular C4d and was moderately (0.2 microg/mL) increased in patients with antibodies but C4d-negative. In contrast, only a slight elevation of soluble C4d (<0.1 microg/mL) was detected in patients without HLA-specific antibodies. CONCLUSIONS: The association of HLA-specific antibodies with vascular C4d deposition and soluble C4d in BAL, in addition to the reduced pulmonary function, might constitute a diagnostic triad for antibody-mediated rejection in lung transplant patients.

Management of inherited von Willebrand disease in 2006.

The aim of treatment of von Willebrand disease (VWD) is to correct the dual defect of hemostasis (i.e., the abnormal platelet adhesion due to reduced and/or dysfunctional von Willebrand factor [VWF] and the abnormal coagulation expressed by low levels of factor [F] VIII). Desmopressin acetate (DDAVP) is the treatment of choice for type 1 VWD because it can induce release of normal VWF from cellular compartments. Prospective studies on biological response versus clinical efficacy of DDAVP in VWD types 1 and 2 are in progress to explore its benefits and limits as a therapeutic option. In type 3 and in severe forms of type 1 and 2 VWD, DDAVP is not effective, and for these patients plasma virally inactivated concentrates containing VWF and FVIII are the mainstay of treatment. Several intermediate- and high-purity VWF/FVIII concentrates are available and have been shown to be effective in clinical practice (bleeding and surgery). New VWF products almost devoid of FVIII are now under evaluation in clinical practice. Although thrombotic events are rare in VWD patients receiving repeated infusions of concentrates, there is some concern that sustained high FVIII levels may increase risk of postoperative venous thromboembolism. Dosage and timing of VWF/FVIII administrations should be planned to keep the FVIII level between 50 and 150 IU/dL. Appropriate dosage and timing in repeated infusions are also very important in patients exposed to secondary long-term prophylaxis for recurrent bleedings.

Monoclonal anti-D antibodies to prevent alloimmunization: lessons from clinical trials.

In the past 20 years, numerous monoclonal anti-D antibodies have been developed in order to replace the human plasma derived anti-D immunoglobulins, using different in vitro functional assays as screening methods. Some of these monoclonal antibodies have been evaluated in exploratory in vivo clinical trials, notably for their ability to mediate the clearance of D-positive red cells. A review of these reported trials is presented and the results are analyzed in the light of the newly published hypothesis conferring an important role to some Fc-FcgammaR interactions and to the glycosylation-dependent potency of the monoclonal anti-D antibodies.

Anti-CD28 antibody-induced kidney allograft tolerance related to tryptophan degradation and TCR class II B7 regulatory cells.

B7/CTLA-4 interactions negatively regulate T-cell responses and are necessary for transplant tolerance induction. Tolerance induction may therefore be facilitated by selectively inhibiting the B7/CD28 pathway without blocking that of B7/CTLA-4. In this study, we selectively inhibited CD28/B7 interactions using a monoclonal antibody modulating CD28 in a rat model of acute kidney graft rejection. A short-term treatment abrogated both acute and chronic rejection. Tolerant recipients presented few alloantibodies against donor MHC class II molecules, whereas untreated rejecting controls developed anti-MHC class I and II alloantibodies. PBMC from tolerant animals were unable to proliferate against donor cells but could proliferate against third-party cells. The depletion of B7+, non-T cells fully restored this reactivity whereas purified T cells were fully reactive. Also, NK cells depletion restored PBMC reactivity in 60% of tolerant recipients. Conversely, NK cells from tolerant recipients dose-dependently inhibited alloreactivity. PBMC anti-donor reactivity could be partially restored in vitro by blocking indoleamine-2,3-dioxygenase (IDO) and iNOS. In vivo, pharmacologic inhibition of these enzymes led to the rejection of the otherwise tolerated transplants. This study demonstrates that an initial selective blockade of CD28 generates B7+ non-T regulatory cells and a kidney transplant tolerance sustained by the activity of IDO and iNOS.

Humoral immunity to vimentin is associated with cardiac allograft injury in nonhuman primates.

Immunity to autologous protein has not previously been described following nonhuman primate cardiac transplant. Native hearts and cardiac allografts from cynomolgus monkeys were assessed by immunohistology for vimentin, a highly conserved intermediate filament protein. IgM and IgG to vimentin were measured in serial sera from untreated (n = 4) or cyclosporine (CsA)-treated (n = 8, 2 with ATG) cardiac allograft recipients, and in groups treated with anti-CD154 antibody with (n = 6) or without ATG (n = 28). IgM or IgG reactive with vimentin was elaborated within 30 days with unmodified acute rejection (3/4) or in CsA-treated animals (5/6). CD154 blockade did not prevent anti-vimentin IgM (14/28) but tended to delay the IgG response during therapy (anti-CD154: 8/28, p = 0.10 vs. CsA; anti-CD154+ATG: 2/6). CAV and alloantibody were seen in 25 of 26 animals with grafts surviving over 30 days, including seven animals without increasing anti-vimentin antibody. Anti-vimentin antibodies and vascular complement deposition were found in rejected hearts. Acute and chronic alloimmunity disrupt modulation of autoreactivity to vimentin through pathways, which are resistant to CsA, but may be partially regulated by CD154.