Recipient Origin Research Articles

Role of innate and acquired immune mechanisms in clinical intestinal transplant rejection.

Long-term outcomes of intestinal transplantation are limited by infection and rejection. To understand the underlying immune mechanisms, graft infiltrating and peripheral blood cells were analyzed using multiple ex vivo assays in intestinal transplantation recipients. Infiltrating cells from rejected (graft enterectomy for rejection) and accepted or quiescent (stoma closure in stable transplant recipients) grafts were isolated and phenotypically characterized as to subsets and Toll-like receptor expressions as well as functionally tested for antimicrobial and antidonor immune responses. Multiparameter antidonor immunity was also assessed serially in the peripheral blood. The graft infiltrating lymphocytes were mostly of recipient origin in all patients tested. In rejecting grafts, the predominant populations were TcRαβ(+)CD3(+)CD8(+) T cells, and CD14(+) monocytes that coexpressed Toll-like receptor-2, receptor-3, receptor-4, receptor-5, and receptor-9, suggesting innate immune activation. In quiescent allografts the major cell subsets were CD13(+)CD14(-) monocytes and CD4(+)CD25(+) T cells with possible regulatory functions. Infiltrating cells from rejected but not quiescent grafts proliferated in response to enteric bacterial and donor antigens as well as killed donor targets. Serial follow-up of peripheral blood indicated donor-specific posttransplant unresponsiveness in micro-cell-mediated lympholysis (m-CML) and mixed lymphocyte reaction (MLR) in recipients with quiescent grafts, but not in recipients with multiple rejection episodes. Enzyme-Linked ImmunoSpot assays yielded parallel results: granzyme-B with micro-cell-mediated lympholysis and interferon-γ with MLR tests. These results were consistent with the notion that rejection was associated with innate and acquired antimicrobial and antidonor immune reactivity and that patients with stable grafts were free from these deleterious effects.

Dendritic Cells in Kidney Transplant Biopsy Samples Are Associated with T Cell Infiltration and Poor Allograft Survival.

Progress in long-term renal allograft survival continues to lag behind the progress in short-term transplant outcomes. Dendritic cells are the most efficient antigen-presenting cells, but surprisingly little attention has been paid to their presence in transplanted kidneys. We used dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin as a marker of dendritic cells in 105 allograft biopsy samples from 105 kidney transplant recipients. High dendritic cell density was associated with poor allograft survival independent of clinical variables. Moreover, high dendritic cell density correlated with greater T cell proliferation and poor outcomes in patients with high total inflammation scores, including inflammation in areas of tubular atrophy. We then explored the association between dendritic cells and histologic variables associated with poor prognosis. Multivariate analysis revealed an independent association between the densities of dendritic cells and T cells. In biopsy samples with high dendritic cell density, electron microscopy showed direct physical contact between infiltrating lymphocytes and cells that have the ultrastructural morphologic characteristics of dendritic cells. The origin of graft dendritic cells was sought in nine sex-mismatched recipients using XY fluorescence in situ hybridization. Whereas donor dendritic cells predominated initially, the majority of dendritic cells in late allograft biopsy samples were of recipient origin. Our data highlight the prognostic value of dendritic cell density in allograft biopsy samples, suggest a new role for these cells in shaping graft inflammation, and provide a rationale for targeting dendritic cell recruitment to promote long-term allograft survival.

CMV promotes recipient T-cell immunity following reduced-intensity T-cell–depleted HSCT, significantly modulating chimerism status

AbstractCytomegalovirus (CMV) remains a significant cause of morbidity after allogeneic hematopoietic stem cell transplantation (HSCT). Clinical risk varies according to a number of factors, including recipient/donor CMV serostatus. Current dogma suggests risk is greatest in seropositive recipient (R+)/seronegative donor (D−) transplants and is exacerbated by T-cell depletion. We hypothesized that in the setting of reduced-intensity T-cell-depleted conditioning, recipient-derived CMV-specific T cells escaping deletion may contribute significantly to CMV-specific immunity and might therefore also influence chimerism status. We evaluated 105 recipients of alemtuzumab-based reduced-intensity HSCT and collated details on CMV infection episodes and T-cell chimerism. We used CMV-specific HLA multimers to enumerate CMV-specific T-cell numbers and select cells to assess chimerism status in a subset of R+/D− and R+/seropositive donor patients. We show that in R+/D− patients, CMV-specific T cells are exclusively of recipient origin, can protect against recurrent CMV infections, and significantly influence the chimerism status toward recipients. The major findings were replicated in a separate validation cohort. T-cell depletion in the R+/D− setting may actually, therefore, foster more rapid reconstitution of protective antiviral immunity by reducing graft-vs-host directed alloreactivity and the associated elimination of the recipient T-cell compartment. Finally, conversion to donor chimerism after donor lymphocytes is associated with clinically occult transition to donor-derived immunity.

