Prolonged Graft Survival Research Articles

An approach to induction of tolerance to pig cardiac xenografts in neonates.

There is a continuing need for donor hearts for infants with complex congenital heart defects. The transplantation of hearts from neonatal pigs would be an alternative to human organs, particularly if donor-specific immunological tolerance could be achieved. The great majority of infant humans do not make natural (preformed) antibodies against triple-knockout (TKO) pigs (that do not express any of the three known pig antigens against which humans have natural anti-pig antibodies). The transplantation of a heart from a TKO pig into an infant would therefore minimize any risk of early antibody-mediated rejection, and, with adequate immunosuppressive therapy, prolonged graft survival may well be achieved. Total host thymectomy (commonly carried out at the time of orthotopic heart transplantation in this age group)±residual T-cell depletion and donor-specific pig thymus tissue transplantation might induce T-cell tolerance and allow immunosuppressive therapy to be discontinued (if there is in vitro evidence of T-cell and B-cell nonresponsiveness to donor-specific pig cells). Even if tolerance were not achieved, with continuing immunosuppressive therapy, the graft would likely "bridge" the patient until a suitable allograft became available or be associated with prolonged xenograft function.

Advances in T follicular helper and T follicular regulatory cells in transplantation immunity.

B cells play a crucial role in alloreactivity of organ transplant rejection and graft versus host diseases (GVHD). Over the past decade, it has been well recognized that B-cell infiltration in allografts and de novo donor-specific antibodies (DSA) were strongly associated with severe graft rejection and loss, as well as glucocorticoid resistance. Emerging evidence has demonstrated that Follicular T helper (Tfh) cells are key effectors to promote the proliferation and differentiation of B cells into antibody-producing plasmablasts and memory B cells. T-follicular regulatory (Tfr) cells are a recently recognized cell population that has a negative regulatory role on Tfh cells in the follicle, preventing incessant antibody production. It is still less clear how those humoral immunoreactive cells affect transplant rejection and allograft loss. This review focuses on the production and function of Tfr/Tfh cells in the transplant environment. Better understanding of the functions and mechanisms of Tfr/Tfh cells will help to design new strategies to prevent allograft rejection and prolong graft survival.

Should We Revisit HLA Matching to Improve Long-Term Graft Outcomes?

We present here an overview of the needs and requirements for human leukocyte antigen (HLA) matching, explain and explore the potential benefits of epitope matching, and discuss the benefits of allele-level high-resolution matching at all HLA loci, i.e. HLA-A, -B, -DR, -DQ and -DP, for donor selection needs during renal transplantation. Donor recipient HLA matching has a substantial impact on organ transplantation by prolonging graft survival, minimizing rejection episodes, preventing sensitization, providing a better chance for retransplantation and reducing the percentage of deaths due to graft dysfunction. Despite these advantages, the utility of HLA matching has been challenged, mainly because of the availability of highly effective immunosuppressive agents clubbed with steady improvements in short-term graft survival. Moreover, the risk associated with prolonged periods on dialysis while waiting for an optimally matched donor is minimized. There is a need to strike a balance between the benefits of HLA matching and problems associated with finding such a donor. A more viable alternative would be to find a donor with mismatches having a low probability of inducing humoral responses.

Administration of donor splenocytes via the respiratory tract generates CD8α+ regulatory dendritic cells and induces hyporesponsiveness to fully allogeneic cardiac grafts

BackgroundWe previously showed that pretreatment with intratracheal delivery (ITD) of alloantigen induced prolonged cardiac allograft survival and generated regulatory T cells (Tregs) in mice. In this study, we examined the role of splenic dendritic cells (DCs) in the ITD model. MethodsCBA mice were treated with ITD from C57BL/10 splenocytes and 7 days later received transplantation of C57BL/10 hearts. In adoptive transfer studies, splenic DCs from ITD-treated mice were transferred into naïve CBA recipients that received C57BL/10 hearts immediately after the transfer. In addition, to determine the role of splenic DCs isolated from ITD-treated mice, the cells were incubated under stimulation with lipopolysaccharide (LPS). ResultsITD-treated CBA recipients had markedly prolonged allograft survival (median survival time [MST], 67 days) while naïve recipients rejected allografts acutely (MST, 8 days). In adoptive transfer studies, CBA recipients of the transfer of splenic DCs from ITD-treated mice had prolonged allograft survival (MST, 85 days), while CBA recipients of the transfer of splenic DCs from naïve mice did not have prolonged allograft survival (MST, 8 days). In another transfer study, CBA recipients of the transfer of splenic CD8α+ DCs from ITD-treated mice had prolonged allograft survival (MST, 79 days), while those receiving splenic CD8α− DCs from ITD-treated mice did not have prolonged allograft survival (MST, 8 days). In vitro studies showed that ITD-treated splenic DCs produced more IL-10 and less IL-12 than naïve splenic DCs under stimulation with LPS. ConclusionsITD pretreatment induces regulatory DCs, which produce high amounts of IL-10 resulting in the prolongation of graft survival in our model.

