Donor-specific Transfusion Research Articles

Donor-Specific Blood Transfusion in Lung Transplantation

Lung transplantation is still hindered by a high rate of chronic rejection necessitating profound immunosuppression with its associated complications. Donor-specific blood transfusion is a pre-transplant strategy aimed at improving graft acceptance. In contrast with standard stored blood or donor-specific regulatory T cells transfusions, this approach utilizes fresh whole blood from the donor prior to allograft transplantation, encompassing all cell types and plasma. The precise mechanisms underlying donor-specific blood transfusion-induced tolerance remain incompletely understood. Associations with regulatory/helper T cells, modulation of mononuclear phagocytic cells or microchimerism have been suggested. While numerous (pre-)clinical studies have explored its application in solid organ transplants like liver, kidney, and intestine, limited attention has been given to the setting of lung transplantation. This comprehensive review summarizes existing knowledge on the mechanisms and outcomes of donor-specific blood transfusion in solid organ transplants both in preclinical and clinical settings. We also address the potential benefits and risks associated with donor-specific blood transfusion in the field of lung transplantation, offering insights into future research directions.

(676) - Donor-Specific Blood Transfusion is Feasible and Safe in Mice: A Proof-of-Concept Study

(676) - Donor-Specific Blood Transfusion is Feasible and Safe in Mice: A Proof-of-Concept Study

Induction of allograft tolerance by adoptive transfer of donor B cells: an immune regulatory strategy for transplantation using MHC-matched iPS cells.

For cellular or tissue transplantation using induced pluripotent stem cells (iPSCs), from the viewpoint of time and economic cost, the use of allogeneic ones is being considered. Immune regulation is one of the key issues in successful allogeneic transplantation. To reduce the risk of rejection, several attempts have been reported to eliminate effects of the major histocompatibility complex (MHC) on the iPSC-derived grafts. On the other hand, we have shown that minor antigen-induced rejection is not negligible even when the MHC's impact is mitigated. In organ transplantation, it is known that donor-specific transfusion (DST) can specifically control immune responses to the donor. However, whether DST could control the immune response in iPSC-based transplantation was not clarified. In this study, using a mouse skin transplantation model, we demonstrate that infusion of donor splenocytes can promote allograft tolerance in the MHC-matched but minor antigen-mismatched conditions. When narrowing down the cell types, we found that infusion of isolated splenic B cells was sufficient to control rejection. As a mechanism, the administration of donor B cells induced unresponsiveness but not deletion in recipient T cells, suggesting that the tolerance was induced in the periphery. The donor B cell transfusion induced allogeneic iPSC engraftment. These results suggest for the first time a possibility that DST using donor B cells could induce tolerance against allogeneic iPSC-derived grafts.

Vγ2+ γδ T Cells in the Presence of Anti-CD40L Control Surgical Inflammation and Promote Skin Allograft Survival

γδ T cells represent a small fraction of total T cells in the body and do not use classical polymorphic major histocompatibility complex‒loaded peptides for mounting an immune response. The importance of the effector and regulatory function of γδ T cells in infections, autoimmunity, and tumor models are well characterized. In this study, we investigated the mechanistic role of γδ T cells in costimulatory blockade‒induced transplantation tolerance. We used donor-specific transfusion and anti-CD40L treatment in C57BL/6 mice to induce tolerance to BALB/c skin allografts. We show that depletion of γδ T cells, specifically Vγ2+ γδ T cells, led to the acute rejection of skin allografts despite tolerogen treatment. Tolerogen treatment promoted CD39+Vγ2+ γδ T cells and suppressed IFN-γ‒producing Vγ2+ γδ T cells in the spleen and allografts. Vγ2+ γδ T cells isolated from tolerized mice suppress T helper type 1 cell differentiation. Adoptive transfer of these regulatory Vγ2+ γδ T cells prolonged the survival of allografts in an untreated recipient and Tcrδ‒/‒ mice. Together, our data show that the Vγ2+ subset promotes costimulatory blockade‒induced survival of skin allografts and that tolerogenic Vγ2+ T cells can be used as an adoptive cellular therapy to promote the survival of allografts.

The use of organ donor blood in liver transplantation.