Donor-Type Aplastic Anemia after Allogeneic Hematopoietic Stem Cell Transplantation: A Common Cause of Late Graft Failure in Patients with Complete Donor Chimerism

Late graft failure (LGF) without evidence of residual recipient cells is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-SCT). Various factors, such as infections and chronic graft-versus-host disease (GVHD), are involved in the development of such donor-type LGF, and it is therefore difficult to identify the exact cause and to restore the graft function without performing a second transplant. One exceptionally treatable condition is aplastic anemia (AA), which occurs during donor-derived hematopoiesis and can be diagnosed by the identification of disease-specific markers in the peripheral blood. These include glycosylphosphatidyl inositol-anchored protein (GPI-AP)-deficient blood cells (Mochizuki, el al. Blood, 2008) and HLA-allele lacking leukocytes (HLA-LLs) due to copy-number neutral loss of heterozygosity in the short arm of chromosome 6 (6pLOH, Katagiri, et al. Blood, 2011) or missense mutations in the HLA alleles, which can be found when the pancytopenia develops. During the six-year period from 2007 through 2013, 12 patients with LGF after allo-SCT were referred to our clinic for the diagnosis of the pathogenesis of LGF. Three of the 12 patients were diagnosed to have late graft rejection due to the residual lymphocytes of recipient origin. None of these patients and other three patients with complete chimerism showed either GPI-AP- cells or HLA-LLs. On the other hand, either of the markers were detected in the remaining six patients with complete donor chimerims who developed LGF two to 84 months after allo-SCT after they had achieved complete hematological recovery with a platelet count > 100 x109/L. The original diagnoses included severe AA in one patient and hematological malignancies in the other five patients (Table). A range of 0.016% to 0.2% GPI-AP- granulocytes were detected in four patients, while 6.5%-80% HLA-LLs were detected in two patients (Figure). All patients first developed thrombocytopenia and gradually progressed to pancytopenia. The donors of the three patients possessed one of the four HLA class I alleles (A*02:01 and A*02:06) that we previously identified as genes associated with a susceptibility to AA. The HLA-A allele (A*02:01) that was lost in the donor’s granulocytes in Case 6 was not shared by the recipient. ATG was given to two patients (cases 1 and 2) and induced complete resolution of the LGF. The thrombocytopenia of Case 6 stopped progressing at three months after allo-SCT following a dose-escalation of tacrolimus and became stable thereafter. These findings suggest that donor-type AA is a common cause of LGF with complete donor chimerism after allo-SCT. This complication occurs regardless of the recipient’s original disease, and is potentially curable by immunosuppressive therapy without the need for a second SCT. It is therefore important to screen the peripheral blood for the presence of GPI-AP- cells and HLA-LLs when donor-type LGF or thrombocytopenia occurs after a patient has achieved complete hematological recovery. Possessing HLA class I alleles associated with an AA susceptibility by donors may confer a risk of developing AA to recipients. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Donor or Recipient Origin of Posttransplant Lymphoproliferative Disorders Following Solid Organ Transplantation

Previous studies of donor or recipient origin of posttransplant lymphoproliferative disorders (PTLDs) following solid organ transplantation (SOT) have either been small or with selected patient groups. We studied tumor origin in a population-based cohort of 93 patients with PTLD following SOT. Tumor origin of PTLD tissue was analyzed by fluorescence in situ hybridization of the sex chromosomes in cases of sex mismatch between donor and recipient (n = 41), or HLA genotyping in cases of identical sex but different HLA type (n = 52). Tumor origin of PTLD could be determined in 67 of the 93 cases. All 67 PTLDs were of recipient origin. They were found in recipients of kidney (n = 38), liver (n = 12), heart (n = 10) and lung (n = 7). The most common recipient-derived lymphomas were monomorphic B-cell PTLDs (n = 45), monomorphic T cell PTLDs (n = 9), indolent lymphomas (n = 6), and polymorphic PTLD (n = 4). Half of the recipient-derived PTLDs were Epstein-Barr virus-positive. Twelve of the recipient-derived PTLDs were located in the grafts: in four cases exclusively and in eight cases in combination with disseminated disease outside the graft. Tumor origin was indeterminable in 26 cases, probably due to low DNA quality. We conclude that the vast majority of PTLDs after SOT was of recipient origin.

