Histologic and molecular features of antibody-mediated rejection.

PURPOSE OF REVIEW: This review aims to summarize the highlights from recent research that involved pathological and molecular analysis of kidney allografts. RECENT FINDINGS: As the research on antibody-mediated rejection (AMR) continues to evolve, studies are focused on identification through transcript studies of pathogenetic pathways involved in the development of AMR as well as refinement of diagnostic methods either by correlating Banff pathologic lesions with clinical and molecular data or by machine learning. Of note, the past year has generated high impact research that underscore the importance of pathologic and molecular correlations and detection of transcripts or gene sets that would aid prognostication. The studies involving refinement of pathologic criteria also highlight the continuous efforts to achieve diagnostic accuracy and standardization. SUMMARY: Research involving histologic and molecular characteristics that define AMR are central to identification and understanding of pathogenetic pathways and remain critical in the development of diagnostic criteria.

Banff Human Organ Transplant Transcripts Correlate with Renal Allograft Pathology and Outcome: Importance of Capillaritis and Subpathologic Rejection.

BACKGROUND: To seek insights into the pathogenesis of chronic active antibody-mediated rejection (CAMR), we performed mRNA analysis and correlated transcripts with pathologic component scores and graft outcomes. METHODS: We utilized the NanoString nCounter platform and the Banff Human Organ Transplant gene panel to quantify transcripts on 326 archived renal allograft biopsy samples. This system allowed correlation of transcripts with Banff pathology scores from the same tissue block and correlation with long-term outcomes. RESULTS: The only pathology score that correlated with AMR pathways in CAMR was peritubular capillaritis (ptc). C4d, cg, g, v, i, t, or ci scores did not correlate. DSA-negative CAMR had lower AMR pathway scores than DSA-positive CAMR. Transcript analysis in non-CAMR biopsies yielded evidence of increased risk of later CAMR. Among 108 patients without histologic CAMR, 23 developed overt biopsy-documented CAMR within 5 years and as a group had higher AMR pathway scores (P=3.4 × 10-5). Random forest analysis correlated 3-year graft loss with elevated damage, innate immunity, and macrophage pathway scores in CAMR and TCMR. Graft failure in CAMR was associated with TCMR transcripts but not with AMR transcripts, and graft failure in TCMR was associated with AMR transcripts but not with TCMR transcripts. CONCLUSIONS: Peritubular capillary inflammation and DSA are the primary drivers of AMR transcript elevation. Transcripts revealed subpathological evidence of AMR, which often preceded histologic CAMR and subpathological evidence of TCMR that predicted graft loss in CAMR.

Long-term Kinetics of Intragraft Gene Signatures in Renal Allograft Tolerance Induced by Transient Mixed Chimerism.

BACKGROUND: Renal allograft tolerance (TOL) has been successfully induced in nonhuman primates (NHPs) and humans through the induction of transient mixed chimerism. To elucidate the mechanisms of TOL, we compared local immunologic responses in renal allografts with those in T-cell-mediated rejection (TCMR) and chronic antibody-mediated rejection (CAMR) in NHPs. METHODS: Using the NanoString nCounter platform, we retrospectively studied 52 mRNAs in 256 kidney allograft samples taken from NHP kidney recipients of donor BMT. No immunosuppression was given after 1-month post-donor BMT. Recipients who achieved TOL (n = 13) survived for >1840 ± 1724 days with normal kidney function, while recipients with CAMR (n = 13) survived for 899 ± 550 days with compromised graft function, and recipients with TCMR (n = 15) achieved only short-term survival (132 ± 69 days). RESULTS: The most prominent difference between the groups was FOXP3, which was significantly higher in TOL than in CAMR and TCMR, both early (<1 y, P < 0.01) and late (≥1 y, P < 0.05) after transplant. Other mRNAs related to regulatory T cells (Treg), such as IL10, TGFB, and GATA3, were also high in TOL. In contrast, transcripts of inflammatory cytokines were higher in TCMR, while activated endothelium-associated transcripts were higher in CAMR than in TOL. The receiver operating characteristic analyses revealed that intragraft FOXP3 and CAV1 can reliably distinguish TOL from CAMR. CONCLUSIONS: High FOXP3 and other Treg-related mRNAs together with suppressed inflammatory responses and endothelial activation in renal allografts suggest that intragraft enrichment of Treg is a critical mechanism of renal allograft TOL induced by transient mixed chimerism.