Mouse Model Established by Early Renal Transplantation After Skin Allograft Sensitization Mimics Clinical Antibody-Mediated Rejection.

Antibody-mediated rejection (AMR) is the main barrier to renal graft survival, and mouse renal AMR models are important to study this process. Current mouse models are established by priming the recipient to donor skin for over 7 days before kidney transplantation. The robustness of AMR in these cases is too strong to mimic clinical AMR and it is unclear why altering the priming times ranging from 7 to 91 days fails to reduce the AMR potency in these models. In the present study, we found that the donor-recipient combination and skin graft size were determinants of donor-specific antibody (DSA) development patterns after skin transplantation. DSA-IgG was sustained for over 100 days after skin challenge, accounting for an identical AMR robustness upon different skin priming times over 7 days. However, decreasing the skin priming time within 7 days attenuated the robustness of subsequent renal allograft AMR in C3H to Balb/c mice. Four-day skin priming guaranteed that recipients develop acute renal AMR mixed with a high ratio of graft-infiltrating macrophages, renal grafts survived for a mean of 6.4 ± 2.1 days, characterized by typical AMR histological changes, such as glomerulitis, peritubular capillary (PTC) dilation, and capillaritis, deposition of IgG and C3d in PTCs, but less prevalence of microthrombus, whereas the cellular rejection histological change of tubulitis was absent to mild. With this scheme, we also found that the renal AMR model can be developed using common mouse strains such as C57BL/6 and Balb/c, with mean prolonged renal graft survival times of 14.4 ± 5.0 days. Finally, we proved that donor-matched skin challenge after kidney transplantation did not strongly affect DSA development and kidney graft outcome. These findings may facilitate an understanding and establishment of mouse renal allograft AMR models and promote AMR-associated studies.

Costimulatory Blockade with CTLA4-Ig Abrogates Prolonged Graft Survival in Old Recipients

Introduction Although they represent a rapidly growing population, elderly organ transplant recipients are underrepresented in clinical trials. Age-specific aspects of established immunosuppressants are therefore poorly understood. Methods Here, we assessed the impact of immunosuppressive treatment with CTLA4-Ig, a fusion protein blocking costimulatory signaling between APCs and T cells through CD28, on alloimmune responses in old and young recipients (2-3 months vs. 16 months) in a fully MHC-mismatched murine transplantation models. Results While treatment with CTLA4-Ig prolonged skin graft survival in young recipients, the same treatment was unable to prolong graft survival in old recipients. Conversely, cardiac allografts in young mice treated with CTLA4-Ig survived indefinitely, while 80% of old recipients treated with CTLA4-Ig had lost their graft after 100 days (log-rank test, p<0.001; n=5/group; Fig. 1).{{AbstractFigure.1}} CTLA4-Ig reduced the in-vivo proliferation of CD4+ and CD8+ T cells (as assessed by BrdU incorporation) uniformly in both young and old recipients; in contrast, CTLA4-Ig reduced CD4+ central-memory and effector-memory T cells only in young but not old recipients. Moreover, systemic frequencies of CD4+IFN-γ+ T cells and systemic levels of IFN-γ cytokine production were reduced in young recipients, but remained unchanged in old. CTLA4-Ig caused significant perturbation in the Treg compartments with reduced frequencies and compromised proliferation of CD4+CD25+Foxp3+ cells. These differences correlated with a significant reduction in expression of CD28 on T cells in old mice, while levels of CTLA4 remained stable.Conclusion Immunosuppressive effects of costimulatory blockade with CTLA4-Ig showed distinct age-dependent effects in both skin and cardiac transplantation. Reduced expression of CD28 with aging may represent an escape mechanism for old alloreactive T cells, with unique clinical consequences for immunosuppression in the growing population of elderly transplant recipients.

Tofacitinib Halts Progression of Graft Dysfunction in a Rat Model of Mixed Cellular and Humoral Rejection.

