Acceptance Of Skin Grafts Research Articles

Deleting Runx3 in CD4+ T Cells Disrupts Effector Functions and Facilitates Tolerance Induction in Solid Organ Transplantation

Abstract Long-term transplantation tolerance depends on the control of graft-reactive T cells. In particular, the alloreactivity of CD4+ T cells needs to be suppressed as these cells are both necessary and sufficient for cardiac allograft rejection. Although experimental protocols such as anti-CD154-mediated co-stimulation blockade can stably induce donor-specific tolerance in MHC-mismatched cardiac allografts, other more immunogenic organs such as skin are less susceptible to tolerogenic treatments, in part due to additional reactivity of the host against donor commensals that accompany the graft. The transcriptional mechanisms that enable CD4+ T cells to drive rejection and whose targeting can promote tolerance are not well understood. RNAseq comparison of the transcriptome in graft-reactive T cells from rejecting versus tolerant heart allograft recipients revealed several transcription factors differentially expressed between rejected and tolerant mice in both spleen and graft. Here, we report that the transcription factor Runx3 in alloreactive CD4+ T cells promotes Th1 and cytotoxic response against the allograft and resistance to tolerance induction. Conversely, ablating Runx3 in alloreactive CD4+ T cells augments the efficacy of anti-CD154 blockade treatment enabling skin graft acceptance.

Oral alloantigen exposure promotes donor-specific tolerance in a mouse model of minor-mismatched skin transplantation

Oral antigen exposure is a powerful, non-invasive route to induce immune tolerance to dietary antigens, and has been modestly successful at prolonging graft survival in rodent models of transplantation. To harness the mechanisms of oral tolerance for promoting long-term graft acceptance, we developed a mouse model where the antigen ovalbumin (OVA) was introduced orally prior to transplantation with skin grafts expressing OVA. Oral OVA treatment pre-transplantation promoted permanent graft acceptance and linked tolerance to skin grafts expressing OVA fused to the additional antigen 2W. Tolerance was donor-specific, as secondary donor-matched, but not third-party allografts were spontaneously accepted. Oral OVA treatment promoted an anergic phenotype in OVA-reactive CD4+ and CD8+ conventional T cells (Tconvs) and expanded OVA-reactive Tregs pre-transplantation. However, skin graft acceptance following oral OVA resisted partial depletion of Tregs and blockade of PD-L1. Mechanistically, we revealed a role for the proximal gut draining lymph nodes (gdLNs) in mediating this effect, as an intestinal infection that drains to the proximal gdLNs prevented tolerance induction. Our study extends previous work applying oral antigen exposure to transplantation and serves as proof of concept that the systemic immune mechanisms supporting oral tolerance are sufficient to promote long-term graft acceptance.

Oral alloantigen exposure promotes donor-specific tolerance in a mouse model of minor mismatched skin transplantation

Abstract Oral tolerance is a powerful, non-invasive route to induce immune tolerance, yet it has been unsuccessful to promote graft acceptance in rodent models of transplantation. To this end, we developed a mouse model where the model antigen ovalbumin (OVA) was introduced orally, in a regimen designed to improve bioavailability, duodenal lymph node drainage and prolong duration of antigen exposure, prior to transplantation with skin grafts constitutively expressing membrane-bound OVA. We found that oral OVA treatment pre-transplantation promoted permanent graft acceptance as well as linked tolerance to skin grafts expressing OVA coupled to the additional model antigen 2W. Tolerance was donor-specific, as secondary donor-matched, but not third-party grafts were spontaneously accepted in the absence of additional immunosuppression. Treatment with this oral OVA regimen promoted an anergic phenotype in OVA-reactive CD8+ T cells, generation of iTregs, and dysfunction of OVA-reactive CD4+ T cells pre-transplantation. The tolerance induced by oral OVA was robust, as anti-CD25/anti-PD-L1 at the time of transplantation failed to prevent skin graft acceptance. Mechanistically, we revealed a critical role for the proximal gut draining lymph nodes (gLNs) in mediating this effect, as an intestinal infection that drains to the proximal gLNs prevented the induction of transplantation tolerance. Our study extends previous work applying oral antigen exposure to transplantation and serves as proof of concept that the systemic immune mechanisms supporting oral tolerance are sufficient to promote long-term graft acceptance. Regimens that improve bioavailability and duration of oral alloantigen administration may hold promise in the clinic. This work was supported by a NIAID grant to ASC and MLA (P01AI-97113). Peter Wang was supported by undergraduate research grants from the Katen Scholars Program and the Dean's Fund for Undergraduate Research.