A Peripheral Blood Gene Expression Signature to Diagnose Subclinical Acute Rejection.

BACKGROUND: In kidney transplant recipients, surveillance biopsies can reveal, despite stable graft function, histologic features of acute rejection and borderline changes that are associated with undesirable graft outcomes. Noninvasive biomarkers of subclinical acute rejection are needed to avoid the risks and costs associated with repeated biopsies. METHODS: We examined subclinical histologic and functional changes in kidney transplant recipients from the prospective Genomics of Chronic Allograft Rejection (GoCAR) study who underwent surveillance biopsies over 2 years, identifying those with subclinical or borderline acute cellular rejection (ACR) at 3 months (ACR-3) post-transplant. We performed RNA sequencing on whole blood collected from 88 individuals at the time of 3-month surveillance biopsy to identify transcripts associated with ACR-3, developed a novel sequencing-based targeted expression assay, and validated this gene signature in an independent cohort. RESULTS: Study participants with ACR-3 had significantly higher risk than those without ACR-3 of subsequent clinical acute rejection at 12 and 24 months, faster decline in graft function, and decreased graft survival in adjusted Cox analysis. We identified a 17-gene signature in peripheral blood that accurately diagnosed ACR-3, and validated it using microarray expression profiles of blood samples from 65 transplant recipients in the GoCAR cohort and three public microarray datasets. In an independent cohort of 110 transplant recipients, tests of the targeted expression assay on the basis of the 17-gene set showed that it identified individuals at higher risk of ongoing acute rejection and future graft loss. CONCLUSIONS: Our targeted expression assay enabled noninvasive diagnosis of subclinical acute rejection and inflammation in the graft and may represent a useful tool to risk-stratify kidney transplant recipients.

Pretransplant transcriptomic signature in peripheral blood predicts early acute rejection.

Commonly available clinical parameters fail to predict early acute cellular rejection (EAR, occurring within 6 months after transplant), a major risk factor for graft loss after kidney transplantation. We performed whole-blood RNA sequencing at the time of transplant in 235 kidney transplant recipients enrolled in a prospective cohort study (Genomics of Chronic Allograft Rejection [GoCAR]) and evaluated the relationship of pretransplant transcriptomic profiles with EAR. EAR was associated with downregulation of NK and CD8+ T cell gene signatures in pretransplant blood. We identified a 23-gene set that predicted EAR in the discovery (n = 81, and AUC = 0.80) and validation (n = 74, and AUC = 0.74) sets. Exclusion of recipients with 5 or 6 HLA donor mismatches increased the AUC to 0.89. The risk score derived from the gene set was also significantly associated with acute cellular rejection after 6 months, antibody-mediated rejection and/or de novo donor-specific antibodies, and graft loss in a cohort of 154 patients, combining the validation set and additional GoCAR patients with surveillance biopsies between 6 and 24 months (n = 80) posttransplant. This 23-gene set is a potentially important new tool for determination of the recipient's immunological risk before kidney transplantation, and facilitation of an individualized approach to immunosuppressive therapy.

When acute kidney injury in the intensive care unit is not acute tubular necrosis: A case report of κ-light chain crystalline tubulopathy
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INTRODUCTION: This case highlights the importance of getting a thorough workup for acute kidney injury before assigning a diagnosis. CASE PRESENTATION: A 68-year-old male was referred to our clinic after a recent outside hospitalization for septic knee arthritis and acute kidney injury requiring hemodialysis. He had chronic kidney disease presumed secondary to diabetes with baseline GFR 50 mL/min. He complained of fatigue and weight loss. Vital signs were within normal limits. Exam was notable for trace ankle edema, healed right knee scar, and right internal jugular hemodialysis catheter. Medications included amlodipine, aspirin, atorvastatin, furosemide, sevelamer, and cephalexin. Calculated creatinine clearance was 6 mL/min with urine output 2 L/day. Urinalysis showed 1+ protein, 2+ glucose, and fine granular casts. Clinical impression was ischemic acute tubular necrosis in recovery phase. However, when he did not improve and continued requiring dialysis, further workup showed elevated serum κ free light chains and urine Bence-Jones protein. Renal biopsy showed κ light chain crystalline tubulopathy, interstitial inflammation, and extensive fibrosis. Subsequent bone marrow biopsy showed 15% κ-restricted plasma cells. Multiple myeloma was diagnosed, and chemotherapy initiated. With decrease in κ light chain burden, kidney function improved, and patient was able to come off dialysis. CONCLUSION: This case describes a rare presentation of κ light chain crystalline tubulopathy and illustrates the value of a comprehensive evaluation for acute kidney injury to enable prompt diagnosis and therapy.
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Induced regulatory T cells in allograft tolerance via transient mixed chimerism.