The progression from acute to chronic antibody-mediated rejection in kidney transplant recipients is usually not prevented by current therapeutic options. Here, we investigated whether the use of tofacitinib (TOFA), a Janus kinase 3 inhibitor, was capable of preventing the progression of allograft dysfunction in a Fisher-to-Lewis rat model of kidney transplantation. Rats were treated from the third week after transplantation to allow the development of rejection. Treatment was based on cyclosporin A, rapamycin or TOFA. Renal function was assessed at 1, 4, 8, and 12 weeks after transplantation, whereas rat survival, histological lesions, and infiltrating lymphocytes were analyzed at 12 weeks. Tofacitinib prolonged graft survival, preserved tubular and glomerular structures and reduced humoral damage characterized by C4d deposition. Tofacitinib was able to reduce donor-specific antibodies. In addition, T and natural killer cell graft infiltration was reduced in TOFA-treated rats. Although rapamycin-treated rats also showed prolonged graft survival, glomerular structures were more affected. Moreover, only TOFA treatment reduced the presence of T, B and natural killer cells in splenic parenchyma. Tofacitinib is able to reduce the immune response generated in a rat model of kidney graft rejection, providing prolonged graft and recipient survival, better graft function, and less histological lesions.

A Simple and Promising Strategy for Pancreatic Islet Preconditioning to Reduce Immunogenicity and Improve Islet Graft Longevity

Introduction An advantage of pancreatic islet transplantation is that the pretransplant in vitro culture period provides an opportunity for cell modification; however, no “islet preconditioning” method has been established yet. We previously reported that the pretreatment of donor islets with an antitumoral antibiotic, mitomycin-C (MMC), and subsequent culture significantly prolonged graft survival in murine renal subcapsular islet transplantations. Here, we introduce an “islet preconditioning” strategy for reducing the immunogenicity of islets and for preventing the activation of proinflammatory events by using MMC in an intraportal islet transplantation model. Methods Freshly isolated BALB/c islets, either untreated or treated for 30 min with 10 μg/mL MMC, were cultured for 20 hours. Subsequently, 600 allogenic islets were transplanted via the portal vein into the liver of C57BL/6N diabetic mice. Recipients did not receive any immunosuppressive treatment. To demonstrate the specific mechanism responsible for prolonging graft survival by islet preconditioning, immunohistochemical studies and inflammatory cytokine assays were conducted. Microarrays were employed to comprehensively analyze the genes expressed in untreated or MMC-treated islets that were subsequently cultured and to identify the biological processes that were most affected by islet preconditioning. Results On islet transplantation, the allograft was rejected by all control mice (n = 11), with a mean survival time (MST) of 13.0 ± 8.3 days. Islet conditioning with MMC significantly prolonged islet allograft survival to an MST of 41.4 ± 7.3 days (n = 14) (P < 0.005). Notably, 42.8% of the recipients accepted islet allografts pretreated with MMC for more than 60 days without any immunosuppression. As shown by immunohistochemical studies, compared with untreated grafts, MMC-treated grafts showed a mild inflammatory cell response and minimal infiltration of immunocompetent cells. In an in vitro study, the expression of IL-6, MCP-1, ICAM-1, and IL-1β was significantly decreased in MMC-treated islets compared with untreated islets. Microarray gene expression analysis identified a significant downregulation of annotated functions associated with the movement of cells and revealed significant downregulation of multiple genes encoding proinflammatory mediators with chemotactic activity. Validation studies also showed that MMC-treated islets secreted lower levels of chemoattractants that reduced the immunogenic potential of islets. Conclusion Islet preconditioning with MMC and subsequent culture can prolong graft survival in intraportal islet transplantations by inhibiting the activation of proinflammatory events and by provoking the suppression of chemoattractants that are responsible for inducing unresponsiveness to islet grafts. This simple and promising islet preconditioning method could be an effective strategy inducing a breakthrough in establishing advanced islet transplantation.

Tomotherapy and Hematopoietic Stem Cells for Tolerance to Kidney Transplants in Rhesus Macaques