Experimental model for assessing the readiness of the wound surface to accept skin grafts

The aim of the study, performed on 65 Wistar rats, was to develop a new model and, as an example, use it to identify the extent to which quercetin and the quercetin-2-hydroxypropyl-β-cyclodextrin nanocomplex, applied to the wound, affect the engraftment of skin autografts. Grafts were obtained from the ear shells of animals, and graft’s survival rate was assessed by the degree of inhibition of wound contraction. It is shown that the latter depends on the area of grafts and the time after which they were located on the wound surface after its creation. It was shown that quercetin worsens, and quercetin-2- hydroxypropyl-β-cyclodextrin does not impair the ability of the wound surface to accept the graft.

T cell exhaustion is associated with antigen abundance and promotes transplant acceptance.

Exhaustion of T cells limits their ability to clear chronic infections or eradicate tumors. Here, in the context of transplant, we investigated whether T cell exhaustion occurs and has a role in determining transplant outcome. A peptide/MHC tetramer-based approach was used to track exhausted CD8+ T cells in a male-to-female skin transplant model. Transplant of large whole-tail skins, but not small tail skins (0.8cm×0.8cm), led to exhaustion of anti-male tetramer+ CD8+ T cells and subsequently the acceptance of skin grafts. To study CD4+ T cell exhaustion, we used the TCR-transgenic B6 TEa cells that recognize a major transplant antigen I-Eα from Balb/c mice. TEa cells were adoptively transferred either into B6 recipients that received Balb/c donor skins or into CB6F1 mice that contained an excessive amount of I-Eα antigen. Adoptively transferred TEa cells in skin-graft recipients were not exhausted. By contrast, virtually all adoptively transferred TEa cells were exhausted in CB6F1 mice. Those exhausted TEa cells lost ability to reject Balb/c skins upon further transfer into lymphopenic B6.Rag1-/- mice. Hence, T cell exhaustion develops in the presence of abundant antigen and promotes transplant acceptance. These findings are essential for better understanding the nature of transplant tolerance.

Vascularized composite allotransplantation combined with costimulation blockade induces mixed chimerism and reveals intrinsic tolerogenic potential.

Vascularized composite allotransplantation (VCA) has become a valid therapeutic option to restore form and function after devastating tissue loss. However, the need for high-dose multidrug immunosuppression to maintain allograft survival is still hampering more widespread application of VCA. In this study, we investigated the immunoregulatory potential of costimulation blockade (CoB; CTLA4-Ig and anti-CD154 mAb) combined with nonmyeoablative total body irradiation (TBI) to promote allograft survival of VCA in a fully MHC-mismatched mouse model of orthotopic hind limb transplantation. Compared with untreated controls (median survival time [MST] 8 days) and CTLA4-Ig treatment alone (MST 17 days), CoB treatment increased graft survival (MST 82 days), and the addition of nonmyeloablative TBI led to indefinite graft survival (MST > 210 days). Our analysis suggests that VCA-derived BM induced mixed chimerism in animals treated with CoB and TBI + CoB, promoting gradual deletion of alloreactive T cells as the underlying mechanism of long-term allograft survival. Acceptance of donor-matched secondary skin grafts, decreased ex vivo T cell responsiveness, and increased graft-infiltrating Tregs further indicated donor-specific tolerance induced by TBI + CoB. In summary, our data suggest that vascularized BM-containing VCAs are immunologically favorable grafts promoting chimerism induction and long-term allograft survival in the context of CoB.