Successful induction of allograft tolerance has been achieved in nonhuman primates (NHPs) and humans via induction of transient hematopoietic chimerism. Since allograft tolerance was achieved in these recipients without durable chimerism, peripheral mechanisms are postulated to play a major role. Here, we report our studies of T cell immunity in NHP recipients that achieved long-term tolerance versus those that rejected the allograft (AR). All kidney, heart, and lung transplant recipients underwent simultaneous or delayed donor bone marrow transplantation (DBMT) following conditioning with a nonmyeloablative regimen. After DBMT, mixed lymphocyte culture with CFSE consistently revealed donor-specific loss of CD8+ T cell responses in tolerant (TOL) recipients, while marked CD4+ T cell proliferation in response to donor antigens was found to persist. Interestingly, a significant proportion of the proliferated CD4+ cells were FOXP3+ in TOL recipients, but not in AR or naive NHPs. In TOL recipients, CD4+FOXP3+ cell proliferation against donor antigens was greater than that observed against third-party antigens. Finally, the expanded Tregs appeared to be induced Tregs (iTregs) that were converted from non-Tregs. These data provide support for the hypothesis that specific induction of iTregs by donor antigens is key to long-term allograft tolerance induced by transient mixed chimerism.

Biopsy transcriptome expression profiling to identify kidney transplants at risk of chronic injury: a multicentre, prospective study.

BACKGROUND: Chronic injury in kidney transplants remains a major cause of allograft loss. The aim of this study was to identify a gene set capable of predicting renal allografts at risk of progressive injury due to fibrosis. METHODS: This Genomics of Chronic Allograft Rejection (GoCAR) study is a prospective, multicentre study. We prospectively collected biopsies from renal allograft recipients (n=204) with stable renal function 3 months after transplantation. We used microarray analysis to investigate gene expression in 159 of these tissue samples. We aimed to identify genes that correlated with the Chronic Allograft Damage Index (CADI) score at 12 months, but not fibrosis at the time of the biopsy. We applied a penalised regression model in combination with permutation-based approach to derive an optimal gene set to predict allograft fibrosis. The GoCAR study is registered with ClinicalTrials.gov, number NCT00611702. FINDINGS: We identified a set of 13 genes that was independently predictive for the development of fibrosis at 1 year (ie, CADI-12 ≥2). The gene set had high predictive capacity (area under the curve [AUC] 0·967), which was superior to that of baseline clinical variables (AUC 0·706) and clinical and pathological variables (AUC 0·806). Furthermore routine pathological variables were unable to identify which histologically normal allografts would progress to fibrosis (AUC 0·754), whereas the predictive gene set accurately discriminated between transplants at high and low risk of progression (AUC 0·916). The 13 genes also accurately predicted early allograft loss (AUC 0·842 at 2 years and 0·844 at 3 years). We validated the predictive value of this gene set in an independent cohort from the GoCAR study (n=45, AUC 0·866) and two independent, publically available expression datasets (n=282, AUC 0·831 and n=24, AUC 0·972). INTERPRETATION: Our results suggest that this set of 13 genes could be used to identify kidney transplant recipients at risk of allograft loss before the development of irreversible damage, thus allowing therapy to be modified to prevent progression to fibrosis. FUNDING: National Institutes of Health.

Increased transfusion-free survival following auxiliary pig liver xenotransplantation.