NIH NHP Transplantation Tolerance Cooperative Study Group. The goal of this project was to develop a tolerance induction protocol for kidney transplantation (tx) in a rhesus macaque model that would permit complete elimination of all immunosuppression (IS). We hypothesized that tolerance to MHC 1-haplotype mismatched kidney tx between living related donor/recipient pairs can be achieved by establishing a mixed chimeric state. The chimeric state would be achieved using a newly established post-kidney tx, non-myeloablative, helical tomotherapy-based total lymphoid irradiation (TLI)-based conditioning regimen followed by donor mobilized peripheral blood-CD34+ hematopoietic stem cell (HSC)/T cell (CD3+) infusions (Figure 1). The hypotheses was tested in a series of 5 HSC-only txs followed by 18 kidney txs with (n=11) and without (n=7) donor HSC infusions. The kidney allografts came from blood group compatible, T and B cell flow cytometric crossmatch negative donors. All recipient animals underwent IS withdrawal according to the same schedule and were followed for rejection. We determined the proportion of recipients that achieved chimerism and that could be withdrawn from all IS for greater than 3 years, while maintaining normal allograft function. Mixed chimerism in the recipient was assessed by quantifying the proportion and duration of donor/recipient DNA, using STR, in various immune cellular lineages circulating within the peripheral blood post-tx. Results are shown in There were 11 kidney+HSC txs. Two achieved transient multilineage chimerism for at least 3 months and both were long-term tolerant survivors (still alive and off IS for more than 3 years). Nine animals in the kidney+HSC group did not achieve any evidence of chimerism – all lost the kidney tx before or during the IS weaning (5 due to rejection, 4 with engraftment syndrome, 1 each from infection and PTLD). All animals in the kidney tx-alone control group lost kidney function, with one having prolonged graft survival without IS, but eventually succumbed to chronic antibody mediated rejection. Furthermore, we assessed the correlation between the presence (n=5, including 3 with HSC-only infusions) or absence (n=8, including 2 with HSC-only infusions and excluding the 4 engraftment syndrome cases) of achieving chimerism and the development of de novo donor specific antibody (DSA) (Figure 3). DSA was absent in all 5 animals that achieved chimerism. Development of early de novo DSA, primarily against Class II MHC, was associated with lack of development of mixed chimerism, and therefore, inability to successfully eliminate IS. The study demonstrated that this TLI-based tolerance induction protocol can result in chimerism and operational tolerance in the rhesus macaque model across a 1-haplo mismatch. However, challenges remain to enhance the frequency of achieving chimerism. Future studies to extend the protocol to animals that are more MHC disparate will include modifications of the conditioning regimen to minimize development of DSA and engraftment syndrome. Funding supported by NIH NIAID.

Immunomodulation with SA-FasL-Engineered Microgels Achieves Long-Term Survival of Allogeneic Islet Grafts

Introduction and Objective Allogenic islet transplantation has shown efficacy in the clinic for the treatment of type 1 diabetes. However, sustained survival of allogeneic islet grafts requires chronic immunosuppression that has significant adverse effects. T effector (Teff) cells recognizing and responding to allograft antigens are the major culprit of graft rejection. Upon activation, Teff cells upregulate Fas death receptor on their surface and become sensitive to FasL-mediated apoptosis. Fas pathway, therefore, presents an important target for immunomodulation to block alloreactive responses with significant therapeutic potential. Herein, we assessed the efficacy of PEG microgels engineered with a novel form of FasL chimeric with a modified form of core streptavidin, SA-FasL, in achieving sustained survival of allogeneic islet grafts in the absence of chronic immunosuppression. Materials and Methods Microgels presenting biotin on their surface were produced by reacting biotin-PEG-thiol with a maleimide-terminated 4-arm poly(ethylene) glycol macromer, and generating 200 μm diameter microgels crosslinked with dithiothreitol. Biotinylated microgels were engineered with SA-FasL (1 μg/103 microgels) taking the advantage of the high affinity interaction between biotin and streptavidin. The apoptotic activity of SA-FasL-engineered microgels was tested on mouse A20 B cell lymphocytes in vitro. The immunomodulatory function of microgels for the prevention of graft rejection was tested by mixing ~500 BALB/c naïve islets with 1000 SA-FasL-engineered microgels and transplanting under the kidney capsule of streptozotocin-induced diabetic C57BL/6 recipients. A group of recipients were also treated transiently with rapamycin (0.2 mg/kg daily for 15 days starting the day of transplantation). Results and Discussion Biotin-PEG microgels were efficiently coupled with SA-FasL, and showed a dose-dependent apoptotic activity in A20 cells. Co-transplantation of SA-FasL-engineered microgels with naïve islets led to prolonged survival of grafts as compared with the control group (islets + unmodified microgels) with 20% surviving for a 200-day observation period. Transient use of low dose rapamycin, enhanced survival to > 90% of the grafts. In marked contrast, only 20% of islets co-transplanted with PEG microgels, and short-course rapamycin, survived long-term. Importantly, CD4+CD25+FoxP3+ Treg cells were required for graft survival as depletion of this cell population on day 50 post-transplantation resulted in prompt rejection. Conclusion These results provide strong proof-of-efficacy and feasibility for the use of SA-FasL-engineered microgels as an off-the-shelf product for the modulation of alloreactive responses with significant therapeutic potential. Funded in part by NIH (grants R21EB020107, R21AI113348, 185 R56AI121281, and F30AR069472) and the Juvenile Diabetes Research Foundation (2-SRA-186 2014-287-Q-R).

