Untreated Recipients Research Articles

Longterm Multilineage Human Hematopoietic Repopulation of Untreated KIT-Mutant Immunodeficient Mice

Background: Assessment of the growth and differentiation of human hematopoietic cells in immunodeficient mice has become vital to much basic and translational research. Historically, the primary focus has been to quantify different types of repopulating cells, based on the diversity and longevity of their clonal outputs in transplanted mice, assuming the results would be relevant to clinical transplants in humans. With the development of mice that lack B, T and NK cells on a variety of backgrounds and that have normal life expectancies but differences in cellular DNA repair ability, an increasing application is to use mice repopulated with human cells to interrogate and perturb mechanisms controlling normal, genetically modified and malignant cell behavior. We have previously shown that C57Bl6 mice homozygous for the W41 mutation of c-kit are fertile with a normal lifespan but have a functionally compromised hematopoietic stem cell (HSC) population. This enables single transplanted syngeneic HSCs to be detected at high frequency in these mice when they have been given a sublethal irradiation dose. Importantly, the HSC-derived clones produced in these mice display the same growth, self-renewal and differentiation abilities as in myeloablated recipients that require a co-transplant of normal mouse bone marrow (BM) cells to support their survival. We now report the development and improved repopulation by human cord blood (CB) CD34+ cells of mice that have the same genetically determined B, T, NK immunodeficiency as NOD/Rag1-/--IL2Rγc-/- (NRG) into which a homozygous W41 gene has been introduced.This was achieved by crossing and backcrossing the progeny of NRG x C57Bl6-W41/W41 matings and selecting mice that were homozygous for the Sirpα allele of the NOD mouse, the null Rag1 and null IL2Rgamma chain, genes of the NRG mouse and the W41 gene to obtain all of these on an otherwise mixed NODxC57Bl/6 background (NRG-W41 mice). The NRG mouse was chosen because the Rag1 KO has no effect on the radiosensitivity of other tissues as is the case with the scid (S) gene in the NSG mouse. As a result, use of the NRG mouse allows exploitation of the radioprotective effect of a reduced irradiation dose rate and hence delivery of a selectively higher dose to the HSCs of the host.Results: Initial studies showed that parental NRG mice given 900 cGy split or spread continuously over 3 hrs show similar repopulation by human CD34+ CB cells as NSG mice given 315 cGy, but are more robust with consistent longterm survival. We then performed a pilot experiment using the same transplant design (2x104 CD34+ CB cells/mouse) to compare chimerism obtained in NRG-W41 mice given an estimated “equivalent” radiosensitizing regimen of 150 cGy. The levels of multiple lineages of human cells measured in the BM and spleen 20 weeks post-transplant revealed these were greatly increased in the NRG-W41 mice (>95% human CD45+ cells in the BM vs 40% in NRG mice). Kinetic analysis of human cells in the blood also showed an enhanced output of human myeloid and B-lymphoid cells over time (5-fold higher in the NRG-W41 mice after >3 weeks). Particularly notable was the selectively increased (20-fold) and sustained output of human glycophorin A+ (GPA+) erythroid cells in the NRG-W41 mice (5% human GPA+ cells in the BM of 20-week NRG-W41 mice given 150 cGy and 5x104 CD34+ CB cells/mouse vs 0.25% in the BM of the matched NRG mice given 900 cGy). A similar marked increase (20-fold) was seen on the level of circulating human platelets (SSClowCD41+ CD61+ cells) in comparable groups of transplanted NRG-W41 and NRG mice. We then investigated the extent of repopulation achievable in untreated NRG-W41 recipients. We therefore transplanted mice of both strains with 5x104 CD34+CB cells each and have now followed the levels of human cells in their circulation and BM for up to 20 weeks. Human cells were barely detectable at 3 weeks post-transplant in either strain, but then in the unirradiated NRG-W41 mice only, their levels (all lineages) increased to close to those attained in NRG mice given 900 cGy.Conclusion: NRG-W41 mice support robustly enhanced and long term generation in vivo of a wide range of human hematopoietic cell types including erythrocytes and platelets, with high levels of chimerism achieved even in unirradiated primary recipients transplanted with relatively low numbers of human CD34+ CB cells. DisclosuresNo relevant conflicts of interest to declare.

The Role of the Cytoplasmic RNA Sensor Retinoic Acid Inducible Gene- I (RIG-I) in Allogeneic Stem Cell Transplantation