BACKGROUND: Pig to baboon liver xenotransplantation typically results in severe thrombocytopenia and coagulation disturbances, culminating in death from hemorrhage within 9 days, in spite of continuous transfusions. We studied the contribution of anticoagulant production and clotting pathway deficiencies to fatal bleeding in baboon recipients of porcine livers. METHODS: By transplanting liver xenografts from α1,3-galactosyltransferase gene-knockout (GalT-KO) miniature swine donors into baboons as auxiliary organs, leaving the native liver in place, we provided the full spectrum of primate clotting factors and allowed in vivo mixing of porcine and primate coagulation systems. RESULTS: Recipients of auxiliary liver xenografts develop severe thrombocytopenia, comparable to recipients of conventional orthotopic liver xenografts and consistent with hepatic xenograft sequestration. However, baboons with both pig and native livers do not exhibit clinical signs of bleeding and maintain stable blood counts without transfusion for up to 8 consecutive days post-transplantation. Instead, recipients of auxiliary liver xenografts undergo graft failure or die of sepsis, associated with thrombotic microangiopathy in the xenograft, but not the native liver. CONCLUSION: Our data indicate that massive hemorrhage in the setting of liver xenotransplantation might be avoided by supplementation with primate clotting components. However, coagulation competent hepatic xenograft recipients may be predisposed to graft loss related to small vessel thrombosis and ischemic necrosis.

Anti-LAMP-2 antibodies are not prevalent in patients with antineutrophil cytoplasmic autoantibody glomerulonephritis.

Lysosomal membrane protein 2 (LAMP-2) is a target of antineutrophil cytoplasmic autoantibodies (ANCA) in addition to the more commonly known targets proteinase 3 and myeloperoxidase. The prevalence of anti-LAMP-2 antibodies and their relationship to disease in ANCA glomerulonephritis are not well described. We measured anti-LAMP-2 reactivity in 680 sera samples (two academic centers) from patients with ANCA glomerulonephritis (n=329); those with ANCA-negative glomerulonephritis (n=104); those with fimbriated, gram-negative Escherichia coli urinary tract infection (n=104); disease controls (n=19); and healthy volunteers (n=124). With levels in healthy controls used to define a reference range, anti-LAMP-2 reactivity was present in 21% of ANCA sera from two of the centers; reactivity was present in 16% of the control group with urinary tract infection. Western blotting and immunofluorescence microscopy did not verify positivity. Titers of anti-myeloperoxidase and anti-proteinase 3 antibodies were 1500-fold and 10,000-fold higher than anti-LAMP-2 titers, respectively. There was no correlation between anti-LAMP-2 antibodies and disease activity. Furthermore, Wistar Kyoto rats injected with anti-LAMP-2 antibodies did not develop glomerulonephritis. In conclusion, antibodies that react with LAMP-2 may exist at very low titers in a minority of patients with ANCA disease. These data do not support a mechanistic relationship between anti-LAMP-2 antibodies and ANCA glomerulonephritis.

Host alloreactive memory T cells influence tolerance to kidney allografts in nonhuman primates.

Transplant tolerance, defined as indefinite allograft survival without immunosuppression, has been regularly achieved in laboratory mice but not in nonhuman primates or humans. In contrast to laboratory mice, primates regularly have high frequencies of alloreactive memory T cells (TMEMs) before transplantation. These TMEMs are poorly sensitive to conventional immunosuppression and costimulation blockade, and the presence of donor-reactive TMEMs in primates may account for their resistance to transplant tolerance protocols that have proven consistently effective in mice. We measured the frequencies of anti-donor TMEMs before and after transplantation in a series of rejecting and tolerant monkeys that underwent nonmyeloablative conditioning, short-term immunosuppression, and combined allogeneic kidney/cell transplantation. Transplants were acutely rejected in all the monkeys with high numbers of donor-specific TMEMs before transplantation. In contrast, long-term survival was observed in the recipients harboring lower frequencies of anti-donor TMEMs before transplantation. Similar amounts of TMEM homeostatic expansion were recorded in all transplanted monkeys upon hematopoietic reconstitution; however, only the tolerant monkeys had no expansion or activation of donor-reactive TMEMs after transplantation. These results indicate that the presence of high frequencies of host donor-reactive TMEMs before transplantation impairs tolerance induction to kidney allografts in this nonhuman primate model. Indeed, recipients harboring a low anamnestic reactivity to their donor before transplantation were successfully rendered tolerant via infusion of donor cells and short-term immunosuppression. This suggests that selection of allogeneic donors with low memory responses in recipients may be essential to successful transplant tolerance induction in patients.