Blood removed from organs during deceased donor organ procurement is routinely discarded but is a potential resource for donor-specific transfusion (DST) in subsequent liver transplantation (LT). This study retrospectively analyses the impact of DST on intraoperative bank blood product usage, long-term graft, and patient survival, as well as frequency of rejection post-LT. A total of 992 adult LT performed from 1993 to 2018 in a single quaternary center were included. Intraoperative blood product usage, patient, and graft survival, as well as acute and chronic rejection were assessed in patients who received blood retrieved from the organ donor, the "donor blood" (DB) group (n=437) and patients who did not, the "no donor blood" (NDB) group (n=555). Processing of DB ensured safe levels of potassium, magnesium, and insulin. There were fewer units of bank red blood cells transfusion required in the DB group compared to NDB group (2vs. 4 units, P=.01). Graft survival was significantly superior in the DB group (10-year survival 75%vs. 69%, respectively, P=.04) but DST was not an independent predictor of graft survival. There was no significant difference in patient survival or rejection between the groups. There was no difference in treated, biopsy-proven rejection between the two groups. This is the first large-cohort study assessing long-term outcomes of intraoperative DST in LT. The collection of organ donor blood and subsequent use in LT recipients appeared feasible with appropriate quality checks ensuring safety. DST resulted in a reduction in the use of packed red blood cells. There was no difference in the rate of rejection or graft or patient survival.

Cellular Therapies in Solid Organ Allotransplantation: Promise and Pitfalls.

Donor specific transfusions have been the basis of tolerance inducing protocols since Peter Medawar showed that it was experimentally feasible in the 1950s. Though trials of cellular therapies have become increasingly common in solid organ transplantation, they have not become standard practice. Additionally, whereas some protocols have focused on cellular therapies as a method for donor antigen delivery—thought to promote tolerance in and of itself in the correct immunologic context—other approaches have alternatively focused on the intrinsic immunosuppressive properties of the certain cell types with less emphasis on their origin, including mesenchymal stem cells, regulatory T cells, and regulatory dendritic cells. Regardless of intent, all cellular therapies must contend with the potential that introducing donor antigen in a new context will lead to sensitization. In this review, we focus on the variety of cellular therapies that have been applied in human trials and non-human primate models, describe their efficacy, highlight data regarding their potential for sensitization, and discuss opportunities for cellular therapies within our current understanding of the immune landscape.

Use of donor-specific red blood cell transfusions for patients undergoing liver transplantation during the COVID-19 pandemic

Increased risks have been found for patients undergoing liver transplantation due to the blood supply shortage following the ongoing coronavirus disease 2019 (COVID-19) pandemic. Hence, exploring a method to alleviate this dilemma is urgent. This phase I, nonrandomized, prospective trial aimed to evaluate the safety and feasibility of using donor-specific red blood cell transfusion (DRBCT) as an urgent measurement to alleviate the blood supply shortage in deceased donor liver transplantation (DDLT). The outcomes of 26 patients who received DRBCT and 37 patients in the control group who only received 3rd party packed red blood cells (pRBCs) transfusion between May 2020 and January 2021 were compared. Patients receiving DRBCT did not develop transfusion-related complications, and the incidence of postoperative infection was similar to that in the control group (23.1% vs. 18.9%, P=0.688). Because the patients received the red blood cells from organ donors, the median volume of intraoperative allogeneic red blood cell transfusion from blood bank was 4.0 U (IQR 1.1-8.0 U) in the DRBCT group, which is significantly lower than that (7.5 U, IQR 4.0-10.0 U) in the control group (P=0.018). The peak aspartate aminotransferase (AST) level was significantly lower in the DRBCT group than in the control group (P=0.008) and so were the AST levels in the first two days after the operation (P=0.006 and P=0.033). DRBCT is a safe and effective procedure to lower the need for blood supply and is associated with a reduction in AST levels after transplantation. DRBCT is beneficial to patients receiving life-saving transplantation without sufficient blood supply during the COVID-19 pandemic.

Effect of donor-specific blood transfusions on allotransplanted teeth in a monkey model: Histoquantification of periodontal healing.