DNA profiling in peripheral blood, buccal swabs, hair follicles and semen from a patient following allogeneic hematopoietic stem cells transplantation.

Allogeneic peripheral blood stem cells transplantation (allo-PBSCT) or allogeneic bone marrow transplantation (allo-BMT) have been widely used to treat patients exhibiting certain severe illnesses. However, previous studies have shown that the biological materials of allo-PBSCT or allo-BMT recipients may not constitute credible materials for personal identification. In the present study, four types of commonly used samples were collected from a male individual following gender-matched allo-BMT. Autosomal short tandem repeat (STR) and Y-STR markers analysis, based on polymerase chain reaction, were used to evaluate the chimerism status. The results showed that the blood sample were all donor type, the buccal swab sample were mixed chimerism, and the sperm and hair follicle samples maintained a recipient origin of 100%. In conclusion, identical results were obtained by the two methods and it was confirmed that DNA extracted from hair follicles and sperm can be used as a reference for the pre-transplant genotype DNA profile of the recipient in the gender-match allo-BMT or -PBSCT.

Orchestration of transplantation tolerance by regulatory dendritic cell therapy or in-situ targeting of dendritic cells.

Extensive research in murine transplant models over the past two decades has convincingly demonstrated the ability of regulatory dendritic cells (DCregs) to promote long-term allograft survival. We review important considerations regarding the source of therapeutic DCregs (donor or recipient) and their mode of action, in-situ targeting of DCregs, and optimal therapeutic regimens to promote DCreg function. Recent studies have defined protocols and mechanisms whereby ex-vivo-generated DCregs of donor or recipient origin subvert allogeneic T-cell responses and promote long-term organ transplant survival. Particular interest has focused on how donor antigen is acquired, processed and presented by autologous dendritic cells, on the stability of DCregs, and on in-situ targeting of dendritic cells to promote their tolerogenic function. New evidence of the therapeutic efficacy of DCregs in a clinically relevant nonhuman primate organ transplant model and production of clinical grade DCregs support early evaluation of DCreg therapy in human graft recipients. We discuss strategies currently used to promote dendritic cell tolerogenicity, including DCreg therapy and in-situ targeting of dendritic cells, with a view to improved understanding of underlying mechanisms and identification of the most promising strategies for therapeutic application.

Immune Monitoring in BK Viurs Nephropathy: How to Identify Patients at the Highest Risk.

Quantification of BKV-load and BKV-specific immunity have been evaluated to monitor BKV-replication and progression to BKV-associated nephropathy. Previous studies, however, failed to identify immune markers characterizing kidney transplant recipients (KTRs) at increased risk of BK viremia. Here, we studied all adult KTRs at our single transplant center transplanted between 2007 and 2011. 42 (10%) of 425 KTRs were diagnosed with BK viremia within the first 6 months after renal transplantation. An age- and gender-matched control group of 127 KTRs without BK viremia was used for comparison. Samples were collected before transplantation and at +1, +2, and +3 months posttransplantation. BKV-specific T-cells directed to VP1 and Large T-antigen and CMV-specific T-cells directed to pp65 and IE-1, were measured using an interferon-γ Elispot assay. The extent of immunosuppression was quantified by measures of immune function including lymphocyte subpopulations and serum cytokine levels. KTRs developing early BKV-replication showed significantly increased frequencies of BKV-specific T-cells directed to Large T-antigen prior to transplantation compared to the control group (p<0.001). Interestingly, BKV-specific T-cells directed to Large T-antigen were significantly higher compared to VP1 (p=0.042). Shortly after transplantation, however, BKV-specific T-cells were significantly decreased or undetectable in KTRs developing BK viremia (p<0.001). In addition CD3+, CD4+, and CD8+ T-cell counts, and interferon-γ serum levels were significantly lower in KTRs with BK viremia at +1 month after transplantation (p<0.05). KTRs developing BK viremia showed lower CMV-specific T-cells prior to transplantation compared to the control group (p=0.048). Our results suggest that BKV-specific cellular immunity is triggered by subclinical activation of BKV infection prior to transplantation. In KTRs developing BK viremia identified risk factors and overimmunosuppression result in a decline of BKV-specific T-cells insufficient to further regulate BKV-replication. Our data indicate that BK viremia may result at least in part from recipient origin, and supports immune monitoring prior to transplantation to identify KTRs at the highest risk.

