The intratumoral CXCR3 chemokine system is predictive of chemotherapy response in human bladder cancer.

Chemotherapy has direct toxic effects on cancer cells; however, long-term cancer control and complete remission are likely to involve CD8+ T cell immune responses. To study the role of CD8+ T cell infiltration in the success of chemotherapy, we examined patients with muscle invasive bladder cancer (MIBC) who were categorized on the basis of the response to neoadjuvant chemotherapy (NAC). We identified the intratumoral CXCR3 chemokine system (ligands and receptor splice variants) as a critical component for tumor eradication upon NAC in MIBC. Through characterization of CD8+ T cells, we found that stem-like T cell subpopulations with abundant CXCR3alt, a variant form of the CXCL11 receptor, responded to CXCL11 in culture as demonstrated by migration and enhanced effector function. In tumor biopsies of patients with MIBC accessed before treatment, CXCL11 abundance correlated with high numbers of tumor-infiltrating T cells and response to NAC. The presence of CXCR3alt and CXCL11 was associated with improved overall survival in MIBC. Evaluation of both CXCR3alt and CXCL11 enabled discrimination between responder and nonresponder patients with MIBC before treatment. We validated the prognostic role of the CXCR3-CXCL11 chemokine system in an independent cohort of chemotherapy-treated and chemotherapy-naïve patients with MIBC from data in TCGA. In summary, our data revealed stimulatory activity of the CXCR3alt-CXCL11 chemokine system on CD8+ T cells that is predictive of chemotherapy responsiveness in MIBC. This may offer immunotherapeutic options for targeted activation of intratumoral stem-like T cells in solid tumors.

Association between Subcutaneous Adipose Tissue Inflammation, Insulin Resistance, and Calorie Restriction in Obese Females.

The worldwide epidemic of overweight and obesity has led to an increase in associated metabolic comorbidities. Obesity induces chronic low-grade inflammation in white adipose tissue (WAT). However, the function and regulation of both innate and adaptive immune cells in human WAT under conditions of obesity and calorie restriction (CR) is not fully understood yet. Using a randomized interventional design, we investigated postmenopausal overweight or obese female subjects who either underwent CR for 3 mo followed by a 4-wk phase of weight maintenance or had to maintain a stable weight over the whole study period. A comprehensive immune phenotyping protocol was conducted using validated multiparameter flow cytometry analysis in blood and s.c. WAT (SAT). The TCR repertoire was analyzed by next-generation sequencing and cytokine levels were determined in SAT. Metabolic parameters were determined by hyperinsulinemic-euglycemic clamp. We found that insulin resistance correlates significantly with a shift toward the memory T cell compartment in SAT. TCR analysis revealed a diverse repertoire in SAT of overweight or obese individuals. Additionally, whereas weight loss improved systemic insulin sensitivity in the intervention group, SAT displayed no significant improvement of inflammatory parameters (cytokine levels and leukocyte subpopulations) compared with the control group. Our data demonstrate the accumulation of effector memory T cells in obese SAT and an association between systemic glucose homeostasis and inflammatory parameters in obese females. The long-standing effect of obesity-induced changes in SAT was demonstrated by preserved immune cell composition after short-term CR-induced weight loss.

Comprehensive Characterization of a Next-Generation Antiviral T-Cell Product and Feasibility for Application in Immunosuppressed Transplant Patients.