Pre-transplant blood transfusions have previously shown a positive effect on organ allograft survival in humans and various animal species. The aims of this study were, first, to evaluate the effect of pre-transplant donor-specific blood transfusions on periodontal healing of fully developed allotransplanted teeth in monkeys; and second, to investigate the immune response against donor antigens and to determine a possible correlation between alloimmune reactions and histopathological signs of rejection. Twenty monkeys (Cercopithecus aethiops) were divided into ten pairs with similar sizes of incisors. One monkey in each pair gave three transfusions to the other monkey with 1-week intervals. One week after the last transfusion, each pair exchanged simultaneously a central maxillary incisor and a lateral mandibular incisor. The corresponding central maxillary and mandibular lateral incisors were autotransplanted to the contralateral sockets. All teeth were treated endodontically per-operatively. Histocompatibility was evaluated by mixed lymphocyte culture before the first transfusion, while alloantibodies and cell-mediated alloresponses were measured before transfusions and at 2 and 8weeks after transplantation. All animals were sacrificed 8weeks after tooth transplantation. Serial sections of the transplanted teeth were quantified histologically. Mixed lymphocyte cultures showed positive reactions in 19 of 20 cases, indicating incompatibility. The majority of the monkeys developed antibodies towards the tooth donor. Cell-mediated cytotoxicity was negative in all monkeys. Histoquantification revealed a mean score of 70% normal periodontal ligament (PDL) in autotransplanted teeth, with 5% ankylosis. The allografts had a mean score of 17% normal PDL and 63% ankylosis, with no significant influence of transfusion. However, in the mandibular grafts, a tendency towards a positive transfusion effect was seen. Donor-specific blood transfusion did not reduce ankylosis in tooth allografts. The healing of mandibular incisor tooth allografts was improved, but not that of maxillary incisors. Donor-specific antibodies showed no effect on the survival of allograft PDL.

Suppression of liver transplant rejection by anti-donor MHC antibodies via depletion of donor immunogenic dendritic cells.

BackgroundWe previously found two distinct passenger dendritic cell (DC) subsets in the rat liver that played a central role in the liver transplant rejection. In addition, a tolerance-inducing protocol, donor-specific transfusion (DST), triggered systemic polytopical production of depleting alloantibodies to donor class I MHC (MHCI) antigen (DST-antibodies).MethodsWe examined the role of DST-antibodies in the trafficking of graft DC subsets and the alloresponses in a rat model. We also examined an anti-donor class II MHC (MHCII) antibody that recognizes donor DCs more selectively.ResultsPreoperative transfer of DST-antibodies or DST pretreatment eliminated all passenger leukocytes, including both DC subsets and depleted the sessile DCs in the graft to ~20% of control. The CD172a+CD11b/c+ immunogenic subset was almost abolished. The intrahost direct or semi-direct allorecognition pathway was successfully blocked, leading to a significant suppression of the CD8+ T-cell response in the recipient lymphoid organs and the graft with delayed graft rejection. Anti-donor MHCII antibody had similar effects without temporary graft damage. Although DST pretreatment had a priming effect on the proliferative response of recipient regulatory T cells, DST-primed sera and the anti-donor MHCII antibody did not.ConclusionDST-antibodies and anti-donor MHCII antibodies could suppress the CD8+ T-cell-mediated liver transplant rejection by depleting donor immunogenic DCs, blocking the direct or semi-direct pathways of allorecognition. Donor MHCII-specific antibodies may be applicable as a selective suppressant of anti-donor immunity for clinical liver transplantation without the cellular damage of donor MHCII– graft cells and recipient cells.

Triazolopyrimidine derivative NK026680 and donor-specific transfusion induces CD4+CD25+Foxp3+ T cells and ameliorates allograft rejection in an antigen-specific manner

We have previously demonstrated the unique properties of a new triazolopyrimidine derivative, NK026680, which exerts immunosuppressive effects in rat heart transplant model and confers tolerogeneic properties on ex vivo-conditioned dendritic cells in mice. We herein demonstrate that NK026680 promotes the expansion of regulatory T cells (Tregs) with potent immunoregulatory effects when used in combination with donor-specific transfusion (DST).BALB/c (H-2d) heart graft were transplanted into C57BL/6 (H-2b) mice following intravenous injection of donor splenocytes (DST) and oral administration of NK026680. The NK026680 plus DST treatment markedly prolonged the survival time of the donor-graft, but not that of the 3rd party-graft (C3H; H-2k). Treg cells in the recipient spleen on day 0 expanded when stimulated with donor-antigens in vivo and in vitro. After heart transplantation, Treg cells accumulated into the graft and increased in the spleen. NK026680 plus DST also decreased activated CD8+ T cells in the spleen and inhibited infiltration of CD8+ T cells into the graft. Depletion of CD25+ cells inhibited the graft prolonging effect of the NK026680 plus DST treatment.NK026680 administration together with DST induces potent immunoregulatory effects in an antigen-specific manner, likely due to the in vivo generation of donor-specific Tregs.