Isolated Peritoneal Donor-Related Plasmacytoma 3 Years After Liver Transplantation: A Case Report

Organ transplantation carries a risk of disease transmission from donor to recipient, primarily infection or malignancy. Although donors are thoroughly screened, donor-related malignancies are reported to occur in 0.01% of solid organ transplants. Plasma cell neoplasm, to the best of our knowledge, has not been reported as a donor-transmitted malignancy in liver transplantation. We describe a liver transplant from a donor with unrecognized plasmacytoma requiring retransplantation. Three years after the first transplant a single peritoneal mass was detected on surveillance imaging and radically excised; HLA phenotyping confirmed the mass to be an isolated extra-medullary plasmacytoma of chimeric donor and recipient origin.

Y-chromosome status identification suggests a recipient origin of posttransplant non-small cell lung carcinomas: chromogenic in situ hybridization analysis.

Owing to the need of lifelong immunosuppression, solid-organ transplant recipients are known to have an increased risk of posttransplant malignancies including lung cancer. Posttransplant neoplastic transformation of donor-derived cells giving rise to hematopoietic malignancies, Kaposi sarcoma, and basal cell carcinoma in nongraft tissues has been reported. The goal of this study was to assess the cell origin (donor versus recipient derived) of posttransplant non-small cell lung carcinomas (NSCLCs) in kidney and heart transplant recipients. An institutional database search identified 2557 kidney and heart transplant recipients in 8 consecutive years. Among this cohort, 20 (0.8%) renal and 18 (0.7%) heart transplant recipients developed NSCLC. The study cohort comprised 6 of 38 NSCLCs arising in donor-recipient sex-mismatched transplant patients. The tumor cell origin was evaluated by chromogenic in situ hybridization with Y-chromosome probe on formalin-fixed, paraffin-embedded tissues. Y-chromosome was identified in 97% ± 1% (range from 92% to 99%) of all types of nucleated cells in male control tissues. In all 5 NSCLCs from male recipients of female donor organ, Y-chromosome was identified in 97% ± 2% (range from 92% to 100%) of tumor cells, statistically equivalent to normal control (P < .001). No Y-chromosome was identified in NSCLC cells from a female recipient of male kidney. These findings suggest a recipient derivation of NSCLC arising in kidney and heart transplant recipients. A combination of histologic evaluation and chromogenic in situ hybridization with Y-chromosome analysis allows reliable determination of tissue origin in sex-mismatched solid-organ transplant recipients and may aid in management of posttransplant malignancy in such cases.

Donor origin of BKV replication after kidney transplantation

BackgroundBK virus associated nephropathy (BKVN) leads to renal allograft dysfunction and loss. However, it is still unclear whether BKV replication in the transplant recipient is a result of reactivation in the recipient's native kidneys or whether BKV originates from the donor kidney. Study designUrine of 249 donor/recipient pairs was investigated for the presence of BKV-DNA by qPCR before living transplantation (Tx) and consecutively after Tx. In BKV positive samples, the VP1 typing region (TR) and, in case of the presence of sufficient amount of DNA, the complete VP1 gene, the NCCR and a fragment of the Large T-antigen were sequenced and compared between donors and corresponding recipients before and after Tx. ResultsIn 20 pairs, sequencing of the BKV TR succeeded in donors and corresponding recipients after Tx. The derived sequences were completely identical in donor and post-Tx recipient samples. For comparison, identical TR sequences were detected in only 24% of 1068 randomly assembled pairs. This difference was statistically highly significant (p<0.0001, Fisher's exact test). Furthermore, all VP1, Large T-antigen and NCCR BKV sequences were also identical between donors and corresponding post-Tx recipients. In two of the 20 donor/recipient pairs, VP1 TR sequencing was also successful from the recipient before Tx. In both cases the sequence differed from the sequence detected in donor and recipient after Tx giving further evidence that recipient BKV was replaced by donor BKV after Tx. ConclusionsOur study for the first time provides evidence of BKV donor origin in renal transplant recipients.