Viral infections have a major impact on morbidity and mortality of immunosuppressed solid organ transplant (SOT) patients because of missing or failure of adequate pharmacologic antiviral treatment. Adoptive antiviral T-cell therapy (AVTT), regenerating disturbed endogenous T-cell immunity, emerged as an attractive alternative approach to combat severe viral complications in immunocompromised patients. AVTT is successful in patients after hematopoietic stem cell transplantation where T-cell products (TCPs) are manufactured from healthy donors. In contrast, in the SOT setting TCPs are derived from/applied back to immunosuppressed patients. We and others demonstrated feasibility of TCP generation from SOT patients and first clinical proof-of-concept trials revealing promising data. However, the initial efficacy is frequently lost long-term, because of limited survival of transferred short-lived T-cells indicating a need for next-generation TCPs. Our recent data suggest that Rapamycin treatment during TCP manufacture, conferring partial inhibition of mTOR, might improve its composition. The aim of this study was to confirm these promising observations in a setting closer to clinical challenges and to deeply characterize the next-generation TCPs. Using cytomegalovirus (CMV) as model, our next-generation Rapamycin-treated (Rapa-)TCP showed consistently increased proportions of CD4+ T-cells as well as CD4+ and CD8+ central-memory T-cells (TCM). In addition, Rapamycin sustained T-cell function despite withdrawal of Rapamycin, showed superior T-cell viability and resistance to apoptosis, stable metabolism upon activation, preferential expansion of TCM, partial conversion of other memory T-cell subsets to TCM and increased clonal diversity. On transcriptome level, we observed a gene expression profile denoting long-lived early memory T-cells with potent effector functions. Furthermore, we successfully applied the novel protocol for the generation of Rapa-TCPs to 19/19 SOT patients in a comparative study, irrespective of their history of CMV reactivation. Moreover, comparison of paired TCPs generated before/after transplantation did not reveal inferiority of the latter despite exposition to maintenance immunosuppression post-SOT. Our data imply that the Rapa-TCPs, exhibiting longevity and sustained T-cell memory, are a reasonable treatment option for SOT patients. Based on our success to manufacture Rapa-TCPs from SOT patients under maintenance immunosuppression, now, we seek ultimate clinical proof of efficacy in a clinical study.

In situ detection of CD73+ CD90+ CD105+ lineage: Mesenchymal stromal cells in human placenta and bone marrow specimens by chipcytometry.

Mesenchymal stromal cells (MSCs) support endogenous regeneration and present therefore promising opportunities for in situ tissue engineering. They can be isolated and expanded from various tissues, for example, bone marrow, adipose tissue, or placenta. The minimal consensus definition criteria of ex vivo expanded MSCs requires them to be positive for CD73, CD90, and CD105 expression, while being negative for CD34, CD45, CD14, CD19, and HLA-DR. This study aimed to compare the in situ phenotype of MSCs with that of their culture-expanded progeny. We report for the first time in situ detection of cells expressing this marker combination in human placenta cryosections as well as in bone marrow aspirates using multiplex-immunohistology (Chipcytometry), a technique that allows staining of more than 100 biomarkers consecutively on the same cell. © 2018 International Society for Advancement of Cytometry.

Intragraft and Systemic Immune Parameters Discriminating Between Rejection and Long-Term Graft Function in a Preclinical Model of Intestinal Transplantation.

BACKGROUND: The pathology and diagnosis of acute and chronic rejection in intestinal transplantation (ITX) are far from being completely understood. We established models of acute and chronic intestinal graft rejection and analyzed peripheral and intragraft immune responses. METHODS: We performed ITX from Dark Agouti into Lewis rats applying single-dose tacrolimus (TAC) at varying concentrations. Graft histology and immunohistology were assessed on postoperative day (POD)7, 14, and 45. Intragraft and peripheral gene expressions of inflammatory and anti-inflammatory markers and lipopolysaccharide binding protein (LBP) plasma as well as alloantibodies were measured simultaneously. RESULTS: A 1-mg TAC resulted in acute rejection and recipient death; 3 mg and 5 mg prolonged survival and led to severe or moderate chronic rejection, respectively, with 50% of the 5-mg TAC recipients surviving the observation period. Consequently, we observed severe infiltration on POD7 in untreated and 1-mg TAC recipients compared with minor histological alterations in 3 and 5 mg TAC groups. Only 5-mg TAC treatment prevented accumulation of CD4+ T cells and ED1+ macrophages over the entire observation period. Peripheral and intragraft expressions of T cell activation inhibitor-mitochondrial were stable in long-term surviving 5-mg TAC recipients but declined before acute or chronic rejection in 1 and 3 mg TAC recipients. In contrast, LBP levels increased during acute and chronic rejections. CONCLUSIONS: We studied acute and chronic rejections in a preclinical model of ITX, which recapitulates clinical findings and highlights the importance of monitoring peripheral T cell activation inhibitor-mitochondrial expression, LBP levels, and antidonor antibodies for revealing rejection.

Defective CD8 Signaling Pathways Delay Rejection in Older Recipients.