Stem cell mobilization and collection for induction of mixed chimerism and renal allograft tolerance in cynomolgus monkeys.

BACKGROUND: We have previously observed that donor bone marrow hematopoietic stem cells successfully induce transient mixed chimerism and renal allograft tolerance following non-myeloablative conditioning of the recipient. Stem cells isolated from the peripheral blood (PBSC) may provide similar benefits. We sought to determine the most effective method of mobilizing PBSC for this approach and the effects of differing conditioning regimens on their engraftment. METHODS: A standard dose (10 µg/kg) or high dose (100 µg/kg) of granulocyte colony-stimulating factor (GCSF) with or without stem cell factor (SCF) was administered to the donor, and PBSC were collected by leukapheresis. Cynomolgus monkey recipients underwent a nonmyeloablative conditioning regimen (total body irradiation, thymic irradiation, and ATG) with splenectomy (splenectomy group) or a short course of anti-CD154 antibody (aCD154) (aCD154 group). Recipients then received combined kidney and PBSC transplantation and a 1-mo post-transplant course of cyclosporine. RESULTS: Treatments with either two cytokines (GCSF+SCF) or high dose GCSF provided significantly more hematopoietic progenitor cells than standard dose GCSF alone. Recipients in the aCD154 group developed significantly higher myeloid and lymphoid chimerism (P < 0.0001 and P = 0.0002, respectively) than those in the splenectomy group. Longer term renal allograft survival without immunosuppression was also observed in the aCD154 group, while two of three recipients in the splenectomy group rejected their allografts soon after discontinuation of immunosuppression. CONCLUSIONS: Protocols including administration of two cytokines (GCSF + SCF) or high dose GCSF alone significantly mobilized more PBSC than standard dose GCSF alone. The recipients of PBSC consistently developed excellent chimerism and survived long-term without immunosuppression, when treated with CD154 blockade.

Prolonged survival of GalT-KO swine skin on baboons.

BACKGROUND: Allogeneic skin is currently the best alternative to autologous skin as a temporary treatment for severe burns, but it has several drawbacks. As a potential alternative, we have evaluated GalT-KO swine skin, which lacks expression of the Gal epitope, to investigate the effect of eliminating this epitope on survival of pig-to-baboon skin grafts. METHODS: Two adult baboons that had fully recovered from previous T cell depletion received simultaneous skin grafts from: (i) GalT-KO swine, (ii) Gal-positive swine, (iii) a third-party baboon, and (iv) self (control skin). Recipients were treated with cyclosporin for 12 days and the survival, gross appearance, and histology of the grafts were compared. RESULTS: In both baboons, the GalT-KO skin survived longer than either the Gal-positive swine skin or the allogeneic skin. Early rejection of the Gal-positive skin appeared to be mediated by cytotoxic preformed anti-Gal IgM antibodies, while the rejection of GalT-KO skin appeared to result from cellular mechanisms. CONCLUSIONS: GalT-KO skin may have potential clinical benefits as an alternative to allogeneic skin as a temporary treatment for severe skin injuries.

Bortezomib in kidney transplant recipients with antibody mediated rejection: three case reports.

Here, we report our experience on three patients with AMR who were treated with bortezomib after other therapeutic interventions had failed. Bortezomib was well tolerated by two of the three patients. The third patient developed worsening thrombocytopenia following the second dose. Despite a low adverse event profile, none of the three patients conclusively responded to the bortezomib treatment. As a result of the difference in our results from that of other centers we feel that a larger prospective study is needed to define appropriate guidelines for the use of bortezomib in cases of acute rejection.

Importance of donor- and recipient-derived selectins in cardiac allograft rejection.