The success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by acute graft-versus-host disease (GVHD). Improving the procedure depends on identifying the mechanisms that contribute to this damaging T cell reactivity, while preserving graft-versus-leukemia (GVL) activity against hematopoietic malignancies."Tonic" type I IFN signaling in BMT recipients and therapeutic application of recombinant IFN-α have been shown to play an important role in defining the balance between GVHD and GVL responses, but the molecular mechanisms inducing this protective response remains unknown. In this regard, pattern recognition receptors (PRRs) that detect cytosolic nucleic acids and lead to the production of large amounts of type I interferons (IFN-α/β) such as the family of RIG-I-like receptors (RLRs) are of particular interest. RLRs, a subfamily of the cytoplasmic DExD/H- box family of helicases, consist of three members: retinoic acid inducible gene I (RIG-I), melanoma differentiation factor 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). RIG-I senses viral and bacterial RNA to induce the production of type I interferons, proinflammatory cytokines and inflammasome activation. Double-stranded RNA (dsRNA) carrying a 5′-triphosphate (3pRNA) has been identified as the natural ligand for RIG-I and serves as a selective trigger for RIG-I signaling. Although initially characterized as a main regulator for antiviral host defense, mice deficient in components of the RLR and type I IFN signaling pathway develop inflammatory bowel disease (IBD)-related pathologies. Furthermore, patients suffering from IBD show a highly significant downregulation of RIG-I in ileal epithelial cells and in a recent meta-analysis of genome-wide association studies (GWAS) data, IFNAR1 and MDA-5 were identified as primary candidate genes in susceptible loci for IBD. Together, these results indicate that RLRs and type I IFN signaling have important functions in the suppression of IBD by yet ill defined mechanisms. Given that the pathophysiology of GVHD shares several features with inflammatory bowel disease (IBD), we tested the role of the RIG-I pathway in the context of allo-HSCT.We utilized MAVS-deficient mice, which lack a common adapter for RIG-I signaling, as recipients in an MHC-disparate (BALB/c into B6) model of allo-HSCT. Compared to wild-type (WT) B6 mice, MAVS-KO mice receiving allogeneic BM + T cells displayed significantly worse GVHD mortality (Fig.1 A). Given the enhanced GVHD observed in the absence of RIG-I signaling, we hypothesized that selective engagement of RIG-I by 3pRNA (RIG-I ligand) in vivo would protect recipients from GVHD. Using a B6 into BALB/c model we observed that i.v. administration of a selective RIG-I ligand on d-1 significantly reduced mortality, weight loss and intestinal GVHD histopathology (Fig. 1B and data not shown). In addition, the translocation of LPS and microorganisms from the bowel lumen through the damaged intestinal mucosa to the systemic circulation during pre-transplant conditioning represents a crucial step in GVHD pathophysiology. We observed that administration of RIG-I ligand prior to allo-HSCT augmented intestinal barrier function measured by less fluorescence in the serum after FITC-dextran gavage compared to untreated WT recipients (Fig. 1C). Moreover, RIG-I stimulation augmented epithelial regeneration as determined by organoid formation from freshly isolated crypts (Fig. 1D) and inhibited activation of dendritic cells (DCs) during pre-transplant conditioning (Fig. 1E). To investigate the impact of 3pRNA administration during GVT, we used luciferase+ A20 bioluminescence in B6 into BALB/c tumor challenge recipients demonstrating that RIG-I ligands do not limit GVL (data not shown). Taken together, our results (i) uncover a previously unknown role of the RIG-I-MAVS signaling pathway in GVHD and (ii) offer a novel strategy to foster epithelial regeneration and inhibit antigen presentation during pre-transplant conditioning, while maintaining GVL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hypothermic Oxygenated Perfusion (HOPE) downregulates the immune response in a rat model of liver transplantation.

To evaluate the impact of a novel oxygenated perfusion approach on rejection after orthotopic liver transplantation (OLT). Hypothermic oxygenated perfusion (HOPE) was designed to prevent graft failure after OLT. One of the mechanisms is downregulation of Kupffer cells (in situ macrophages). We, therefore, designed experiments to test the effects of HOPE on the immune response in an allogeneic rodent model of nonarterialized OLT. Livers from Lewis rats were transplanted into Brown Norway rats to induce liver rejection in untreated recipients within 4 weeks. Next, Brown Norway recipients were treated with tacrolimus (1 mg/kg), whereas in a third group, liver grafts from Lewis rats underwent HOPE or deoxygenated machine perfusion for 1 hour before implantation, but recipients received no immunosuppression. In a last step, low-dose tacrolimus treatment (0.3 mg/kg) was assessed with and without HOPE. Allogeneic OLT without immunosuppression led to death within 3 weeks after nonarterialized OLT due to severe acute rejection. Full-dose tacrolimus prevented rejection, whereas low-dose tacrolimus led to graft fibrosis within 4 weeks. HOPE treatment without immunosuppression also protected from lethal rejection. The combination of low-dose tacrolimus and 1-hour HOPE resulted in 100% survival within 4 weeks without any signs of rejection. We demonstrate that allograft treatment by HOPE not only protects against preservation injury but also impressively downregulates the immune system, blunting the alloimmune response. Therefore, HOPE may offer many beneficial effects, not only to rescue marginal grafts but also by preventing rejection and the need for immunosuppression.

Effectiveness of IVIG Prophylaxis Against Antibody Mediated Rejection in Sensitized Renal Transplant Recipients.