THE USE OF ORGAN DONOR BLOOD IN LIVER TRANSPLANTATION

BACKGROUND Blood removed from organs during deceased organ procurement is routinely discarded but is a potential resource for donor-specific transfusion (DST) in subsequent liver transplantation (LT). This study retrospectively analyses the impact of DST on intraoperative bank product usage, long-term graft, and patient survival, as well as frequency of rejection post-LT. METHODS A total of 992 adult LT performed from 1993 to 2018 in a single quaternary center were included. Intraoperative product usage, patient, and graft survival, as well as acute and chronic rejection were assessed in patients who received retrieved from the organ donor, the donor blood (DB) group (n = 437) and patients who did not, the blood (NDB) group (n = 555). RESULTS Processing of DB ensured safe levels of potassium, magnesium, and insulin. There were fewer units of bank red cells transfusion required in the DB group compared to NDB group (2 vs. 4 units, P = .01). Graft survival was significantly superior in the DB group (10-year survival 75% vs. 69%, respectively, P = .04) but DST was not an independent predictor of graft survival. There was no significant difference in patient survival or rejection between the groups. There was no difference in treated, biopsy-proven rejection between the two groups. CONCLUSIONS This is the first large-cohort study assessing long-term outcomes of intraoperative DST in LT. The collection of organ and subsequent use in LT recipients appeared feasible with appropriate quality checks ensuring safety. DST resulted in a reduction in the use of packed red cells. There was no difference in the rate of rejection or graft or patient survival.

Indirectly Activated Treg Allow Dominant Tolerance to Murine Skin-grafts Across an MHC Class I Mismatch After a Single Donor-specific Transfusion.

Tolerance induced in stringent animal transplant models using donor-specific transfusions (DST) has previously required additional immunological manipulation. Here, we demonstrate a dominant skin-allograft tolerance model induced by a single DST across an major histocompatibility class I mismatch in an unmanipulated B6 host. C57BL/6 (H-2) (B6) mice were injected intravenously with splenocytes from B6.C.H-2 (H-2k) (bm1) or F1 (B6 × bm1) mice before skin transplantation. Mice were transplanted 7 days postinjection with donor (bm1 or F1) and third-party B10.BR (H-2) skin grafts. B6 hosts acutely rejected skin grafts from B6.C.H-2 (bm1) and F1 (B6 × bm1) mice. A single transfusion of F1 splenocytes into B6 mice without any additional immune modulation led to permanent acceptance of F1 skin grafts. This graft acceptance was associated with persistence of donor cells long-term in vivo. The more rapid removal of DST bm1 cells than F1 cells was reduced by natural killer-cell depletion. Tolerant grafts survived an in vivo challenge with naive splenocytes. Both CD4CD25 and CD4CD25 T cells from F1 DST treated B6 mice suppressed alloproliferation in vitro. Tolerance was associated with expansion of peripheral Foxp3CD4CD25 regulatory T cells (Treg) and increased forkhead box P3 (Foxp3) expression in tolerant grafts. In tolerant mice, Foxp3 Treg arises from the proliferation of indirectly activated natural Foxp3 Treg (nTreg) and depletion of Foxp3 Treg abrogates skin-graft tolerance. This study demonstrates that the persistence of transfused semiallogeneic donor cells mismatched at major histocompatibility class I can enhance tolerance to subsequent skin allografts through indirectly expanded nTreg leading to dominant tolerance without additional immunological manipulation.