The selectins expressed on activated endothelial cells (E- and P-selectin), leukocytes (L-selectin), and platelets (P-selectin) play crucial roles in the rolling and tethering of leukocytes. We explored the importance of donor and recipient selectins in acute and chronic cardiac allograft rejection using mice deficient in all three selectins (ELP-/-). In BALB/c recipients, survival of fully allomismatched hearts from ELP-/- C57BL/6 donors was almost double that of wild-type grafts. In ELP-/- cardiac allografts, mononuclear cell infiltration and vasculitis of intramyocardial coronary arteries were significantly reduced. Interestingly, ELP-/- grafts were rejected similarly in both the presence and the absence of recipient selectins, and both wild-type and ELP-/- recipients promptly rejected wild-type hearts. Alternative adhesive molecules such as alpha4beta7 integrin may compensate for the lack of selectins and may mediate rejection in ELP-/- recipients. Chronic rejection was evaluated in a major histocompatibility complex (MHC) class II mismatch model using C57BL/6.C-H2(bm12) mice. While lack of selectins in recipients did not offer protection against chronic rejection, luminal stenosis of coronary arteries in ELP-/- grafts was markedly diminished. In conclusion, donor-derived selectins contribute to the development of both acute and chronic cardiac allograft rejection, and targeting donor selectins may open novel therapeutic approaches in clinical transplantation.

Monitoring antidonor alloantibodies as a predictive assay for renal allograft tolerance/long-term observations in nonhuman primates.

BACKGROUND: In an effort to define reliable assays that might predict postimmunosuppressant-withdrawal development of chronic rejection (CR), despite conditioning for tolerance induction, we evaluated various immunological responses in nonhuman primate renal allograft recipients. METHODS: Fourteen Cynomolgus monkeys received low dose total body irradiation, thymic irradiation, antithymocyte globulin, and peritransplant CD154 blockade, followed by a one-month course of cyclosporine. Recipients underwent major histocompatibility complex mismatched kidney transplantation with donor bone marrow infusion (Group A, n=8), without donor cell infusion (Group B, n=2), or with donor splenocyte infusion (Group C, n=4). RESULTS: All Group A recipients developed mixed chimerism and four of them survived long-term without rejection. The remaining four rejected their kidney allografts either chronically or acutely. All recipients in Groups B and C failed to develop chimerism and rejected their allografts. Among various in vitro assays, detection of anti-donor alloantibody (ADA) by flow cytometry (FCM) was the most relevant to long-term outcome. All five recipients that developed both anti-T cell and B cell IgG ADA in Groups A, B and C, developed histological evidence of CR within 200 days of the appearance of ADA. One of two recipients that developed only anti-B cell IgG ADA eventually developed CR over two years following discontinuation of immunosuppression and 1.5 years after ADA development. Another recipient with very low anti-B cell ADA has never developed CR. CONCLUSION: ADA monitoring with FCM assay appears to be useful in predicting the failure of tolerance prior to the development of functional or histologic abnormalities of the renal allograft.

Role of CXC chemokine receptor 3 pathway in renal ischemic injury.

Chemokines play a major role in the recruitment of leukocytes in inflammation and in the regulation of T helper 1 (Th1)/Th2 immune responses. These mechanisms have been recognized to be important in the pathogenesis of renal ischemia-reperfusion (I/R) injury. The interaction of the CXC chemokine receptor 3 (CXCR3) receptor with its ligands is a key pathogenic pathway in promoting inflammation and in enhancing Th1 immune responses. After the induction of ischemia in the mouse model of renal ischemia, an increase in intrarenal expression of CXCR3 and its ligands was observed. Compared with the wild-type (WT) mice, CXCR3-deficient mice (CXCR3-/-) had significantly lower serum creatinine levels, better survival rate, and significantly less acute tubular necrosis and cellular infiltrates. In the kidney, intracellular staining of infiltrating cells that were recovered from kidneys revealed a lower percentage of CD4+IFN-gamma+ cells in the CXCR3-/- mice compared with the WT mice. Furthermore, adoptive transfer of WT CD3+ cells into CXCR3-/- mice before induction of I/R injury abrogated the protection of CXCR3-/- mice from I/R injury. It is concluded that CXCR3 plays an important role in orchestrating the recruitment of Th1 cells to the ischemic kidney and in mediating I/R injury and therefore may serve as a novel target for the therapy of I/R injury.

C4d deposition in cardiac allografts correlates with alloantibody.