CD8+ T cells play a cardinal feature in response to alloantigens and are able to generate effector/memory T cells independently from CD4+ T cells. To investigate the impact of aging on CD8 T cells, we used a fully mismatched mouse skin transplant model. Our findings showed a prolonged allograft survival in older recipients associated with a significant increase of CD4+ and CD8+ CD44high CD62Llow effector/memory T cells and a reduced systemic IFNγ production. When reconstituting young CBA Rag-1 mice that lack mature T and B cells with old CD8+ T cells expressing clonal anti-H2K T cell receptor (TCR) alloreactive for MHC I, graft survival was significantly prolonged and comparable to those receiving young CD8+ T cells. Moreover, our data showed that reduced systemic IFNγ levels observed in old recipients had been linked to a compromised expression of the IL-2R ß subunit (CD122) by old CD8+ T cells. In addition, we observed an impaired IFNγ production on IL-2 receptor activation. At the same time, gene profiling analysis of old CD8 T cells demonstrated reduced chemokine ligand-3 and CD40L expression that resulted in compromised CD8+ T cell/dendritic cell communication, leading to impaired migratory and phagocytic activity of CD11c cells.Collectively, our study demonstrated that aging delays allograft rejection. CD8 T cells play a critical role in this process linked to a compromised production of IFNγ, in addition to a defective IL-2 receptor signaling machinery and a defective communication between CD8 T cells and dendritic cells.

Peripheral blood-derived virus-specific memory stem T cells mature to functional effector memory subsets with self-renewal potency.

Memory T cells expressing stem cell-like properties have been described recently. The capacity of self-renewal and differentiation into various memory/effector subsets make them attractive for adoptive T cell therapy to combat severe virus infections and tumors. The very few reports on human memory stem T cells (T(SCM)) are restricted to analyses on polyclonal T cells, but extensive data on Ag-specific T(SCM )are missing. This might be due to their very low frequency limiting their enrichment and characterization. In this article, we provide functional and phenotypic data on human viral-specific T(SCM), defined as CD8(+)CD45RA(+)CCR7(+)CD127(+)CD95(+). Whereas <1% of total T cells express the T(SCM) phenotype, human CMV-specific T(SCM) can be detected at frequencies similar to those seen in other subsets, resulting in ∼ 1 /10,000 human CMV-specific T(SCM). A new virus-specific expansion protocol of sort-purified T(SCM) reveals both upregulation of various T cell subset markers and preservation of their stem cell phenotype in a significant proportion, indicating both self-renewal and differentiation potency of virus-specific T cells sharing their TCR repertoire. Furthermore, we describe a simplified culture protocol that allows fast expansion of virus-specific T(SCM) starting from a mixed naive T/T(SCM) pool of PBLs. Due to the clinical-grade compatibility, this might be the basis for novel cell therapeutic options in life-threatening courses of viral and tumor disease.

Short-term TNF-alpha inhibition reduces short-term and long-term inflammatory changes post-ischemia/reperfusion in rat intestinal transplantation.

BACKGROUND: Tumor necrosis factor (TNF)-α inhibition was shown to reduce ischemia/reperfusion injury (IRI) after intestinal transplantation (ITX). We studied the effects of different TNFα inhibitors on acute IRI and long-term inflammatory responses in experimental ITX. METHODS: Orthotopic ITX was performed in an isogenic ischemia/reperfusion model in Lewis rats. The TNFα inhibition groups received infliximab post-reperfusion; etanercept pre-reperfusion and at postoperative days (POD) 1, 3, 5, and 7; or pentoxifylline pre-reperfusion and at POD 1 to 5. Tissue samples were taken from proximal and distal graft sections and mesenteric lymph nodes at 20 min, 12 hr, 7 day, and 6 months post-reperfusion for histopathology, immunohistology, terminal deoxyribosyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and real-time RT-PCR. Lung sections were stained for the myeloperoxidase assay. RESULTS: TNFα inhibitors decreased inflammatory changes after IRI in all treatment groups. Infliximab significantly improved 7-day survival and reduced the histological and immunohistochemical signs of IRI, the numbers of graft-infiltrating T cells and ED1 monocytes and macrophages, and pulmonary neutrophil infiltration, and also enhanced the accumulation of cytoprotective markers. Graft injury was more prominent in the distal graft than in the proximal graft in all groups, regardless of TNFα inhibition. CONCLUSION: Infliximab significantly reduced both acute IRI and, as with other TNFα inhibitors, long-term inflammatory responses after rat ITX. TNFα inhibition may help diminish chronic inflammatory long-term effects and avoid chronic allograft enteropathy.