Pediatric Fanconi Anemia With Secondary AML: A Retrospective Outcome Report From The German AML-BFM Group

Abstract Background To date, allogeneic stem cell transplantation (SCT) is the only curative approach for patients with Fanconi anemia (FA) and myeloid malignancy (AML). No data exist on whether cytoreductive chemotherapy prior to SCT impacts outcome. Methods We retrospectively analyzed data retrieved from the AML-BFM database (Germany) regarding children with FA and AML. Results Between January 1993 and May 2013 14 patients were identified, in whom AML was diagnosed at a median age of 11.6 yrs [0.9-19.6] (n=13). In 10 patients (4 not available) the diagnosis FA preceded the occurrence of AML by a median of 32.3 months [0-161]. Six of 14 patients underwent SCT achieving a 5 year overall survival of 42%. Median time to transplant after the diagnosis of AML was 2 months. One patient died after relapse, one due to severe graft versus host disease (GvHD), another of unknown cause. Of the three surviving patients two had chronic GvHD (one limited, one extended). All three surviving patients received a radiation free preparative regimen (1. fludarabine (Flu) and cyclophosphamide (Cy); 2. busulfan (Bu) and Flu; 3. Bu, Flu and Cy.) including antibodies for intensive GVHD prophylaxis. In all three cases a high cell dose of either PBSC (30.1 x 10e6 CD34+) or BM (2.13 and 3.43 x 10e8 MNC) was infused. Two surviving patients received low dose cytoreductive therapy (thioguanin and cytarabin) to bridge time to SCT. The remaining patient did not receive pre-transplant therapy. Eight of 14 patients did not receive a SCT. Median survival for these children was 40 days. Three of 8 patients received low intensity chemotherapy (thioguanin and/or cytarabin). Two were treated with more intensive combinations (e.g. cytarabin, adriamycin and etoposide). The latter all died of complications from long lasting aplasia. Additionally to the 14 patients described above, another patient was identified, in whom AML developed 14 months after successful SCT for bone marrow failure. Blasts were of recipient origin and the patient died of progressive disease. Conclusion The data presented are subject to selection bias. Three of 14 patients survived and 2/3 surviving patients received low dose cytoreductive therapy for disease control before going to transplant. None of them achieved complete remission (CR) before SCT. Graft failure and chronic GVHD were the main complications after transplantation. FA patients with AML should proceed to SCT. Mild cytoreductive therapy with thioguanine and cytarabine may be useful. Achieving CR pre transplant might not be necessary for OS. Disclosures: No relevant conflicts of interest to declare.

Graft TCR-Alpha/Beta Depletion and Peri-Transplant Use Of Eculizumab In Matched Unrelated Donor Transplantation For Severe Aplastic Anemia-Paroxysmal Nocturnal Hemoglobinuria: Report Of Two Cases