Ex vivo Expanded Regulatory T cells Combined with Short-term Costimulation Blockade Prevent Rejection of Vascularized Composite Allografts

Background Reconstructive transplantation represents a valid therapeutic option after devastating tissue loss. Routine clinical application, however, is hampered by the toxicity of long-term maintenance immunosuppression. The current study investigated a novel approach using ex vivo expanded regulatory T cells combined with a short-term immunomodulatory strategy in a murine hind limb transplantation model. Methods Fully MHC-mismatched orthotopic hind limb transplants were performed from Balb/C to C57BL/6 mice. Recipients in the experimental groups received a combination regimen consisting of 0.5mg CTLA4 Ig on day 0, 2, 4 and 6 post-transplant, 20mg/kg anti-Thy 1.2 mAb on POD-1, and 1mg/kg Rapamycin (POD 0-9), and in one group, 1 wk expanded CD4+CD25+ Treg cells. Allograft survival was monitored and flow cytometric analysis was performed to evaluate mixed chimerism and clonal deletion of alloreactive T cells. Treg activity was assessed in vitro using suppression assays in order to support and supplement in vivo data. Results Combination of T cell depletion and CTLA4-Ig plus short-course of Rapamycin increased VCA survival significantly while untreated controls rejected allografts (MST 105 days; Untreated, MST 9 days; CTLA4 Ig only, MST 17 days, Rapamycin, MST 20 days; T cell depletion, 20 days; p<0.01[GB1]). [GB2] Mixed chimerism was detected in recipients receiving this combined treatment protocol with 5.013 ± 1.23 % of CD11b+ cells being donor-derived on POD 55. Vβ – TCR staining profiles in recipients after full treatment showed 1.570 ± 0.3700 % of νβ5+CD4+ T cells, while naïve C57BL/6 express 3.567 ± 0.3690 % of νβ5+CD4+ T cells, suggesting the actuation of central deletion of developing donor-reactive T cells. In order to further prolong allograft survival, one week expanded Tregs were then included in the combination therapy. The suppressive activity of the CD4+CD25+ Tregs was confirmed with in vitro suppression assays. The addition of ex vivo expanded regulatory T cells further increased VCA survival to >200 days and induced long-term stable mixed chimerism[GB3] with 16.7±1.5 % of CD11b cells being donor-derived on POD 55 after administration of expanded Treg cells. Conclusion The combination of T cell depletion, costimulation blockade, and a short-course of Rapamycin prevents VCA rejection and significantly prolongs graft survival without the need for myeloablative conditioning or maintenance therapy. Moreover, regulatory T cells added in the early post transplant period further optimize immune regulation by inducing sustained mixed chimerism.

Mechanisms of Vascularized Bone Marrow Graft Protection of Vascularized Composite Allografts

Background Co-transplantation of vascularized bone marrow (VBM) promotes vascularized composite allograft (VCA) survival with standard immunosuppression in a non-human primate (NHP) model. We investigated mechanisms of immunomodulation by comparing VCA survival following donor bone marrow cell (BMC) infusion, donor and recipient conditioning strategies, and heterotopic VBM allografts to historical groups. Methods Groups of 3–4 NHP received facial subunit VCA and immunosuppression of tacrolimus (Tac) and mycophenolate mofetil (MMF). Comparative groups, VCA+VBM (n=4) and VCA (no VBM, n=3) were from historical cohorts (Group 1 and 2, respectively). Group 3 received VCA without VBM and transfused donor BMC at 20x 106 cells/kg on d 1. Group 4 received VCA+VBM following donor mandible irradiation (1.5 Gy) on d −1, while Group 5 received irradiated donor mandible followed by BMC transfusion on d 1. Group 6 underwent co-transplantation of heterotopic VBM and VCA without VBM. Group 7 received VCA+VBM after donor T cell depletion with anti-thymocyte globulin (ATGAM, 50 mg/kg) on days −2, and −1. An additional dose of ATGAM was given on d 0 if CD3+ cells remained >50/mm3. Group 8 recipients received ATGAM depletion as above, VCA+VBM, and a 21-d steroid taper. Banff classification was determined by histopathologic analysis. Chimerism was assessed by flow cytometry from peripheral blood and sampled tissues. Results VCA + VBM treated with Tac/MMF demonstrated prolonged graft survival and rejected only after immunosuppression was withdrawn compared to Groups 2–8 (mean survival 348d vs 36d). This effect was statistically significant (p=0.05). Endpoints were met secondary to Banff IV rejection and malignancy. Macrochimerism was present only in Group 1; remaining groups demonstrated only transient microchimerism. Discussion The immunologic advantage of VBM prolonging VCA graft survival is contingent on proximity of the VBM to the VCA allograft. Donor conditioning nullifies this benefit and it is not reconstituted by transfusion of donor BMC. Recipient T cell depletion mirrors the early graft loss observed with donor T cell depletion. Conclusion Our data elucidate a mechanism for VCA graft survival that is mediated by locally acting, radiosensitive T cell populations in both donor and recipient and is dependent on the unique immunologic and structural niche of VBM. Astellas Pharma, Inc.