Intro: With increasing transplantation of highly sensitized patients, we explored methods to decrease the incidence of antibody mediated rejection (AMR) in these high risk patients. Renal transplantation in highly sensitized patients is associated with as increased rate of rejection and poorer graft survival compared to unsensitized patients. In 2010, our center implemented IVIG prophylaxis against AMR in renal transplant recipients using 0.5gm/kg IVIG monthly for 4 doses. Initially, recipients with cPRA>80% received IVIG, which was revised in 2013 to cPRA>50 due to the positive impact of IVIG. We followed cross-match compatible, sensitized patients cPRA>50% for development of donor specific antibody (DSA), biopsy proven rejection, viral infections, neutropenia, graft loss, and death. Steroid-free immunosuppression (tacrolimus and mycophenolate) with Induction Thymoglobulin® and 2 doses of methylprednisolone was used unless patients developed rejection or new DSA. Minimum followup is 6 months. Results: IVIG prophylaxis significantly decreased the rate of AMR from 35% (15/43) to 9% (3/33) p=0.019 in treated vs. untreated recipients. Untreated patients include cPRA>50% 2006-2010 and cPRA 50-79% 2010-2013. In treated vs. untreated recipients, one year graft survival was 94% vs 91%. New DSAs decreased from 35% (15/43) to 21% (7/33). Patients with cPRA 50%-79% accounted for significant numbers of total positive DSAs (41%) and AMR (50%). IVIG prophylaxis was associated with a higher incidence of BK viremia (24% v 19%) and neutropenia (36% v 23%), but not CMV viremia (18% v 16%).Table: No Caption available.Data expressed as n(%) except N and Mean cPRA. Graft Loss defined as within 1yr. Conclusions: 1) In sensitized renal transplant recipients (cPRA>50%), 4 monthly doses of IVIG was effective as prophylaxis against AMR over more than 1 year post-transplantation. 2) IVIG prophylaxis also appears to decrease the incidence of new DSAs. 3) IVIG prophylaxis did not impact the incidence of ACR.

B7-H1 Expression Is Critical to Cardiac Allograft Tolerance Induced By the Combination Therapy of Mesenchymal Stem Cells and Rapamycin.

Background: Mesenchymal stem cells (MSCs) become an attractive tool to suppress immune response. We have previously reported that MSC-based therapy in combination with rapamycin induced cardiac allograft tolerance in a mouse model. The present study investigated the role of MSC-expressing B7-H1 in MSC-mediated cardiac allograft protection. Methods & Results:MSCs (1×106, i.v.) from C57BL/6 donor mice were injected into BALB/c recipients 24 hours after receiving a heterotopic C57BL/6 heart graft. Grafts in untreated recipients were rapidly rejected in 7.5±0.7 days. Either MSCs or rapamycin monotherapy doubled allograft survival time to 15.8±1.5 days and 16.3±1.1 days, respectively. MSCs in combination with rapamycin induced heart allograft tolerance with normal graft histology and achieved graft mean survival time (MST) for more than 100 days. In contrast, treatment with B7-H1-blocking MSCs was not able to prolong allograft survival (MST: 8.7±1.1 days), and in combination with rapamycin prevented tolerance induction. Graft MST in the recipients treated with B7-H1-blocking MSC-based combination therapy was significantly reduced to 18.5±0.8 days. In addition, Flow cytomytry analysis showed that B7-H1 is required for MSC-mediated downregulation of antibody production and enhancement of the frequency of tolerogeneic dendritic cells (Tol-DCs) and CD4+CD25+Foxp3+ cells in tolerant recipients. In vivo treatment with MSCs expressing B7-H1 is also involved in the modulation of CD83 and cytokine (IL-4 and IL-10) expression of B cells. Furthermore, MSC-mediated suppression of B cells measured by MLR, ELISA and transwell assays requires B7-H1 and contact between CD19+ B cells and MSCs. Conclusion:This study supports a critical role of B7-H1 in MSC-mediated immunosuppression and tolerance induction in transplantation, and would clearly emphasize the significant clinical potential of MSCs.

Attenuation of allograft rejection by intragraft inhibition of class II transcativator in high responder rat liver transplantation.

Major histocompatibility complex-II (MHC-II) plays an important role in graft rejection and class II transactivator (CIITA) is the key regulator for MHC-II expression. The aim of this study was to determine the efficacy of intragraft inhibition of CIITA in attenuating liver transplant rejection. Three plasmids containing small hairpin RNA (shRNA) against rat CIITA (pCIITA-shRNA) and one control plasmid of pHK-shRNA were constructed. In vitro dendritic cell (DC) transfection and liver transfection via portal vein in donor rats (n = 8) by shRNA plasmids were performed to confirm the inhibitory effect of pCIITA-shRNA on CIITA expression. It showed that expressions of CIITA and MHC-II were significantly inhibited by pCIITA-shRNA in both DC in vitro and liver of donor rats in vivo (p < 0.05 vs. control pHK-shRNA treatment). pCIITA1-shRNA was proved to be the best inhibitor among three pCIITA-shRNAs and then used in high-responder rat liver transplantation model (DA donors-to-Lewis recipients). Transplant groups (n = 16/group) include untreated recipients transplanted with donor liver graft pretreated with either saline, or pHK-shRNA, or pCIITA1-shRNA. Cyclosporine-treated (10 mg/kg, im, day 0-7) recipients transplanted with unmodified liver grafts were used as no rejection control. The results showed that the recipient rats survived significantly longer in pCIITA1-shRNA-treated group with markedly attenuated liver graft rejection (p < 0.05 vs. saline and pHK-shRNA-treated groups). Furthermore, significantly decreased intragraft expressions of CIITA, MHC-II, IL-2, and IFN-γ were found in pCIITA1-shRNA-treated group (p < 0.05 vs. saline and pHK-shRNA-treated groups). This study suggests that intragraft inhibition of CIITA could be a novel strategy for attenuating graft rejection in liver transplantation.