Synergistic effects of prolonged warm ischemia and donor age on the immune response following donation after cardiac death kidney transplantation.

BACKGROUND: Organs from DCD (donation after cardiac death) donors are increasingly used for transplantation. The impact of advanced donor age and warm ischemia on the immune response of the recipient has not been studied. We developed a novel and clinically relevant model of DCD kidney transplantation and investigated the effects of donor age and prolonged warm ischemia on the recipient immune response after following DCD kidney transplantation. METHODS: DCD grafts from young and old F-344 donor rats were engrafted into LEW recipients who were nephrectomized bilaterally after a short (20 minutes) or prolonged (45 minutes) warm ischemia time. RESULTS: Analysis of the recipient's immune response early after transplantation showed an enhanced innate and adaptive immune response when old DCD kidneys were engrafted. Next, we studied DCD recipients with a supportive, contralateral native kidney in place, which allowed the recovery of the transplanted DCD kidney. Old DCD kidneys, demonstrated an impaired renal function associated with pronounced histomorphologic graft deterioration and an enhanced immune response by day 100 after transplantation. Interestingly, young DCD kidneys with a long warm ischemic time recovered from acute tubular necrosis and did not stimulate the long-term immune response. CONCLUSION: Our observations emphasize that prolonged warm ischemic time and advanced donor age augment the immune response after transplantation of DCD grafts. These results provide an experimental model and a mechanistic framework of clinically relevant aspects in DCD donation.

Inhibition of WISE preserves renal allograft function.

Wnt-modulator in surface ectoderm (WISE) is a secreted modulator of Wnt signaling expressed in the adult kidney. Activation of Wnt signaling has been observed in renal transplants developing interstitial fibrosis and tubular atrophy; however, whether WISE contributes to chronic changes is not well understood. Here, we found moderate to high expression of WISE mRNA in a rat model of renal transplantation and in kidneys from normal rats. Treatment with a neutralizing antibody against WISE improved proteinuria and graft function, which correlated with higher levels of ß-catenin protein in kidney allografts. In addition, treatment with the anti-WISE antibody reduced infiltration of CD68(+) macrophages and CD8(+) T cells, attenuated glomerular and interstitial injury, and decreased biomarkers of renal injury. This treatment reduced expression of genes involved in immune responses and in fibrogenic pathways. In summary, WISE contributes to renal dysfunction by promoting tubular atrophy and interstitial fibrosis.

Graft-specific immune cells communicate inflammatory immune responses after brain death.

BACKGROUND: Donor brain death (BD) triggers inflammatory graft activation that leads to impaired graft quality and outcome. We used a mouse BD model to investigate graft inflammation in cardiac transplants from immune-competent and immune-deficient donor animals. Effects of donor T-cell depletion were tested in an additional group of cardiac transplant recipients. METHODS: We analyzed systemic and graft-specific inflammatory activation after BD in donors and in syngeneic recipients of hearts retrieved from BD donors. To dissect the role of donor-specific immune cells in communicating BD-triggered inflammation, immune-deficient T-cell-, B-cell-, and natural killer cell-deficient Rag2/double knockout mice and naïve C57BL6 treated with anti-thymocyte globulin (Thymoglobulin; Genzyme Transplant, Cambridge, MA) were observed. RESULTS: Donor BD boosted lymphocyte activation in donors and recipients of syngeneic BD grafts. Lymphocyte activation was mitigated in recipients of immune-deficient and Thymoglobulin-treated BD donor grafts. Likewise, systemic and intra-graft levels of inflammatory cytokines interleukin -1, interleukin-6, interferon-γ, and tumor necrosis factor-α were significantly reduced in immune-deficient and anti-thymocyte globulin-treated recipients. Dense lymphocyte infiltrates were detected in the hearts from untreated BD donors; in contrast, the hearts from donors treated with Thymoglobulin demonstrated a preserved structure with minimal infiltrates comparable with naïve controls. CONCLUSION: BD triggers inflammatory graft activation communicated through intra-graft immune cells. Donor treatment with Thymoglobulin prevented inflammatory immune activation and improved graft quality to levels comparable to living donor organs.