Differential Impact of Autophagy on Innate and Adaptive Immune Cells Characterizes Alloimmune Responses in Aging

Introduction Aging and chronic antigen exposure shape the balance of innate and adaptive immunity. We hypothesized that autophagy will impact the homeostasis of innate and adaptive alloimmune responses in an age-dependent fashion. Methods A mouse model of sequential donor-specific and third party skin grafts was used to dissect innate and adaptive alloimmunity. Results Young and old C57BL/6 recipient mice (2 and 18 mths, respectively) were sensitized with fully mismatched skin grafts from young DBA/2 mice. Graft survival following this set of transplants confirmed prolonged graft survival in old recipients (p=0.0195). Three weeks after the initial transplant, recipient mice were challenged with second skin grafts of either donor (DBA/2) or third-party (CBA/J) origin. Secondary donor-specific grafts accelerated rejection in both groups regardless of recipient age (p=0.0304). Notably, secondary third party grafts were rejected in a dramatically accelerated fashion in old but not young recipients (p=0.0198). Next, we transplanted an additional set of skin transplant recipients three weeks after the primary graft placement with a secondary F1 (C57BL/6-CBA/J) graft, presenting both ‘self’ and ‘non-self’ antigens. Graft survival of secondary F1 grafts was prolonged in in old recipients, supporting the relevance of innate over adaptive immunity with aging. As autophagy has been shown to be compromised in aging, we next probed the expression of major genes of autophagy induction (atg5, atg7, becn1, map1lc3a) in innate and adaptive immune cells. Intriguingly, autophagy levels mimicked the altered balance of innate and adaptive immunity detected in our skin graft experiments: while isolated old CD4+ T cells showed significantly lower baseline levels compared to young cells, innate immune cells displayed either age-independent (natural killer cells) or even increased levels in old monocytes. Conclusion Collectively, our results highlight a predominance of innate immune responses during an aging alloimmune response with a specific autophagy pattern, unraveling a new conceptual approach in understanding age-dependent graft survival and immunosuppression.

The effects of local administration of mesenchymal stem cells on rat corneal allograft rejection

BackgroundMesenchymal stem cells (MSCs) have been reported to promote long-term cellular and organ transplant acceptance due to their immunotherapeutic characteristics. Previous work from our lab using a rat allograft model has shown that systemic infusion of MSCs inhibited corneal allograft rejection and prolonged graft survival. Here, we further investigated the effects of local MSCs administration in the same animal model.MethodsDonor-derived MSCs were isolated and cultured while corneal grafts obtained from Wistar rats were transplanted into Lewis rat hosts. Hosts were then randomly separated into four groups and treated with previously cultured MSCs at different times and doses. Graft survival was clinically assessed using slit-lamp biomicroscopy and the median survival time (MST) was calculated. Grafts were examined histologically using hematoxylin-eosin (H-E) staining and immunohistochemically using antibodies against CD4. A comprehensive graft analysis of IL-2, IL-4, IL-10, and IFN-γ expression was also conducted using both real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).ResultsPostoperative MSCs injection prolonged graft survival time when compared with controls (MST 9.8 ± 1.2 days). Injection twice of MSCs (MST 12.6 ± 1.4 days) was more effective than a single injection (MST 10.8 ± 1.3 days). MSCs-treated groups also showed suppression of inflammatory cell as well as CD4 + T cell infiltration in the allograft region. IL-4 and IL-10 levels were significantly increased in grafts obtained from postoperative twice MSCs-treated rats when compared with controls. There were no significant differences in IL-2 or IFN-γ expression across groups.ConclusionsSubconjunctival injection of MSCs in rats was effective in prolonging corneal allograft survival. This effect was mediated by inhibition of inflammatory and immune responses, indicating an anti-inflammatory shift in the balance of T helper (Th)1 to T helper(Th) 2.