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.

Treg-inducing microparticles promote donor-specific tolerance in experimental vascularized composite allotransplantation

For individuals who sustain devastating composite tissue loss, vascularized composite allotransplantation (VCA; e.g., hand and face transplantation) has the potential to restore appearance and function of the damaged tissues. As with solid organ transplantation, however, rejection must be controlled by multidrug systemic immunosuppression with substantial side effects. As an alternative therapeutic approach inspired by natural mechanisms the body uses to control inflammation, we developed a system to enrich regulatory T cells (Tregs) in an allograft. Microparticles were engineered to sustainably release TGF-β1, IL-2, and rapamycin, to induce Treg differentiation from naïve T cells. In a rat hindlimb VCA model, local administration of this Treg-inducing system, referred to as TRI-MP, prolonged allograft survival indefinitely without long-term systemic immunosuppression. TRI-MP treatment reduced expression of inflammatory mediators and enhanced expression of Treg-associated cytokines in allograft tissue. TRI-MP also enriched Treg and reduced inflammatory Th1 populations in allograft draining lymph nodes. This local immunotherapy imparted systemic donor-specific tolerance in otherwise immunocompetent rats, as evidenced by acceptance of secondary skin grafts from the hindlimb donor strain and rejection of skin grafts from a third-party donor strain. Ultimately, this therapeutic approach may reduce, or even eliminate, the need for systemic immunosuppression in VCA or solid organ transplantation.

Tumescent non-tumescent technique for split thickness skin graft harvesting

Background: Split-thickness skin graft failures can be attributed to flaws in the recipient bed which has to be well prepared. Tissues with limited blood supply such as bone, tendons, cartilage or sites with necrotic tissue or infection do not accept skin grafts. Adrenaline is used to harvest skin grafts due to its vasoconstriction effect which limits blood loss. The aim of our study was to determine skin graft take after tumescent technique compared to a non-tumescent technique for harvesting.Methods: Two treatment groups of patients who fulfilled the inclusion criteria were randomly assigned. Forty patients underwent split-thickness skin graft harvesting with tumescent technique and forty patients underwent non-tumescent split-thickness skin graft harvesting. The recipient site was opened in both groups on the fifth day after surgery and take rate assessed. The donor site was assessed on day ten and if not healed, followed up for three weeks.Results: There was a statistically significant association between skin graft take rate and skin grafting technique (p=0.011). The mean graft take rate was 2.5% higher in the tumescent group compared to the non-tumescent group (96.3% compared to 94%). On day 10, there was no difference in percentage healing of donor sites between the tumescent and non-tumescent groups, p=0.562.Conclusions: Tumescent technique significantly reduced intraoperative blood loss. It is safe, inexpensive and easy to use. The subdermal adrenaline/saline injection creates a smooth, dense surface which assists debridement and donor harvesting.

Stability of Chimerism in Non-Obese Diabetic Mice Achieved By Rapid T Cell Depletion Is Associated With High Levels of Donor Cells Very Early After Transplant.