Inflammatory immune responses in a reproducible mouse brain death model.

BACKGROUND: Brain death impairs donor organ quality and accelerates immune responses after transplantation. Detailed aspects of immune activation following brain death remain unclear. We have established a mouse model and investigated the immediate consequences of brain death and anesthesia on immune responses. METHODS: C57JBl/6 mice (n=6/group) were anesthetized with isoflurane (ISF) or ketamine/xylazine (KX); subsequently, animals underwent brain death induction and were followed for 3h under continuous ventilation. Blood pressure was monitored continuously and animals were resuscitated with normal saline to achieve normotension. Immune activation in brain dead animals was analyzed by IFNγ-ELispot, MLR, and flow-cytometry. Sham-operated and naïve animals served as controls. RESULTS: Blood pressure remained stable in both BD/KX and BD/ISF animals during the 3h observation time. Brain death was linked to systemic immune activation: IFNγ-expression of splenocytes and lymphocyte proliferation rates was significantly elevated subsequent to brain death (p<0.02, <0.01); T-cell activation markers CD28 and CD69 had increased in brain dead animals (p<0.03, <0.02). Isoflurane treatment in sham controls throughout the observation period (3.5h) revealed anesthesia associated IFNγ-expression and lymphocyte activation which were not observed when animals were treated with ketamine/xylazine (p<0.04, <0.009). CONCLUSIONS: This study reports on a reproducible and hemodynamically stable brain death mouse model. Hemodynamic stability was not impacted through either isoflurane or ketamine/xylazine induction. Of clinical relevance, prolonged anesthesia with isoflurane had been linked to pro-inflammatory cytokine activation. Brain death caused systemic immune activation in organ donors.

Modified CD4(+) T-cell response in recipients of old cardiac allografts.

With an increasing demand, organs from elderly donors are more frequently utilized for transplantation. Herein, we analyzed the impact of donor age on CD4(+) T-cell responses with regard to regulatory and effector mechanisms. Young (3months) BM12 recipients were engrafted with young or old (18months) B6 cardiac allografts. Systemic CD4(+) T-cell responses and intragraft changes were monitored and compared to age-matched syngenic transplant controls. While elderly, nonmanipulated hearts contained significantly elevated frequencies of donor-derived leukocytes prior to transplantation, allograft survival was age-independent. T-cell activation, however, was delayed and associated with a compromised immune response in mixed lymphocyte cultures (MLR; P=0.0002) early after transplantation (day 14). During the time course after transplantation, recipients of old grafts demonstrated an augmented immune response as shown by significantly higher frequencies of activated CD4(+) T-cells and a stronger in vitro alloreactivity (MLR; ELISPOT; P<0.01). In parallel, frequencies of regulatory T-cells had increased systemically and overall fewer CD4(+) T-cells were detected intragraft. Interestingly, changes in the CD4(+) T-cell response were not reflected by graft morphology. Of note, transplantation of young and old syngenic hearts did not show age-related differences of the CD4(+) T-cells response suggesting that old grafts can recover from a period of short cold ischemia time. Our data suggest that donor age is associated with an augmented CD4(+) T-cells response which did not affect graft survival in our model. These findings contribute to a better understanding of the immune response following the engraftment of older donor organs.

TIM-3: a novel regulatory molecule of alloimmune activation.