Effective targeting of chronic myeloid leukemia initiating activity with the combination of arsenic trioxide and interferon alpha

Imatinib is the standard of care in chronic meloid leukemia (CML) therapy. However, imatinib is not curative since most patients who discontinue therapy relapse indicating that leukemia initiating cells (LIC) are resistant. Interferon alpha (IFN) induces hematologic and cytogenetic remissions and interestingly, improved outcome was reported with the combination of interferon and imatinib. Arsenic trioxide was suggested to decrease CML LIC. We investigated the effects of arsenic and IFN on human CML cell lines or primary cells and the bone marrow retroviral transduction/transplantation murine CML model. In vitro, the combination of arsenic and IFN inhibited proliferation and activated apoptosis. Importantly, arsenic and IFN synergistically reduced the clonogenic activity of primary bone marrow cells derived from CML patients. Finally, in vivo, combined interferon and arsenic treatment, but not single agents, prolonged the survival of primary CML mice. Importantly, the combination severely impaired engraftment into untreated secondary recipients, with some recipients never developing the disease, demonstrating a dramatic decrease in CML LIC activity. Arsenic/IFN effect on CML LIC activity was significantly superior to that of imatinib. These results support further exploration of this combination, alone or with imatinib aiming at achieving CML eradication rather than long-term disease control.

Th1/Th17-Mediated Interstitial Pneumonia in Chronic Colitis Mice Independent of Intestinal Microbiota

Although intestinal microbiota are essential for the development of T cell-mediated colitis, it remains undetermined whether they enhance or suppress the chronic extraintestinal inflammation that often complicates inflammatory bowel diseases. In this study, we investigate the role of intestinal microbiota in the development of colitis and extraintestinal manifestations in a mouse model in which colitis was induced in SCID mice by adoptive transfer of CD4(+)CD45RB(high) T cells. Under specific pathogen-free conditions, these mice developed both colitis and extraintestinal interstitial pneumonia, whereas mice given a mixture of antibiotics did not develop colitis, but, surprisingly, developed Th1/Th17-mediated IP. Irrespective of antibiotic treatment, cotransfer of CD4(+)CD25(+) regulatory T cells suppressed the development of pneumonitis and colitis, with all local CD4(+)CD45RB(high) T cell-derived cells converted to CD44(high)CD62L(-)IL-7Rα(high) effector-memory T cells. Retransfer of CD4(+) effector-memory T cells from the lungs of antibiotic-treated mice with IP not only induced IP in both antibiotic-treated and -untreated recipients but also induced colitis in the untreated recipients. In summary, we have established a unique model of Th1/Th17-mediated IP in microbiota-free and antibiotic-treated mice. This model may be valuable in investigating the immunological mechanisms underlying extraintestinal disorders in patients with inflammatory bowel disease.

The Potassium Channel KCa3.1 as New Therapeutic Target for the Prevention of Obliterative Airway Disease

The calcium-activated potassium channel KCa3.1 is critically involved in T-cell activation as well as in the proliferation of smooth muscle cells and fibroblasts. We sought to investigate whether KCa3.1 contributes to the pathogenesis of obliterative airway disease (OAD) and whether knockout or pharmacologic blockade would prevent the development of OAD. Tracheas from CBA donors were heterotopically transplanted into the omentum of C57Bl/6J wild-type or KCa3.1 mice. C57Bl/6J recipients were either left untreated or received the KCa3.1 blocker TRAM-34 (120 mg/kg/day). Histopathology and immunologic assays were performed on postoperative day 5 or 28. Subepithelial T-cell and macrophage infiltration on postoperative day 5, as seen in untreated allografts, was significantly reduced in the KCa3.1 and TRAM-34 groups. Also, systemic Th1 activation was significantly and Th2 mildly reduced by KCa3.1 knockout or blockade. After 28 days, luminal obliteration of tracheal allografts was reduced from 89%±21% in untreated recipients to 53%±26% (P=0.010) and 59%±33% (P=0.032) in KCa3.1 and TRAM-34-treated animals, respectively. The airway epithelium was mostly preserved in syngeneic grafts, mostly destroyed in the KCa3.1 and TRAM-34 groups, and absent in untreated allografts. Allografts triggered an antibody response in untreated recipients, which was significantly reduced in KCa3.1 animals. KCa3.1 was detected in T cells, airway epithelial cells, and myofibroblasts. TRAM-34 dose-dependently suppressed proliferation of wild-type C57B/6J splenocytes but did not show any effect on KCa3.1 splenocytes. Our findings suggest that KCa3.1 channels are involved in the pathogenesis of OAD and that KCa3.1 blockade holds promise to reduce OAD development.

Selected Commensals Mediate GvHD Via MyD88-Dependent Signaling in Non-Hematopoietic Cells.