Stable mixed hematopoietic chimerism is a robust method for inducing donor-specific tolerance with the potential to prevent rejection of donor islets in recipients with autoimmune type-1 diabetes. However, with reduced intensity conditioning, fully allogeneic chimerism in a tolerance resistant autoimmune-prone non-obese diabetic (NOD) recipient has rarely been successful. In this setting, successful multilineage chimerism has required either partial major histocompatability complex matching, mega doses of bone marrow, or conditioning approaches that are not currently clinically feasible. Irradiation free protocols with moderate bone marrow doses have not generated full tolerance; donor skin grafts were rejected. We tested whether more efficient recipient T cell depletion would generate a more robust tolerance. We show that a combination of donor-specific transfusion-cyclophosphamide and multiple T cell depleting antibodies could induce stable high levels of fully allogeneic chimerism in NOD recipients. Less effective T cell depletion was associated with instability of chimerism. Stable chimeras appeared fully donor-specific tolerant, with clonal deletion of allospecific T cells and acceptance of donor skin grafts, while recovering substantial immunocompetence. The loss of chimerism months after transplant was significantly associated with a lower level of chimerism and donor T cells within the first 2 weeks after transplant. Thus, rapid and robust recipient T cell depletion allows for stable high levels of fully allogeneic chimerism and robust donor-specific tolerance in the stringent NOD model while using a clinically feasible protocol. In addition, these findings open the possibility of identifying recipients whose chimerism will later fail, stratifying patients for early intervention.

Donor bone marrow cells are essential for iNKT cell‐mediated Foxp3+ Treg cell expansion in a murine model of transplantation tolerance

Mixed chimerism induction is the most reliable method for establishing transplantation tolerance. We previously described a novel treatment using a suboptimal dose of anti-CD40 ligand (anti-CD40L) and liposomal formulation of a ligand for invariant natural killer Tcells administered to sub-lethally irradiated recipient mice after donor bone marrow cell (BMC) transfer. Recipient mice treated with this regimen showed expansion of a Foxp3-positive regulatory T(Treg) cell phenotype, and formation of mixed chimera. However, the mechanism of expansion and bioactivity of Treg cells remains unclear. Here, we examine the role of donor BMCs in the expansion of bioactive Treg cells. The mouse model was transplanted with a heart allograft the day after treatment. The results showed that transfer of spleen cells in place of BMCs failed to deplete host interferon (IFN)-γ-producing CD8+ Tcells, expand host Ki67+ CD4+ CD25+ Foxp3+ Treg cells, and prolong graft survival. Severe combined immunodeficiency mice who received Treg cells obtained from BMC-recipients accepted skin grafts in an allo-specific manner. Myeloid-derived suppressor cells, which were a copious cell subset in BMCs, enhanced the Ki67 expression of Treg cells. This suggests that donor BMCs are indispensable for the expansion of host bioactive Treg cells in our novel treatment for transplant tolerance induction.

The Knife's Edge of Tolerance: Inducing Stable Multilineage Mixed Chimerism but With a Significant Risk of CMV Reactivation and Disease in Rhesus Macaques.

Although stable mixed-hematopoietic chimerism induces robust immune tolerance to solid organ allografts in mice, the translation of this strategy to large animal models and to patients has been challenging. We have previously shown that in MHC-matched nonhuman primates (NHPs), a busulfan plus combined belatacept and anti-CD154-based regimen could induce long-lived myeloid chimerism, but without T cell chimerism. In that setting, donor chimerism was eventually rejected, and tolerance to skin allografts was not achieved. Here, we describe an adaptation of this strategy, with the addition of low-dose total body irradiation to our conditioning regimen. This strategy has successfully induced multilineage hematopoietic chimerism in MHC-matched transplants that was stable for as long as 24 months posttransplant, the entire length of analysis. High-level T cell chimerism was achieved and associated with significant donor-specific prolongation of skin graft acceptance. However, we also observed significant infectious toxicities, prominently including cytomegalovirus (CMV) reactivation and end-organ disease in the setting of functional defects in anti-CMV T cell immunity. These results underscore the significant benefits that multilineage chimerism-induction approaches may represent to transplant patients as well as the inherent risks, and they emphasize the precision with which a clinically successful regimen will need to be formulated and then validated in NHP models.

Immunoglobulin G promotes skin graft acceptance in an immunologically potent rat model.