T cell Ig domain and mucin domain (TIM)-3 has previously been established as a central regulator of Th1 responses and immune tolerance. In this study, we examined its functions in allograft rejection in a murine model of vascularized cardiac transplantation. TIM-3 was constitutively expressed on dendritic cells and natural regulatory T cells (Tregs) but only detected on CD4(+)FoxP3(-) and CD8(+) T cells in acutely rejecting graft recipients. A blocking anti-TIM-3 mAb accelerated allograft rejection only in the presence of host CD4(+) T cells. Accelerated rejection was accompanied by increased frequencies of alloreactive IFN-γ-, IL-6-, and IL-17-producing splenocytes, enhanced CD8(+) cytotoxicity against alloantigen, increased alloantibody production, and a decline in peripheral and intragraft Treg/effector T cell ratio. Enhanced IL-6 production by CD4(+) T cells after TIM-3 blockade plays a central role in acceleration of rejection. Using an established alloreactivity TCR transgenic model, blockade of TIM-3 increased allospecific effector T cells, enhanced Th1 and Th17 polarization, and resulted in a decreased frequency of overall number of allospecific Tregs. The latter is due to inhibition in induction of adaptive Tregs rather than prevention of expansion of allospecific natural Tregs. In vitro, targeting TIM-3 did not inhibit nTreg-mediated suppression of Th1 alloreactive cells but increased IL-17 production by effector T cells. In summary, TIM-3 is a key regulatory molecule of alloimmunity through its ability to broadly modulate CD4(+) T cell differentiation, thus recalibrating the effector and regulatory arms of the alloimmune response.

Identification of a microRNA signature of renal ischemia reperfusion injury.

Renal ischemia reperfusion injury (IRI) is associated with significant morbidity and mortality. Given the importance of microRNAs (miRNAs) in regulating gene expression, we examined expression profiles of miRNAs following renal IRI. Global miRNA expression profiling on samples prepared from the kidneys of C57BL/6 mice that underwent unilateral warm ischemia revealed nine miRNAs (miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805, and miR-194) that are differentially expressed following IRI when compared with sham controls. These miRNAs were also differently expressed following IRI in immunodeficient RAG-2/common gamma-chain double-knockout mice, suggesting that the changes in expression observed are not significantly influenced by lymphocyte infiltration and therefore define a lymphocyte-independent signature of renal IRI. In vitro studies revealed that miR-21 is expressed in proliferating tubular epithelial cells (TEC) and up-regulated by both cell-intrinsic and -extrinsic mechanisms resulting from ischemia and TGF-beta signaling, respectively. In vitro, knockdown of miR-21 in TEC resulted in increased cell death, whereas overexpression prevented cell death. However, overexpression of miR-21 alone was not sufficient to prevent TEC death following ischemia. Our findings therefore define a molecular fingerprint of renal injury and suggest miR-21 may play a role in protecting TEC from death.

Prolonged graft survival in older recipient mice is determined by impaired effector T-cell but intact regulatory T-cell responses.

Elderly organ transplant recipients represent a fast growing segment of patients on the waiting list. We examined age-dependent CD4(+) T-cell functions in a wild-type (WT) and a transgenic mouse transplant model and analyzed the suppressive function of old regulatory T-cells. We found that splenocytes of naïve old B6 mice contained significantly higher frequencies of T-cells with an effector/memory phenotype (CD4(+)CD44(high)CD62L(low)). However, in-vitro proliferation (MLR) and IFNgamma-production (ELISPOT) were markedly reduced with increasing age. Likewise, skin graft rejection was significantly delayed in older recipients and fewer graft infiltrating CD4(+)T-cells were observed. Old CD4(+) T-cells demonstrated a significant impaired responsiveness as indicated by diminished proliferation and activation. In contrast, old alloantigen-specific CD4(+)CD25(+)FoxP3(+) T-cells demonstrated a dose-dependent well-preserved suppressor function. Next, we examined characteristics of 18-month old alloreactive T-cells in a transgenic adoptive transfer model. Adoptively transferred old T-cells proliferated significantly less in response to antigen. Skin graft rejection was significantly delayed in older recipients, and graft infiltrating cells were reduced. In summary, advanced recipient age was associated with delayed acute rejection and impaired CD4(+) T-cell function and proliferation while CD4(+)CD25(+)FoxP3(+) T-cells (Tregs) showed a well-preserved function.

Machine perfusion or cold storage in organ transplantation: indication, mechanisms, and future perspectives.