Abstract 3005Graft-versus-host disease (GVHD) primarily affects the epithelial compartments of skin, liver, and gut and is thought to display tissue tropism for two main reasons: the conditioning regimen can selectively damage these epithelial cell compartments due to rapid cell turnover; and these epithelial organs have high exposure to host commensals and their products that promote tissue-specific inflammation. To understand how commensals mediate GVHD, we first tested the hypothesis that the disruption of the MyD88 signaling pathway, which is critical for host-microbe interaction, would alter GVHD outcomes. We created bone marrow chimeras that received a second allogeneic bone marrow transplant (BMT) and showed that MyD88 deficiency in recipient hematopoietic cells reduced BMT mortality, while deficiency in the non-hematopoietic cells (NHC) increased BMT mortality (p<0.05). These findings indicate the protective role of MyD88 expression in the latter compartment. Furthermore, deficiency of TLR6, but not TLR1,2,4, and 5, in NHC recapitulated similar BMT outcomes, suggesting regulation of MyD88 by TLR6 in our BMT model. Based on a hypothesis that MyD88 signaling on NHC promotes epithelial healing and barrier function, we first determined whether the level of bacterial translocation in MyD88KO BMT recipients are different. 16s DNA, a measurement of bacteria content, was significantly increased in livers of MyD88 knock-out (KO) transplant recipients compared to WT counterparts. We next decontaminated MyD88KO transplant recipients with a cocktail of broad spectrum antibiotics (Abx) prior to BMT and maintained these mice in sterile isolators. MyD88KO mice treated with Abx and untreated WT BMT recipients had similar survival; in contrast, untreated MyD88KO recipients died rapidly within the first 2 weeks following BMT (p<0.05). 16S DNA level was reduced in MyD88KO mice treated with Abx prior to BMT compared to their untreated MyD88KO counterpart. Furthermore, MyD88KO radiation controls treated with Abx had improved survival, similar to WT radiation controls, compared to untreated MyD88KO controls. These series of findings suggest a critical role of MyD88 in promoting barrier protection and healing via the NHC following BMT. Interestingly, similar Abx decontamination in WT BMT recipients did not change survival, suggesting that broad microbial decontamination in normal recipients without significant risk for bacterial translocation is not beneficial. Based on these findings, we next hypothesized that selective versus broad decontamination of commensal populations would improve GVHD outcomes following allogeneic BMT in WT mice. In MHC-matched and mismatched BMT murine models, we used different antibiotic cocktails to selectively remove subsets of commensals from recipients pre- and peri-BMT. Transplant recipients which received vancomycin (V) alone had significantly improved GVHD measures and survival compared to those that were administered more broad spectrum antibiotic cocktails (p <0.05). Flow cytometric analysis of the colon lamina propria and intraepithelial layer showed a significant increase of Foxp3-expressing lymphocytes in mice given V vs other groups, suggesting that the reduced GVHD in the former may be due to a higher regulatory cell population that suppresses local inflammation in the gut. In sum, our studies suggest a critical interaction between commensals and host cells via MyD88 signaling in mediating GVHD and BMT outcomes, and support consideration of selective versus broad microbial decontamination with BMT. Disclosures:No relevant conflicts of interest to declare.

Effect of CD8+ T Cell Depletion in Acute Lung Allograft Rejection in Mice

Background: Despite contemporary immunosuppression protocols, acute allograft rejection remains a major obstacle to successful lung transplantation. We and others previously showed that, unlike in other solid organs, acute allograft rejection of lungs is independent of CD4+ T cell allorecongnition pathways. We investigated the role of CD8+ T cells predominantly found in rejected lungs. Methods: Lung allografts from C57BL/6 mice were transplanted orthotopically into fully MHC-mismatched BALB/c (H-2d) recipients. Recipients received anti-CD4mAb or anti-CD8 mAb (100μg i.p., d-2, d-1, d0). Depletion was confirmed by FACS analysis of blood and spleen. In the syngeneic group, C57BL/6 grafts were transplanted into C57BL/6 recipients. Rejection was confirmed by direct visualization after thoracotomy. On day 7 post transplantation, grafts were examined by immunohistology and flow cytometry. Titers of immunoglobulin were measured by flow cytometry at different time-points in recipient groups. Results: In contrast to syngeneic grafts, which revealed normal appearance at day 7, allografts displayed severe acute rejection with extensive perivascular and interstitial infiltrates of mononuclear cells. CD4+ depleted recipients rejected their grafts at the same time as the untreated recipients. In the untreated recipients, the percentage of infiltration of CD8+ T cells was significantly superior to that of CD4+ T cells. However, CD8+ depleted recipients displayed no enhancement of graft survival compared to wild-type control recipients. Conclusion: This is the first report indicating that both CD4+ and CD8+T cells can independently reject lung allografts in mice.

No Inhibitory Effect of Pre-Transplant Desensitization Treatment with Rituximab or Splenectomy on de Novo HLA Antibody Production in Renal Transplantation