IntroductionInhibition of terminal complement components provides effective control of severe hemolytic PNH, but remains prohibitively expensive in countries with limited resources. Allogeneic hematopoietic stem cell transplantation offers a chance of permanent cure at the expense of increased risk of transplant-related morbidity and mortality in severe PNH. We report two patients with hemolytic PNH, associated with severe aplastic anemia, who were treated with limited eculizumab course to reduce the risk of complications of matched unrelated donor (MUD) transplantation.Patient #1 is a 22 y.o. young man. Severe aplastic anemia (SAA) was diagnosed at the age of 13 y.o. He received combined IST with ATG and cyclosporine A (CsA) and remained transfusion–free with complete hematologic response until 17 y.o., when SAA relapsed. CsA was re-administered as monotherapy due to severe anaphylaxis to ATG. At the age of 20 y.o. episodic hemolysis and severe abdominal pain bouts developed. PNH was diagnosed with standard flow cytometric assay. Two years later 10/10 matched unrelated donor was found and a decision was made to proceed to allogeneic transplantation. The preparative regimen included total doses of fludarabine - 150mg/m2, cyclophosphamide - 100mg/m2, thoracoabdominal irradiation - 6Gy and alemtuzumab 30 mg/m2. Eculizumab was administered weekly since day -14 till +14 at 600mg x2 and 300mg x3 resulting in prompt resolution of hemolysis. PBSC graft processing included TCR alpha/beta and CD19 depletion on CliniMACS instrument according to manufacturer's instructions. The final graft contained 6,4*106 per kg NC, 6,6*106 per kg CD34+, 15*104 per kg TCR alpha/beta+ lymphocytes. GVHD prophylaxis included tacrolimus till day +30 and Mtx at +1,3,6,10. Engraftment was stable with Plt and WBC on day + 18. PNH clone in granulocytes and monocytes remained around detection limit. A peculiar feature of engraftment was stable split chimerism with all hematopoietic lineages except CD3 originating from the donor. T cells remained almost completely of recipient origin until thymic function recovered with dominant production of donor T cells (Fig. 1). Independent of this T-cell chimerism dynamics, the hematopoiesis remains stable and of donor origin for one year from transplant. No manifestations of acute or chronic GVHD were observed. Transplant toxicity included CMV reactivation controlled with standard antiviral treatment. [Display omitted] Patient#2 is a 15 y.o. girl. The diagnosis of SAA was established at the age of 8 y.o. Combined IST with ATG and CsA induced partial hematologic response. Three years later she developed SAA relapse and hemolytic PNH complicated by cerebral sinus thrombosis and severe ischemic attack. She recovered upon endovascular thrombectomy and systemic thrombolysis eculizumab was started at 600 mg/biweekly leading to resolution of hemolysis. Four months later 10/10 MUD was identified and the patient proceeded to transplantation. The preparative regimen included fludarabine - 150mg/m2, cyclophosphamide - 100mg/m2, thoracoabdominal irradiation - 6Gy and ATG(horse) - 90 mg/m2. Eculizumab was administered weekly till day +21 at 300mg/week. Graft processing included TCR alpha/beta and CD19 depletion. The graft contained 13,8*106 per kg NC, 13,5*106 per kg CD34+, 31*104 per kg TCR alpha/beta+ lymphocytes. GVHD prophylaxis included tacrolimus till day +30 and Mtx at +1,3,6. Engraftment was rapid with Plt and WBC on day + 12 and + 15 respectively. Graft function is stable for 100 days post-transplant and PNH clone in granulocytes and monocytes is at the limits of detection. T-cell chimerism remains mixed with 10% of recipient cells. Grade 1-2 skin aGVHD was observed and controlled by short steroid course. No other early transplant-associated toxicity was observed. ConclusionCombined use of peri-transplant eculizumab and TCR alpha/beta depletion of the unrelated graft potentially protects patients with severe PNH from transplant-related toxicity. Disclosures:Boyakova:Alexion: Research Funding.

Effect of Reverse Chimerism on Rejection in Clinical Transplantation

Chimerism may enable allografts to survive when immunosuppressive therapy is administered at low levels or is even absent. Reverse chimerism (RC) is focused on intragraft chimerism that repopulates the allograft with cells of recipient origin. We aimed to identify and analyze current clinical evidence on RC and the presence of endothelial RC and tissue-specific RC. A total of 33 clinical reports on cardiac, kidney, liver, and lung transplants published between 1972 and 2012 that focused on RC were included in a systematic review. Liver allografts presented with the highest percentage of endothelial RC and lung allografts by far the lowest. Tissue-specific RC was present in most of the recipients, but at very low levels. There were also cardiac and kidney allografts with chimerism, but the functionality of the cells of recipient origin was questionable. We were unable to determine whether RC was a trigger for or a result of acute rejection. Further clinical research should focus on outcomes to evaluate the clinical relevance of this form of chimerism in transplantation.

Renal Heparan Sulfate Proteoglycans Modulate Fibroblast Growth Factor 2 Signaling in Experimental Chronic Transplant Dysfunction

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.