Local immunomodulation Fas ligand-engineered biomaterials achieves allogeneic islet graft acceptance.

Islet transplantation is a promising therapy for type 1 diabetes. However, chronic immunosuppression to control rejection of allogeneic islets induces morbidities and impairs islet function. T-effector cells are responsible for islet allograft rejection and express Fas death receptor following activation, becoming sensitive to Fas-mediated apoptosis. Here, we report that localized immunomodulation using microgels presenting an apoptotic form of Fas ligand (SA-FasL) results in prolonged survival of allogeneic islet grafts in diabetic mice. A short course of rapamycin treatment boosted the immunomodulatory efficacy of SA-FasL-microgels, resulting in acceptance and function of allografts over 200 days. Survivors generated normal systemic responses to donor antigens, implying immune privilege of the graft, and had increased CD4+CD25+FoxP3+ T-regulatory cells in the graft and draining lymph nodes. Deletion of T-regulatory cells resulted in acute rejection of established islet allografts. This localized immunomodulatory biomaterial-enabled approach may provide an alternative to chronic immunosuppression for clinical islet transplantation.

Gut microbes contribute to variation in solid organ transplant outcomes in mice

BackgroundSolid organ transplant recipients show heterogeneity in the occurrence and timing of acute rejection episodes. Understanding the factors responsible for such variability in patient outcomes may lead to improved diagnostic and therapeutic approaches. Rejection kinetics of transplanted organs mainly depends on the extent of genetic disparities between donor and recipient, but a role for environmental factors is emerging. We have recently shown that major alterations of the microbiota following broad-spectrum antibiotics, or use of germ-free animals, promoted longer skin graft survival in mice. Here, we tested whether spontaneous differences in microbial colonization between genetically similar individuals can contribute to variability in graft rejection kinetics.ResultsWe compared rejection kinetics of minor mismatched skin grafts in C57BL/6 mice from Jackson Laboratory (Jax) and Taconic Farms (Tac), genetically similar animals colonized by different commensal microbes. Female Tac mice rejected skin grafts from vendor-matched males more quickly than Jax mice. We observed prolonged graft survival in Tac mice when they were exposed to Jax mice microbiome through co-housing or fecal microbiota transplantation (FMT) by gastric gavage. In contrast, exposure to Tac mice did not change graft rejection kinetics in Jax mice, suggesting a dominant suppressive effect of Jax microbiota. High-throughput sequencing of 16S rRNA gene amplicons from Jax and Tac mice fecal samples confirmed a convergence of microbiota composition after cohousing or fecal transfer. Our analysis of amplicon data associated members of a single bacterial genus, Alistipes, with prolonged graft survival. Consistent with this finding, members of the genus Alistipes were absent in a separate Tac cohort, in which fecal transfer from Jax mice failed to prolong graft survival.ConclusionsThese results demonstrate that differences in resident microbiome in healthy individuals may translate into distinct kinetics of graft rejection, and contribute to interpersonal variability in graft outcomes. The association between Alistipes and prolonged skin graft survival in mice suggests that members of this genus might affect host physiology, including at sites distal to the gastrointestinal tract. Overall, these findings allude to a potential therapeutic role for specific gut microbes to promote graft survival through the administration of probiotics, or FMT.

Late night salivary cortisol, stress-resilience and Type 2 diabetes mellitus. Findings from population-based KORA Age study

The main limitation to successful transplantation is the antigraft response developed by the recipient immune system, and the adverse side effects of immunosuppressive agents which are associated with significant toxicity and counter indications such as infection and cancer. Furthermore, immunosuppressants do little to prevent ischemia-reperfusion injury during the transplantation procedure itself hence there is a growing need to develop novel immunosuppressive drugs specifically aimed at prolonging graft survival. Linear tetrapyrroles derived from the breakdown of mammalian heme have been shown in numerous studies to play a protective role in allograft transplantation and ischemia-reperfusion injury; however, commercial sources of these products have not been approved for use in humans. Plants and algae produce equivalent linear tetrapyrroles called bilins that serve as chromophores in light-sensing. One such marine-derived tetrapyrrole, phycocyanobilin (PCB), shows significant structural similarity to mammalian biliverdin (BV) and may prove to be a safer alternative for use in the clinic if it can exert direct effects on human immune cells. Using a mixed lymphocyte reaction, we quantified the allogeneic responses of recipient cells to donor cells and found that PCB, like BV, effectively suppressed proliferation and proinflammatory cytokine production. In addition, we found that BV and PCB can directly downregulate the proinflammatory responses of both innate dendritic cells and adaptive T cells. We therefore propose that PCB may be an effective therapeutic drug in the clinical setting of transplantation and may also have wider applications in regulating inappropriate inflammation.