Introduction: De novo HLA antibody (Ab) production has been reported to be associated with chronic antibody mediated rejection (CAMR) in renal transplantation (RTx), resulting in poor graft outcome. B cell-targeted therapy has been suggested as a potential strategy. The effect of immunosuppressive treatment including pre-transplant splenectomy (SPX) or rituximab (RIT) on de novo HLA Ab production was examined. Methods: 337 living donor renal transplants were performed between 2005 and 2009. As pre-transplant DSA-positive recipients (n=17) were excluded, 320 transplants including 228 ABO-identical/compatible RTx (ABO-Id/C) and 92 ABO-incompatible RTx (ABO-In) were analyzed. Immunosuppressive regimen consisted of basiliximab, calcineurin inhibitor (CSA or TAC), antimetablite (MMF or mizoribine: MZR) and steroid. The dose was adjusted according to pharmacokinetics (AUC0-4 or 0-12). Recipients in ABO-In were additionally pre-treated with antimetablite (cyclophosphamide or MMF for 2W), plasmapheresis and splenectomy (SPX) or rituximab (RIT; from 2008) or none (because of low anti-A/B Ab titer). In 2010 and 2011, DSA was examined in 293 recipients using LABScreen single antigen beads (MFI>1000), after HLA Ab screening using FlowPRA or LABScreen Mixed. Results: Death-uncensored 5-year graft survivals were 98.1% in ABO-Id/C and 90.9% in ABO-In (Table). During the study period, only 2 developed CAMR. De novo HLA Ab including DSA (n=24) and NDSA (N=15) were detected in 39 (13.3%) recipients. Most of DSA were directed to class II (22/24), while a majority of NDSA were directed to class I (10/15). Rates of de novo HLA Ab were 13.9% in ABO-Id/C and 11.7% in ABO-In (14.3% in RIT-treated and 13.2% in post-SPX recipients). De novo HLA Abs were produced in 13.8% of CSA-, 12.0% of TAC-, 13.0% of MMF- and 19.0% of MZR-treated recipients. However, renal function remained stable even in recipients with de novo HLA Ab (eGFR: 47.2±16.3).Table: [Graft survival, de novo HLA antibody and eGFR]Conclusion: We obtained the excellent graft outcome in living donor RTx, whereas graft survival of ABO-In was somewhat lower than that of ABO-Id/C. The incidence of CAMR was very low within 5 years, although de novo HLA Ab was observed in SPX or RIT-pretreated recipients (ABO-In) as well as untreated recipients (ABO-Id/C). Neither pre-transplant SPX nor RIT treatment seemed to have an inhibitory effect on de novo HLA antibody production. Chronic AMR could be mainly controlled by optimizing immunosuppressive therapy based on pharmacokinetics and therapeutic drug monitoring. Careful, long-term follow-up would be essential.

Alloantigen-Driven Regulatory T Cells Are More Effective at Regulating Rejection in Immunocompetent Recipients than Freshly Isolated or Expanded nTreg

Introduction: Regulatory T cells (Treg) are being considered for evaluation as a cellular therapy in clinical transplantation and most suggested protocols will examine polyclonally-expanded naturally occurring populations (nTreg). Whilst both mouse and human nTreg can regulate rejection in adoptive transfer mouse models, it is less clear whether they have the same impact in immunocompetent recipients, a question of considerable relevance to the clinical situation. Using an approach that enriches for alloantigen-experienced Treg in vitro, we have been able to compare directly the effectiveness of freshly isolated nTreg, expanded nTreg and alloantigen-driven adaptive Treg in immunocompetent mouse heart allograft recipients. Methods: Recipient (CBA H-2k) splenic CD4+CD25+ nTreg were isolated by MACS (purity >95% CD25+) and were used either after isolation or expanded with plate-bound anti-CD3 and anti-CD28 antibody plus IL-2 for 5 days. Antigen-experienced Treg (IFN-γ Treg) were enriched in vitro by stimulating naïve CBA CD4+ T cells with modulated donor-strain (C57BL/6 H-2b) antigen-presenting cells in the presence of interferon-γ. All Treg populations were examined phenotypically for expression of FoxP3 and evaluated for their ability to control rejection of fully MHC-mismatched heterotopic cardiac allografts. Treg were given on day -1 i.v. together with co-stimulation blockade deliberately designed to be sub-optimal (CTLA-4 Ig, 25μg day 0, 1, 5), in order to reveal any Treg-dependent impact on graft outcome. Results: Untreated CBA recipients rejected C57BL/6 hearts acutely (median survival time, MST, 8 days n=10). Sub-optimal CTLA-4 Ig treatment prolonged graft survival to an MST of 50 days, (n=8). When given in combination with sub-optimal CTLA-4 Ig, neither 2×106 freshly isolated nTreg (90-95% FoxP3+), nor 2×106 expanded-nTreg (90% FoxP3+) prolonged graft survival long-term and in fact, freshly isolated nTreg appeared to partially over-ride the effect of CTLA-4 Ig (MST=24 days n=8; MST=40 days n=5, fresh and expanded nTreg respectively). In clear contrast, delivery of 2×106 IFN-γ Treg led to a median survival time of 95 days (n=6), with 50% graft survival >100 days. The effect was dose-dependent (MST=66 days n=4; MST=100 days n=3, 0.5×106 and 4×106 IFN-γ Treg, respectively) and occurred despite the fact that this non-selected population was only between 40-50% FoxP3+. Furthermore, preliminary results indicate that the optimal time of IFN-γ Treg delivery is before rather than after transplant (MST 100 days, n=3 vs MST 18 days, n=4; delivery on day -3 vs. day +3 respectively) suggesting that early homing to the graft may be critical determinant in Treg efficacy. Conclusions: Although initial attempts to utilise Treg cell therapy in clinical transplantation will focus predominantly on expanded nTreg, our data indicate that efforts to develop alloantigen-driven adaptive Treg should not be overlooked. Whilst the generation of alloantigen-driven Treg will be challenging in terms of logistics, the data suggest that such cells may provide the most effective populations for cellular therapy and may offer a route to drug-minimisation without compromising transplant outcome.