Resilience of T cell-intrinsic dysfunction in transplantation tolerance

Following antigen stimulation, naïve T cells differentiate into memory cells that mediate antigen clearance more efficiently upon repeat encounter. Donor-specific tolerance can be achieved in a subset of transplant recipients, but some of these grafts are rejected after years of stability, often following infections. Whether T cell memory can develop from a tolerant state and whether these formerly tolerant patients develop antidonor memory is not known. Using a mouse model of cardiac transplantation in which donor-specific tolerance is induced with costimulation blockade (CoB) plus donor-specific transfusion (DST), we have previously shown that systemic infection with Listeria monocytogenes (Lm) months after transplantation can erode or transiently abrogate established tolerance. In this study, we tracked donor-reactive T cells to investigate whether memory can be induced when alloreactive T cells are activated in the setting of tolerance. We show alloreactive T cells persist after induction of cardiac transplantation tolerance, but fail to acquire a memory phenotype despite becoming antigen experienced. Instead, donor-reactive T cells develop T cell-intrinsic dysfunction evidenced when removed from the tolerant environment. Notably, Lm infection after tolerance did not rescue alloreactive T cell memory differentiation or functionality. CoB and antigen persistence were sufficient together but not separately to achieve alloreactive T cell dysfunction, and conventional immunosuppression could substitute for CoB. Antigen persistence was required, as early but not late surgical allograft removal precluded the acquisition of T cell dysfunction. Our results demonstrate transplant tolerance-associated T cell-intrinsic dysfunction that is resistant to memory development even after Lm-mediated disruption of tolerance.

Novel Targeting to XCR1+ Dendritic Cells Using Allogeneic T Cells for Polytopical Antibody Responses in the Lymph Nodes.

Vaccination strategy that induce efficient antibody responses polytopically in most lymph nodes (LNs) against infections has not been established yet. Because donor-specific blood transfusion induces anti-donor class I MHC antibody production in splenectomized rats, we examined the mechanism and significance of this response. Among the donor blood components, T cells were the most efficient immunogens, inducing recipient T cell and B cell proliferative responses not only in the spleen, but also in the peripheral and gut LNs. Donor T cells soon migrated to the splenic T cell area and the LNs, with a temporary significant increase in recipient NK cells. XCR1+ resident dendritic cells (DCs), but not XCR1− DCs, selectively phagocytosed donor class I MHC+ fragments after 1 day. After 1.5 days, both DC subsets formed clusters with recipient CD4+ T cells, which proliferated within these clusters. Inhibition of donor T cell migration or depletion of NK cells by pretreatment with pertussis toxin or anti-asialoGM1 antibody, respectively, significantly suppressed DC phagocytosis and subsequent immune responses. Three allogeneic strains with different NK activities had the same response but with different intensity. Donor T cell proliferation was not required, indicating that the graft vs. host reaction is dispensable. Intravenous transfer of antigen-labeled and mitotic inhibitor-treated allogeneic, but not syngeneic, T cells induced a polytopical antibody response to labeled antigens in the LNs of splenectomized rats. These results demonstrate a novel mechanism of alloresponses polytopically in the secondary lymphoid organs (SLOs) induced by allogeneic T cells. Donor T cells behave as self-migratory antigen ferries to be delivered to resident XCR1+ DCs with negligible commitment of migratory DCs. Allogeneic T cells may be clinically applicable as vaccine vectors for polytopical prophylactic antibody production even in asplenic or hyposplenic individuals.

Oscar Salvatierra: helped draft the US National Organ Transplant Act of 1984 and fought for its passage

Lucile Packard Foundation for Children’s Health Oscar Salvatierra Jr was widely acknowledged as an outstanding paediatric kidney transplantation surgeon. He approached each patient with one goal in mind: to make...

Role of regulatory T cells in CD47/donor-specific transfusion-induced immune tolerance in skin-heart transplantation mice.

To explore the role of regulatory T (Treg ) cells in the establishment of immune tolerance induced by donor-specific transfusion (DST) in mice with skin-heart transplantation. C57BL/6 mice received DST of splenocytes from CD47+/+ or CD47-/- H-2bm1 mice or no DST 7days before skin-heart transplantation from major histocompatibility complex class I-mismatched H-2bm1 donors. The number and proportion of Treg cells in graft and lymphoid organs were measured by flow cytometry (FACS) and immunohistochemistry (IHC). The inhibitory function of Treg cells and anti-donor T-cell responses were assessed by mixed lymphocyte reaction. We observed that mean survival time (MST) of skin or heart graft was significantly longer in C57BL/6 mice which received DST from CD47+/+ H-2bm1 mice than from CD47-/- H-2bm1 mice. By FACS, we found that the number of Treg cells in spleen was increased significantly in mice which received CD47-/- DST compared to mice which received CD47+/+ DST. However, the percentages of Treg cells in total splenocytes and lymph node cells were significantly higher in mice that received CD47+/+ DST than mice which received CD47-/- DST. Immunohistochemistry showed an increased heart grafts infiltration of Treg cells in the recipients with CD47-/- DST, but not CD47+/+ DST. Supporting this, we found that donor T-cell proliferation was significantly suppressed in mice which received CD47+/+ DST compared to mice which received CD47-/- DST. There was no difference of inhibitory function of Treg cells between these two groups. Our results indicated that CD47 expression on DST cells plays an important role in the induction of immune tolerance in mice with skin-heart transplantation. Increased percentage of Treg cells may contribute to immune tolerance induced by CD47+/+ DST.