Subcutaneous administration of a neutralizing IL-1β antibody prolongs limb allograft survival.

Cytokine‐expression profiles revealed IL‐1ß highly upregulated in rejecting skin of limb allografts. We investigate the effect of intragraft treatment with a neutralizing IL‐1β antibody in limb transplantation. Following allogenic hind‐limb transplantation, Lewis rats were either left untreated1 or treated with anti‐lymphocyte serum + tacrolimus (baseline)2; baseline immunosuppression + anti‐IL‐1β (1 mg/kg once/week, 6‐8 subcutaneous injections) into the transplanted3 or contralateral4 limb. Endpoint was rejection grade III or day 100. Graft rejection was assessed by histology, immunohistochemistry, flow cytometry phenotyping of immune cells, and monitoring cytokine expression. Anti‐IL‐1β injections into the allograft or contralateral limb resulted in a significant delay of rejection onset (controls: 58.60 ± 0.60; group 3: 75.80 ± 10.87, P = .044; group 4: 73.00 ± 6.49, P = .008) and prolongation of graft survival (controls: 64.60 ± 0.87; group 3: 86.60 ± 5.33, P = .002; group 4: 93.20 ± 3.82, P = .002), compared to controls. Although the phenotype of the graft infiltrating immune cells did not differ between groups, significantly decreased skin protein levels of IL‐1β, IL‐4, IL‐13, IP‐10, MCP‐1, and MCP‐3 in long‐term‐survivors indicate an overall decrease of chemoattraction and infiltration of immune cells as the immunosuppressive mechanism of anti‐IL‐1β. Inhibition of IL‐1β with short‐term systemic immunosuppression prolongs limb allograft survival and represents a promising target for immunosuppression in extremity transplantation.

SA-FasL-engineered PEG microgels as a novel means of modulating immune response to allogeneic islet grafts

Abstract Allogenic islet transplantation is an effective therapy for type 1 diabetes in the clinic. Sustained graft survival requires chronic immunosuppression that has adverse effects. Islet graft rejection is initiated and perpetuated by T effector cells (Teffs). Teffs upregulate Fas death receptor on their surface and undergo apoptosis following engagement with FasL. Therefore, FasL has the potential as an immunomodulator to establish immune tolerance to islet grafts by eliminate alloreactive Teffs. This study used PEG microgels engineered with a novel form of FasL, SA-FasL, for the prevention of allogeneic islet graft rejection. Poly(ethylene glycol) microgels were produced with biotin on their surface. Biotinylated microgels were engineered with SA-FasL (1 μg/103 microgels) by taking advantage of the high affinity interaction between biotin and the streptavidin (SA) domain of the molecule. SA-FasL-PEGs induced apoptosis in Fas expressing A20 lymphoid cells in vitro. Co-transplantation of SA-FasL-PEGs with naïve islets led to prolonged survival of grafts as compared with the control group (islets + unmodified PEGs), with 20% surviving for a 200-day observation period. A short (15 days) treatment with low dose rapamycin further improved the immunomodulatory efficacy of SA-FasL-PEGs, with &amp;gt; 90% of grafts surviving for a 200-day observation period. CD4+CD25+FoxP3+ Tregs were required for graft survival, as depletion of this cell population on day 50 post-transplantation resulted in prompt rejection. These findings provide strong proof-of-efficacy and feasibility for the use of SA-FasL-engineered microgels as an off-the-shelf product for the modulation of alloreactive responses with significant therapeutic potential.

Limbokeratoplasty to Treat Lattice and Granular Corneal Dystrophies

The surgical technique of limbokeratoplasty (limbo-KP) was initially established for the treatment of severe limbal deficiencies. Besides improving visual acuity, surgery is aimed at ensuring complete, long-lasting epithelialization of the ocular surface. Due to the extension of the indication spectrum, limbo-KPs are also used in various forms of epithelial/stromal corneal dystrophies such as lattice and granular (transforming growth factor beta-induced [TGFBI] gene mutation associated) dystrophies. The objective of limbo-KP is to ensure prolonged clear graft survival without a recurrence of the dystrophy as otherwise observed after conventional keratoplasty.