Evaluation of FR104, a Treg sparing antagonist anti-CD28 monovalent Fab’ antibody in kidney transplantation in non-human primates

Methods and results Here we evaluated in a non-human primates this “Treg sparing strategy” with FR104, a novel monovalent humanized and pegylated Fab’ anti-CD28 antibody fragment. PK/PD studies in monkeys revealed that FR104 presented an elimination half-life of 8 days and 100% target saturation over at least a month after a single iv injection of 5 mg/kg. FR104 was next evaluated in a baboon kidney allograft model at the dose of 5 mg/kg at day 0, 4, 14 and then every two-week until 3 months. Monotherapy modestly but significantly prolonged allograft survival (MST: 18.5 days for monotherapy vs 6 days for untreated recipients). FR104 synergized with low doses tacrolimus (lowTac, trough: 5-10 ng/ml; MST >100 days for FR104/ lowTac vs. 15 days for lowTac alone) as well as with calcineurin-free regimens: therapeutic doses of MMF or rapamycin (day 0-90) with 1 mg/kg of corticosteroids from day 0-14 (MST >100 days for FR104 + MMF/Rapa vs. 18/15 days for MMF/Rapa alone). Flow cytometry analyses indicated that blood Treg cells of the natural and inducible types were preserved in FR104/MMF or FR104/lowTAC bitherapies and accumulated in FR104 monotherapy and in FR104/Rapa bitherapy, whereas Treg cells were lowered by MMF and lowTac monotherapies. Histology also revealed that CTLA4+ and Foxp3+ T lymphocytes were accumulated into the graft of FR104 treated recipients.

Flt3L Combined with Rapamycin Promotes Cardiac Allograft Tolerance by Inducing Regulatory Dendritic Cells and Allograft Autophagy in Mice

The induction of immune tolerance is still a formidable challenge in organ transplantation. Dendritic cells (DCs) play an important role in orchestrating immune responses by either mediating protective immune responses or inducing antigen specific tolerance. Previous studies demonstrated that the fms-like tyrosine kinase 3 receptor (Flt3) and its ligand (Flt3L) play an essential role in the regulation of DC commitment and development. Here, we report a synergic effect between Flt3L and low-dose rapamycin (Rapa) in the protection of allograft rejction. It was found that Flt3L combined with Rapa significantly prolonged murine cardiac allograft survival time as compared with that of untreated recipients or recipients treated with Rapa or Flt3L alone. Mechanistic studies revealed that Flt3L combined with low-dose of Rapa induced the generation of tolerogenic DCs along with the production of CD25+ Foxp3+ regulatory T cells and IL-10 secretion. We also observed enhanced autophagy in the cardiac allograft, which could be another asset contributing to the enhanced allograft survival. All together, these data suggest that Flt3L combined with low-dose of Rapa could be an effective therapeutic approach to induce tolerance in clinical setting of transplantation.

Patterns of De Novo Allo B Cells and Antibody Formation in Chronic Cardiac Allograft Rejection After Alemtuzumab Treatment

Even though the etiology of chronic rejection (CR) is multifactorial, donor specific antibody (DSA) is considered to have a causal effect on CR development. Currently the antibody-mediated mechanisms during CR are poorly understood due to lack of proper animal models and tools. In a clinical setting, we previously demonstrated that induction therapy by lymphocyte depletion, using alemtuzumab (anti-human CD52), is associated with an increased incidence of serum alloantibody, C4d deposition and antibody-mediated rejection in human patients. In this study, the effects of T cell depletion in the development of antibody-mediated rejection were examined using human CD52 transgenic (CD52Tg) mice treated with alemtuzumab. Fully mismatched cardiac allografts were transplanted into alemtuzumab treated CD52Tg mice and showed no acute rejection while untreated recipients acutely rejected their grafts. However, approximately half of long-term recipients showed increased degree of vasculopathy, fibrosis and perivascular C3d depositions at posttransplant day 100. The development of CR correlated with DSA and C3d deposition in the graft. Using novel tracking tools to monitor donor-specific B cells, alloreactive B cells were shown to increase in accordance with DSA detection. The current animal model could provide a means of testing strategies to understand mechanisms and developing therapeutic approaches to prevent chronic rejection.

Auditory stimulation of opera music induced prolongation of murine cardiac allograft survival and maintained generation of regulatory CD4+CD25+ cells

BackgroundInteractions between the immune response and brain functions such as olfactory, auditory, and visual sensations are likely. This study investigated the effect of sounds on alloimmune responses in a murine model of cardiac allograft transplantation.MethodsNaïve CBA mice (H2k) underwent transplantation of a C57BL/6 (B6, H2b) heart and were exposed to one of three types of music--opera (La Traviata), classical (Mozart), and New Age (Enya)--or one of six different single sound frequencies, for 7 days. Additionally, we prepared two groups of CBA recipients with tympanic membrane perforation exposed to opera for 7 days and CBA recipients exposed to opera for 7 days before transplantation (pre-treatment). An adoptive transfer study was performed to determine whether regulatory cells were generated in allograft recipients. Immunohistochemical, cell-proliferation, cytokine, and flow cytometry assessments were also performed.ResultsCBA recipients of a B6 cardiac graft that were exposed to opera music and Mozart had significantly prolonged allograft survival (median survival times [MSTs], 26.5 and 20 days, respectively), whereas those exposed to a single sound frequency (100, 500, 1000, 5000, 10,000, or 20,000 Hz) or Enya did not (MSTs, 7.5, 8, 9, 8, 7.5, 8.5 and 11 days, respectively). Untreated, CBA mice with tympanic membrane perforations and CBA recipients exposed to opera for 7 days before transplantation (pre-treatment) rejected B6 cardiac grafts acutely (MSTs, 7, 8 and 8 days, respectively). Adoptive transfer of whole splenocytes, CD4+ cells, or CD4+CD25+ cells from opera-exposed primary allograft recipients resulted in significantly prolonged allograft survival in naive secondary recipients (MSTs, 36, 68, and > 100 days, respectively). Proliferation of splenocytes, interleukin (IL)-2 and interferon (IFN)-γ production was suppressed in opera-exposed mice, and production of IL-4 and IL-10 from opera-exposed transplant recipients increased compared to that from splenocytes of untreated recipients. Flow cytometry studies showed an increased CD4+CD25+ Forkhead box P3 (Foxp3)+ cell population in splenocytes from those mice.ConclusionOur findings indicate that exposure to opera music, such as La traviata, could affect such aspects of the peripheral immune response as generation of regulatory CD4+CD25+ cells and up-regulation of anti-inflammatory cytokines, resulting in prolonged allograft survival.