C5aR1 regulates migration of suppressive myeloid cells required for costimulatory blockade-induced murine allograft survival.

Costimulatory blockade-induced murine cardiac allograft survival requires intragraft accumulation of CD11b+ Ly6Clo Ly6G- regulatory myeloid cells (Mregs) that expand regulatory T cells (Tregs) and suppress effector T cells (Teffs). We previously showed that C5a receptor (C5aR1) signaling on T cells activates Teffs and inhibits Tregs, but whether and/or how C5aR1 affects Mregs required for transplant survival is unknown. Although BALB/c hearts survived >60days in anti-CD154 (MR1)-treated or cytotoxic T-lymphocyte associated protein 4 (CTLA4)-Ig-treated wild-type (WT) recipients, they were rejected at ~30days in MR1-treated or CTLA4-Ig-treated recipients selectively deficient in C5aR1 restricted to myeloid cells (C5ar1fl/fl xLysM-Cre). This accelerated rejection was associated with ~2-fold more donor-reactive T cells and ~40% less expansion of donor-reactive Tregs. Analysis of graft-infiltrating mononuclear cells on posttransplant day 6 revealed fewer Ly6Clo monocytes in C5ar1fl/fl xLysM-Cre recipients. Expression profiling of intragraft Ly6Clo monocytes showed that C5aR1 deficiency downregulated genes related to migration/locomotion without changes in genes associated with suppressive function. Cotransfer of C5ar1fl/fl and C5ar1fl/fl xLysM-Cre myeloid cells into MR1-treated allograft recipients resulted in less accumulation of C5ar1-/- cells within the allografts, and in vitro assays confirmed that Ly6Chi myeloid cells migrate to C5a/C5aR1-initiated signals. Together, our results newly link myeloid cell-expressed C5aR1 to intragraft accumulation of myeloid cells required for prolongation of heart transplant survival induced by costimulatory blockade.

Type-1 Interferons Impair the Immunoregulatory Activity of IL-10

A growing body of evidence shows that induction of long term transplant survival by “costimulation blockade” (CoB) regimens is impaired by inflammatory responses. In particular, multiple studies reported that engagement of toll like receptors (TLR) abrogate the tolerogenic effect of CoB. Despite the identification of type-1 interferons (TI-IFN) as mediators of this effect in multiple models, the target population and specific pathway used by TI-IFN to induce this effect, remain unknown. CoB therapies rely on the enhancement of the frequency and suppressive activity of regulatory T cells (Tregs) that use interleukin 10 (IL-10) as a key weapon of suppression. IL-10 is a pleiotropic and immunomodulatory cytokine that in the field of transplant tolerance, despite its anticipated protective function, maintains a controversial role. To better understand how an inflammatory environment, more specifically IFN-β, could interfere with the induction of transplant tolerance, we studied its impact on the immunomodulatory properties of IL-10. Our results showed that the transient blockade of IL-10 signaling had a deleterious impact on the ability of a CoB regimen based on the infusion of donor specific transfusion (DST) and anti-CD154 (MR-1), to prolong the survival of murine full mismatch skin transplants (MST 105d vs 47d with anti-IL-10R administration). Following 48h of in vitro incubation with IFN-β, memory T cells (Tmem) and Tregs presented a dramatic defect in the production of phospho-STAT3 in response to IL-10. This effect was very selective, as IL-6 signaling (post IFN-β exposure) induced normal levels of phospho-STAT3. The reduced accumulation of phospho-STAT3 in conditioned cells resulted in the inhibition of the upregulation of mRNAs for LIGHT, Sphk1 and Tarm-1 – three genes we have discovered are induced by IL-10 in T cells. Encouragingly, this inhibition of IL-10 signaling is slowly reversible with the removal of TI-IFN. Microarray and flow cytometry data indicated that this IL-10-specific unresponsiveness was not associated with any reduction of IL-10 receptor expression or an increase in SOCS (Suppressor of Cytokine Signaling) 1 and 3, nor with reduced STAT3 cytoplasmic availability. Instead, this analysis suggested a novel role for the transcription factor STAT1 in dampening IL-10 signaling. Using T cells from STAT1-KO mice, we show that the absence of STAT1 prevented IL-10 inhibition induced by IFN-β in Treg and Tmem, supporting our new model. Overall, these results highlight the importance of IL-10 signaling in the therapeutic effect of CoB regimens in transplantation. More importantly, they reveal a new molecular mechanism whereby IFN-β interferes with IL-10 signaling, its suppressive function in T cells, and ultimately with regulation of alloreactivity. Identifying a strategy to target this mechanism will be a powerful tool to improve the efficacy of immunomodulatory strategies for transplant tolerance induction. AAI 2016-2017 Careers in Immunology Fellowship. JDRF strategic research agreement 2-SRA-2016-310-S-B.