Injured kidney endothelium is only marginally repopulated by cells of extrarenal origin

The role of bone marrow marrow-derived cells after kidney endothelial injury is controversial. In this study, we investigated if and to what extent extrarenal cells incorporate into kidney endothelium after acute as well as during chronic endothelial injury. Fischer F-344wt (wild type) rat kidney grafts were transplanted into R26-hPAP (human placental alkaline phosphatase) transgenic Fischer F-344 recipient rats to allow identification of extrarenal cells by specific antibody staining. A severe model of renal thrombotic microangiopathy was induced via graft perfusion with antiglomerular endothelial cell (GEN) antibody and resulted in eradication of 85% of the glomerular and 69% of the peritubular endothelium (GEN group). At week 4 after injury, renal endothelial healing as well as recovery of the kidney function was seen. Endothelial chimerism was evaluated by double staining for hPAP and endothelial markers RECA-1 or JG-12. Just 0.25% of the glomerular and 0.1% of the peritubular endothelium was recipient derived. In a second experiment, chronic endothelial injury was induced by combination of kidney transplantation with 5/6 nephrectomy (5/6 Nx group). After 14 wk, only 0.86% of the peritubular and 0.05% of the glomerular endothelium was of recipient origin. In summary, despite demonstration of extensive damage and loss as well as excellent regeneration, just a minority of extrarenal cells were incorporated into kidney endothelium in rat models of acute and chronic renal endothelial cell injury. Our results highlight that kidney endothelial regeneration after specific and severe injury is almost exclusively of renal origin.

Lung cancer following lung transplant: Single institution 10 year experience

BackgroundLung cancer following lung transplantation is an infrequent occurrence of post-transplant neoplasia. Tumors are classified based on donor or recipient origin. Recipient tumors can be diagnosed in explanted specimens or found in contralateral native lungs which remain in place during single lung transplant (SLTx). The aim of our study was to review our institution's incidence of post lung transplant lung cancer, describe tumor histology, and review our experience with their outcomes. MethodsA total of 335 lung transplants from 2001 to 2010 were reviewed. Patients were identified with a post-transplant diagnosis of lung cancer, neoplasia, or mass. Fifteen patients were identified; two were excluded due to concomitant cancers with which the lung cancer would represent a metastasis. Retrospective chart review was undertaken for thirteen patients for descriptive statistics, tumor characteristics and overall survival. ResultsOverall incidence of lung cancer following transplant was 13 cases (3.88%). Six tumors were found in native explanted lungs and six developed subsequently in native lungs. One tumor was confirmed to be of donor origin.Histology included squamous cell in five (38.4%), adenocarcinoma in four (30.7%), and one patient each with adenosquamous (7.6%), carcinoid (7.6%), small cell (7.6%), or malignant solitary fibrous tumor (7.6%). Mean age at transplant was 65±3 years. Mean time from transplant to diagnosis is reported as 241±7 days (range 1–1170). Each patient had at least a 20 pack year smoking history with a mean of 45±3 years. One-year survival for those with lung cancer following transplant was 42.8% while 1 year survival of all lung transplants at our institution is 85.7%. ConclusionLung cancer incidentally found at the time of transplant or following transplantation is a serious complication with a noted effect on overall survival. The infrequent occurrence of donor tumors represents an adequate screening process of potential young donor lungs. The recognition of cancers in explanted specimens brings to question policies regarding screening of potential recipients with extensive smoking history. A high index of suspicion for native tumors is needed when conducting post-transplant surveillance as these tumors tend to be stage 4 at time diagnosis.

Expansion of recipient‐derived antiviral T cells may influence donor chimerism after allogeneic stem cell transplantation

Donor chimerism (DC) analysis is an important marker in the hematopoietic stem cell transplant follow-up. Here, we present evidence for a possible relationship of infectious complications and declines in DC. We analyzed the DC in patients experiencing cytomegalovirus (CMV) reactivation. In addition, in some patients chimerism analyses of T-cell subsets were performed. CMV-specific cytotoxic T-lymphocytes (CMV-CTL) were monitored using human leukocyte antigen-restricted multimer staining. Interestingly, CMV reactivation was accompanied by changes in DC in 11 of 67 patients transplanted. For example, DC declined in a cord blood recipient, in both total leukocytes and CD4 and CD8 T-cell subsets upon CMV reactivation. The latter was controlled after only 5 days through expanding CMV-CTL of 96% recipient origin, according to chimerism analysis of CMV-CTL (enriched beyond 50%). In another patient, transplanted after reduced-intensity conditioning from a DQB1 mismatched, CMV seronegative donor, incipient CMV reactivation was completely aborted by CMV-CTL of recipient origin. However, at the same time, mixed chimerism dropped from 51% to 0% donor type, resulting in late graft rejection. Our data indicate that chimerism analyses in subset populations lead to a better understanding of declining total leukocyte chimerism. Furthermore, recipient-derived CMV-CTL may be able to control CMV reactivation after reduced-intensity conditioning. We speculate that autologous CMV-CTL may be instrumental to overcome recurrent CMV reactivations, especially in patients transplanted from CMV-seronegative donors. In addition, the expansion of recipient-derived CMV-CTL may contribute to both, graft failure or to conversion to full DC.