Immunoglobulin G (IgG) has been shown to protect graft rejection after transplantation, whereas the molecular mechanism of IgG in promoting graft acceptance has not been well established. In this study, we tested the effectiveness of IgG in preventing rejection of transplanted skin graft in an immunologically potent rat model, and studied the mechanism of this protection. We found that systemic or local administration of IgG significantly prolonged the survival of skin grafts with the immune tolerance induced by IgG and subcutaneous local injection of 1mg IgG to adult SD rat yielded the longest survival of skin grafts from 5.8 to 17.3 days. We also found that IgG reduced the number of pro-inflammatory cells especially lymphocytes, neutrophils and basophils, increased the seral levels of anti-inflammatory factors including IL-10 and IL-4, and activated CD4+CD25+Foxp3+ regulatory T cells, unveiling the mechanisms of this protective effect. These findings provide new insight to support clinical application of IgG in treating transplantation.

Both rejection and tolerance of allografts can occur in the absence of secondary lymphoid tissues.

In this study, we showed that aly/aly mice, which are devoid of lymph nodes and Peyer's patches, acutely rejected fully allogeneic skin and heart grafts. They mounted potent inflammatory direct alloresponses but failed to develop indirect alloreactivity after transplantation. Remarkably, skin allografts also were rejected acutely by splenectomized aly/aly (aly/aly-spl(-)) mice devoid of all secondary lymphoid organs. In these recipients, the rejection was mediated by alloreactive CD8(+) T cells presumably primed in the bone marrow. In contrast, cardiac transplants were not rejected by aly/aly-spl(-) mice. Actually, aly/aly-spl(-) mice that spontaneously accepted a heart allotransplant and displayed donor-specific tolerance also accepted skin grafts from the same, but not a third-party, donor via a mechanism involving CD4(+) regulatory T cells producing IL-10 cytokine. Therefore, direct priming of alloreactive T cells, as well as rejection and regulatory tolerance of allogeneic transplants, can occur in recipient mice lacking secondary lymphoid organs.

Mixed chimerism and transplant tolerance are not effectively induced in C3a-deficient mice

Mixed chimerism, a phenomenon involved in the development of specific alloantigen tolerance, could be achieved through the transplantation of hematopoietic stem cells into properly prepared recipients. Because the C3a complement component modulates hematopoietic cell trafficking after transplantation, in the present study, we investigated the influence of the C3a deficiency on mixed chimerism and alloantigen tolerance induction. To induce mixed chimerism, C57BL/6J (wild-type strain; H-2K(b); I-E(-)) and B6.129S4-C3(tm1Crr)/J (C3a-deficient) mice were exposed to 3 G total body irradiation (day -1). Subsequently, these mice were treated with CD8-blocking (day -2) and CD40L-blocking (days 0 and 4) antibodies, followed by transplantation with 20 × 10(6) Balb/c (H-2K(d); I-E(+)) bone marrow cells (day 0). The degree of mixed chimerism in peripheral blood leukocytes was measured several times during the 20-week experiment. The tolerance to Balb/c mouse antigens was assessed based on the number of lymphocytes expressing Vβ5 and Vβ11 T-cell receptor and on skin-graft (day 0) acceptance. Applying our experimental model, mixed chimerism and alloantigen tolerance were effectively induced in C57BL/6J (wild-type) mice, but not in C3a(-/-) animals. The present study is, to our knowledge, the first to demonstrate that C3a is vital for achieving stable mixed chimerism and related to this induction of transplant tolerance.

Transplantation of tail skin to study allogeneic CD4 T cell responses in mice.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. In general, both CD4(+) and CD8(+) allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-A(bm12)) in the antigen-presenting groove of the MHC-class II (I-A(b)) molecule is sufficient to induce strong allogeneic CD4(+) T cell activation in C57BL/6 mice. Skin grafts from I-A(bm12) mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3(-/-) and Rag2(-/-) mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 10(4) wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-γ in I-A(bm12)-transplanted Rag2(-/-) mice.

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-γ in I-Abm12-transplanted Rag2-/- mice.