Effects of an agonist interleukin-2/Fc fusion protein, a mutant antagonist interleukin-15/Fc fusion protein, and sirolimus on cardiac allograft survival in non-human primates

To tilt the immunologic balance toward tolerance and away from rejection, non-human primate recipients of cardiac allografts were treated with interleukin (IL)-2/Fc, mutant (m) antagonist type mIL-15/Fc, and sirolimus. Heterotopic heart transplants were performed on 8 fully mismatched cynomolgus macaques. An untreated control recipient rejected its graft by post-operative Day 6. The remaining 7 animals received oral or intramuscular immunosuppression with sirolimus. A recipient treated with sirolimus alone rejected at the end of 28 days of immunosuppression. The remaining 6 monkeys also received IL-2/Fc and mIL-15/Fc intramuscularly until 28 days after transplant. One animal received a second 28-day course of fusion protein starting at Day 50. In these 6 animals, sirolimus was continued for 28 days (n = 4) or until protein levels were low (n = 2). In the 4 monkeys treated with a 28-day course of sirolimus and fusion proteins, mean graft survival was 51.5 days (range, 28-76 days). The animal receiving a second course of fusion protein rejected its graft on Day 177, despite detectable levels of the fusion proteins and sirolimus. The central memory, effector memory, and naïve CD4(+) and CD8(+) T-cell populations in the peripheral blood did not change significantly during fusion protein administration. A 2.5-fold expansion in CD4(+)CD25(+) lymphocytes occurred in recipients treated with fusion proteins and sirolimus that was not observed in the recipient treated with sirolimus alone. Although IL-2/Fc, mIL-15/Fc, and sirolimus administered in this manner permitted modest prolongation of graft survival and expansion of CD4(+)CD25(+) T cells, tolerance was not achieved.

Intratumoral IL-12 Gene Therapy Results in the Crosspriming of Tc1 Cells Reactive Against Tumor-associated Stromal Antigens

HLA-A2 transgenic mice bearing established HLA-A2(neg) B16 melanomas were effectively treated by intratumoral (i.t.) injection of syngeneic dendritic cells (DCs) transduced to express high levels of interleukin (IL)-12, resulting in CD8(+) T cell-dependent antitumor protection. In this model, HLA-A2-restricted CD8(+) T cells do not directly recognize tumor cells and therapeutic benefit was associated with the crosspriming of HLA-A2-restricted type-1 CD8(+) T cells reactive against antigens expressed by stromal cells [i.e., pericytes and vascular endothelial cells (VEC)]. IL-12 gene therapy-induced CD8(+) T cells directly recognized HLA-A2(+) pericytes and VEC flow-sorted from B16 tumor lesions based on interferon (IFN)-γ secretion and translocation of the lytic granule-associated molecule CD107 to the T cell surface after coculture with these target cells. In contrast, these CD8(+) T effector cells failed to recognize pericytes/VEC isolated from the kidneys of tumor-bearing HHD mice. The tumor-associated stromal antigen (TASA)-derived peptides studied are evolutionarily conserved and could be recognized by CD8(+) T cells harvested from the blood of HLA-A2(+) normal donors or melanoma patients after in vitro stimulation. These TASA and their derivative peptides may prove useful in vaccine formulations against solid cancers, as well as, in the immune monitoring of HLA-A2(+) cancer patients receiving therapeutic interventions, such as IL-12 gene therapy.

Establishment of a Chronic Rejection Model for Orthotopic Liver Transplantation in the Rat

Background: This study aims to establish a stable chronic rejection model for orthotopic liver transplantation in the rat and to describe the pathological features of this model. Methods: The livers from different strains of rats were transplanted in various allogeneic donor-recipient combinations with appropriate syngeneic grafts used as controls. The rats were untreated after surgery (acute rejection model) or treated with cyclosporin A (1 mg/kg) and hydrocortisone (0.75 mg/kg) to establish chronic rejection as determined by rejection activity index scores from pathological examination of liver specimens. Results: Acute rejection occurred in all of the untreated recipients of allogeneic orthotopic liver transplants, and all died within 30 days. Among the treated recipients, the combinations of Lewis with brown Norway and dark agouti with Lewis rats developed acute rejection, and no changes characteristic of chronic rejection were observed in the few rats that survived beyond 30 days. In contrast, the treated Lewis recipients of livers from Sprague-Dawley rats exhibited chronic rejection in the liver specimens. Conclusion: We have established an animal model for chronic rejection after transplantation of livers from Sprague-Dawley to Lewis rats under short-term immunosuppression induced by small doses of cyclosporin A and hydrocortisone.