Prolongation of the Rat Composite Tissue Allograft Survival by the Combination of Tolerogenic Immature Dendritic Cells and Short-Term Treatment With FK506

ObjectivePrevention of rejection in composite tissue allotransplantation without continuous immunosuppression is of paramount importance in the field of transplantation. Recently dendritic cells (DCs) have gained considerable attention as antigen-presenting cells that are also capable of tolerance induction. This study assessed the effect of interleukin (IL)-10–supplemented, alloantigen-pulsed immature tolerogenic DC to increase survival of orthotopic hind limb transplantations in rats. Materials and MethodsHind limbs from Sprague-Dawley (SD) hosts were transplanted to Fischer 344 (F344) rats. Peripheral blood mononuclear cells (PBMC) isolated from F344 were cultured to generate immature DCs (imDCs); IL-10 was added for tolerogenic DC induction. Flow cytometric analysis were performed to characterize the DC phenotype. IL-10-imDCs cocultured with donor mononuclear cells for 24 hours were reinjected into recipients subcutaneously 1 day before transplantation. Recipient animals were divided into 4 groups, each comprising 6 rats (n = 6): group I (untreated controls), group II (IL-10-imDCs alone), group III (FK-506 [Tacrolimus, 2 mg/kg] for 2 weeks postoperative), and group IV (recipient origin IL-10-imDCs combined with FK-506 for 2 weeks postoperative). Observation of graft appearance, cytokine production assays, and confocal immunofluorescence were performed at postoperative 1 week. ResultsThe combination of IL-10–treated imDCs and FK-506 produced a significantly prolonged median allograft survival (46.7 days) compared with groups I (4.7), II (5.3), or III (26.3). Splenocytes isolated from rats treated with IL-10-imDCs plus and FK-506 produced significant amounts of IL-10 and IL-4 cytokines upon alloantigen stimulation, as confirmed using ELISPOT and ELISA. ConclusionsWe demonstrated that IL-10–treated imDCs induced T-cell hyporesponsiveness, skewing the immune response to Th2 elements, resulting in long-term survival of composite tissue allografts.

Stable renal engraftment in a patient following successful tandem autologous/reduced-intensity conditioning allogeneic transplantation for treatment of multiple myeloma with del(17p) that developed as a post-transplantation lymphoproliferative disease following renal transplantation

Multiple myeloma (MM) developing after renal transplantation is rare. From January 1972 to December 2011, a total of 1,485 patients underwent renal transplantation in Nagoya Daini Red Cross Hospital; 14 (0.9%) of these recipients developed post-transplantation lymphoproliferative disorders (PTLDs) including two plasma cell neoplasms. Here, we report the clinical course of a 35-year-old male with immunoglobulin G k-type MM of recipient origin that developed 5 years after renal transplantation from a human leukocyte antigen (HLA)-haploidentical female sibling donor, which was performed to address dialysis-dependent chronic glomerulonephritis. Cytogenetic analysis revealed significant del(17p) abnormalities in myeloma cells. After non-response to bortezomib treatment, the patient achieved partial response with a thalidomide-containing salvage regimen and underwent successful tandem autologous/reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation (HSCT) from an unrelated male donor matched for seven of eight HLAs. At the 8-month follow-up time point, the patient's performance status remained good, and the transplanted kidney remains functional without rejection. To the best of our knowledge, this is the first report of a successful use of allogeneic HSCT for a patient who developed MM as a PTLD after renal transplantation. This patient has a transplanted kidney and transplanted hematopoietic cells that currently coexist without rejection.