The enigmatic impact of donor T cell subsets on the therapeutic efficacy of tolerogenic protocols for transplant rejection

Abstract Costimulation blockade (CoB) regimens are a promising immunomodulatory strategy to prevent transplant rejection. However, we are learning that their efficacy is affected by multiple factors. Recent reports have started highlighting the unexpected capacity of passenger donor lymphocytes to enhance the recipient’s anti-graft response. New studies are necessary to understand this novel phenomenon and how it can affect the induction of transplant tolerance. In this study we assessed if T lymphocytes contained in the donor specific transfusion (DST) inoculum, used in combination with anti-CD154 (MR1) as CoB regimen, impact the regulation of mouse skin transplant rejection. When T cells were depleted from the DST, transplant survival was almost doubled. T cell subset-depletion studies indicated, unexpectedly, that donor CD8 T cells were responsible for limiting the efficacy of the tolerogenic regimen. Even more surprisingly, the presence of CD4 T cells in DST induced a remarkable improvement in transplant survival, beyond that observed with full T cell depletion. Ongoing experiments are determining the correlation between donor T lymphocytes and variations in humoral and cellular anti-donor responses. Overall, these data reveal the existence of a novel and unexpected opposing role of donor passenger lymphocytes that significantly modifies the therapeutic efficacy of DST+MR1 based regimens: a deleterious role for donor CD8, and a beneficial one for CD4 T cells. Identification of the specific mechanisms through which these divergent modulations of the anti-donor alloresponse are exerted could suggest significant changes in how we should approach immunoregulation, both experimentally as well as clinically.

Type-1 interferon impairs the immunoregulatory activity of IL-10: Understanding the mechanisms of abrogation of transplant tolerance

Abstract Long term transplant survival by costimulation blockade (CoB)-based regimens is impaired or eroded by inflammatory responses. Type-1 interferons (TI-IFN) are key mediators of this effect, however, their target population and mechanism of action remain unknown. To better understand how IFN-β interferes with the induction of transplant tolerance, we studied its impact on the recently discovered immunomodulatory properties of interleukin 10 (IL-10) in mouse T cells. We first demonstrate that IL-10 has an unappreciated fundamental role in transplant tolerance, as its transient blockade in recipients of full mismatch skin grafts impairs the efficacy of a peri-transplant CoB regimen based on donor specific transfusion (DST) plus anti-CD154. Moreover, when T cells are exposed to IFN-β in vitro, they develop a selective dramatic defect in their response to IL-10 (but not to IL-6) both at the signaling and gene-induction level. This IL-10-specific unresponsiveness is not associated with any reduction of IL-10R expression or an increase in SOCS1 and 3, nor with a reduced STAT3 cytoplasmic availability. Instead, our gene-expression and functional analysis suggests a novel “cross-regulatory” role for the transcription factor STAT1. Encouragingly, this inhibition is slowly reversible with the removal of TI-IFN, proving the feasibility a pharmacological intervention. Overall, these results reveal a new molecular mechanism whereby IFN-β interferes with the suppressive function of IL-10 in T cells, and potentially with regulation of alloreactivity. Identifying a strategy to target this mechanism will then be a powerful tool to improve the efficacy of CoB-based strategies for transplant tolerance induction.