Most organs are currently preserved by cold storage (CS) prior to transplantation. However, as more so called marginal donor organs are utilized, machine perfusion has regained clinical interest. Recent studies have demonstrated advantages of pulsatile perfusion over CS preservation for kidney transplantation. However, it remains unclear whether there is a significant benefit of one preservation method over the other in general, or, whether the utilization of particular preservation approaches needs to be linked to organ characteristics. Proposed protective mechanisms of pulsatile perfusion remain largely obscure. It can be speculated that pulsatile perfusion may not only provide nutrition and facilitate the elimination of toxins but also trigger protective mechanisms leading to the amelioration of innate immune responses. Those aspects may be of particular relevance when utilizing grafts with suboptimal quality which may have an increased vulnerability to ischemia/reperfusion injury and compromised repair mechanisms. This review aims to enunciate the principles of organ perfusion and preservation as they relate to indication, aspects of organ protection and to highlight future developments.

Cold ischemia does not interfere with tolerance induction.

BACKGROUND: Ischemia/reperfusion injury activates innate immunity, which in turn may prevent tolerance induction. We asked whether prolonged cold ischemia interferes with successful tolerance induction. METHODS: Kidneys from Dark Agouti donors were grafted into binephrectomized Lewis rats after short (20 min) or prolonged (6 hr) cold ischemia. Tolerance was induced by nondepleting anti-CD4 monoclonal antibody RIB 5/2 (10 mg/kg for 5 days). Binephrectomized untreated and cytotoxic T-lymphocyte antigen (CTLA)-4Ig treated recipients served as controls. Animals were followed for 100 days. Adoptive transfer experiments into sublethally irradiated naive Lewis were performed at day 100. Animals received kidneys from Dark Agouti rats subsequently without further immunosuppression and were followed for an additional 20 days. RESULTS: All RIB 5/2-treated recipients survived the first observation period independent of the cold ischemia time. Graft function, morphology, and transferred T-cell numbers were comparable in both groups. Twenty days after transfer amounts of intragraft and peripheral donor-derived cells were significantly reduced in recipients of the initially prolonged cold ischemia group associated with an attenuated immune response. CONCLUSIONS: Our results prove that an initially extended cold ischemia does not interfere with tolerance induced by RIB 5/2. Moreover, we conclude that a "tolerizing conditioning" achieved by prolonged cold ischemia during the tolerance-induction phase may reduce the immune response in recipients of an adoptive cell transfer.

Donor brain death significantly interferes with tolerance induction protocols.

Studies in rodents showed that antibodies are able to induce tolerance of allografts. As clinical results are unsatisfactory and deceased donors are still the main source of organ transplants, we investigated whether donor brain-death impacts on tolerance induction after experimental kidney transplantation. Anti-CD4 monoclonal antibodies (RIB 5/2; 2.5 mg/kg x 5 days) treated and untreated recipients of brain-dead donor grafts were compared with RIB 5/2 treated and untreated recipients of living donor grafts (F344-to-Lewis). All recipients received low-dose CsA (1.5 mg/kg x 10 days). Kidneys were recovered 4, 16 and 40 weeks after transplantation and examined by morphology, immunohistology and flow cytometry. Renal function was monitored monthly. RIB 5/2 treatment significantly decreased proteinuria in recipients of living donor allografts when compared with living donor controls. After 40 weeks, inflammatory cell infiltration and MHC class II expression were reduced while morphologic alterations were minimal. In contrast, treatment of brain-dead graft recipients had no impact on graft function. Structural changes and graft infiltration were comparable to brain-dead donor controls at all time points. RIB 5/2 treatment significantly improved graft function in recipients of living donor grafts; however, it was not effective in recipients of brain-dead donor organs.

Ischemic preconditioning produces systemic protective and adoptively transferable effects.

Ischemic preconditioning directly protects organs from subsequent non-specific injuries. To test for systemic protective effects kidneys from F-344 donor rats went through a short warm ischemic time. Both, clamped and contralateral unclamped kidneys were procured after either a short (15 min) or long (24 h) reperfusion period and transplanted into Lewis rats following a prolonged cold ischemia. To test for transferable effects serum from preconditioned rats was infused either into native donors or recipients. Following a short reperfusion interval protective effects were only evident in previously clamped grafts. However, after a long reperfusion interval protective effects were observed in previously clamped and contralateral unclamped kidneys promoting improved survival, structure, function and reduced inflammation. These effects were not related to heme oxygenase-1 induction or neural transmission as heme oxygenase-1 inhibition or denervation prior to preconditioning did not affect organ protection. These results show that renal ischemic preconditioning is associated with time-dependent local and systemically transferable protection.