BACKGROUND: The presence of C4d along the peritubular capillaries in kidney allografts correlates with the presence of anti-donor serum alloantibodies. We applied C4d staining to cardiac allograft and non-allograft biopsies to determine if C4d staining in heart allografts correlates with anti-donor serum alloantibodies. METHODS: We stained for C4d all available frozen tissue biopsies from cardiac transplant recipients between 1997 and 2002, including autopsies. Two hundred twenty-one tissue samples from 124 patients were analyzed. Included in both groups were a variety of International Society for Heart and Lung Transplantation (ISHLT) grades of rejection plus post-implant cardiac ischemic injury (PIMI), and biopsies from patients who had received OKT3. Patients were matched by age, gender and interval after transplantation. Forty-four additional controls were included from patients biopsied for non-transplant-related cardiac disease. RESULTS: C4d staining of the myocardial capillaries correlated well with the presence of anti-donor alloantibodies. Twenty-one of 25 biopsies from patients with anti-donor alloantibodies showed C4d staining (84%), whereas only 7 of 60 without anti-donor alloantibodies stained for C4d. C4d staining did not correlate with ischemia or OKT3 therapy. Only 4 of 44 non-transplant biopsies stained for C4d (9%). An example of the clinical utility of C4d staining in patient care is presented. CONCLUSIONS: C4d staining of the capillaries in cardiac allografts correlates well with anti-donor serum alloantibodies, is a useful assay to verify alloantibody deposition, and can be used to establish one of the criteria for antibody-mediated cardiac rejections.

CD154 blockade for induction of mixed chimerism and prolonged renal allograft survival in nonhuman primates.

Costimulatory blockade with anti-CD154 monoclonal antibody (aCD154) prolongs allograft survival in nonhuman primates, but has not reliably induced tolerance when used alone. In the current studies, we evaluated the effect of adding CD154 blockade to a chimerism inducing nonmyeloablative regimen in primates. We observed a significant improvement of donor bone marrow (DBM) engraftment, which has been associated with a lower incidence of acute rejection and long-term survival of renal allografts without the need for previously required splenectomy. Among the long-term survivors, four never showed evidence of rejection, with the longest survival exceeding 1700 days following discontinuation of immunosuppression. Nevertheless, late chronic rejection was observed in three of eight recipients, indicating the necessity of further modifications of the regimen. Control recipients receiving no DBM or donor splenocytes in place of DBM rejected their allografts. Thus, DBM engraftment with, at least, transient mixed chimerism appears essential for induction of allograft tolerance using this conditioning regimen. Modification of the original mixed chimerism approach, by the addition of costimulatory blockade, has been shown to enhance mixed chimerism and induce renal allograft tolerance with less morbidity in nonhuman primates.

Differential role of CCR2 in islet and heart allograft rejection: tissue specificity of chemokine/chemokine receptor function in vivo.

Chemokines have a pivotal role in the mobilization and activation of specific leukocyte subsets in acute allograft rejection. However, the role of specific chemokines and chemokine receptors in islet allograft rejection has not been fully elucidated. We now show that islet allograft rejection is associated with a steady increase in intragraft expression of the chemokines CCL8 (monocyte chemoattractant protein-2), CCL9 (monocyte chemoattractant protein-5), CCL5 (RANTES), CXCL-10 (IFN-gamma-inducible protein-10), and CXCL9 (monokine induced by IFN-gamma) and their corresponding chemokine receptors CCR2, CCR5, CCR1, and CXCR3. Because CCR2 was found to be highly induced, we tested the specific role of CCR2 in islet allograft rejection by transplanting fully MHC mismatched islets from BALB/c mice into C57BL/6 wild-type (WT) and CCR2-deficient mice (CCR2-/-). A significant prolongation of islet allograft survival was noted in CCR2-/- recipients, with median survival time of 24 and 12 days for CCR2-/- and WT recipients, respectively (p < 0.0001). This was associated with reduction in the generation of CD8+, but not CD4+ effector alloreactive T cells (CD62L(low)CD44(high)) in CCR2-/- compared with WT recipients. In addition, CCR2-/- recipients had a reduced Th1 and increased Th2 alloresponse in the periphery (by ELISPOT analysis) as well as in the grafts (by RT-PCR). However, these changes were only transient in CCR2-/- recipients that ultimately rejected their grafts. Furthermore, in contrast to the islet transplants, CCR2 deficiency offered only marginal prolongation of heart allograft survival. This study demonstrates the important role for CCR2 in early islet allograft rejection and highlights the tissue specificity of the chemokine/chemokine receptor system in vivo in regulating allograft rejection.