Myeloid Progenitors Can Induce Tolerance in the Context of Autologous HSC Transplantation.

i. Purpose. We have published that tolerance for skin graft transplantation can be consistently induced with Myeloid Progenitor Cells (MP) without long-term high-level donor-derived engraftment. Importantly, MP are in clinical trials to test their ability to lower infections in bone marrow transplantation, making them clinically available. MP can be given either two months prior to or at the time of organ transplantation for tolerance induction. Using MP from B10;B6-Rag2-/-Il2rg-/- mice (devoid of T, B or NK cells) we have established that donor-matched lymphoid cells are not required. Here we test whether tolerance is preserved when MP are combined with host-autologous HSC which would reduce HCT-related complications. ii. Methods. In our model mice (typically BALB/c) were irradiated (8.5Gy) and reconstituted with 500 BALB/c or 4,000 FVB HSC and 100,000 third party MP. MP-matched (or mismatched) skin grafts are placed on the same day and their fate is followed long-term.Figure: No Caption available.iii. Results. Specific tolerance induction in this model can be achieved with autologous HSC (which can rapidly restore the hematopoietic system) and skin graft-matched MP. Tolerance induced by 100,000 skin graft-matched MP is slightly lower (p=0.02, logrank) when combined with 500 host autologous HSC (solid black line) than it is with 4,000 allogeneic HSC which are not matched to the skin graft (solid grey line): At the dose tested approx. 80% of the grafts (35/43) show long-term skin graft acceptance without further immunosuppression. Grafts not matched to host, HSC or MP are all rejected (dashed lines). Ongoing experiments test whether increasing the MP dose will further improve outcome. iv. Conclusion. Organ matched MP can induce tolerance in combination with autologous HSC significantly increasing the potential clinical applicability of this approach to tolerance induction and importantly, indicating that reduced intensity preconditioning regimens should be feasible.

IL-7 receptor blockade following T cell depletion promotes long-term allograft survival

T cell depletion is commonly used in organ transplantation for immunosuppression; however, a restoration of T cell homeostasis following depletion leads to increased memory T cells, which may promote transplant rejection. The cytokine IL-7 is important for controlling lymphopoiesis under both normal and lymphopenic conditions. Here, we investigated whether blocking IL-7 signaling with a mAb that targets IL-7 receptor α (IL-7Rα) alone or following T cell depletion confers an advantage for allograft survival in murine transplant models. We found that IL-7R blockade alone induced indefinite pancreatic islet allograft survival if anti-IL-7R treatment was started 3 weeks before graft. IL-7R blockade following anti-CD4- and anti-CD8-mediated T cell depletion markedly prolonged skin allograft survival. Furthermore, IL-7 inhibition in combination with T cell depletion synergized with either CTLA-4Ig administration or suboptimal doses of tacrolimus to induce long-term skin graft acceptance in this stringent transplant model. Together, these therapies inhibited T cell reconstitution, decreased memory T cell numbers, increased the relative frequency of Tregs, and abrogated both cellular and humoral alloimmune responses. Our data suggest that IL-7R blockade following T cell depletion has potential as a robust, immunosuppressive therapy in transplantation.

Heterologous Immunity Triggered by a Single, Latent Virus in Mus musculus: Combined Costimulation- and Adhesion- Blockade Decrease Rejection

The mechanisms underlying latent-virus-mediated heterologous immunity, and subsequent transplant rejection, especially in the setting of T cell costimulation blockade, remain undetermined. To address this, we have utilized MHV68 to develop a rodent model of latent virus-induced heterologous alloimmunity. MHV68 infection was correlated with multimodal immune deviation, which included increased secretion of CXCL9 and CXCL10, and with the expansion of a CD8dim T cell population. CD8dim T cells exhibited decreased expression of multiple costimulation molecules and increased expression of two adhesion molecules, LFA-1 and VLA-4. In the setting of MHV68 latency, recipients demonstrated accelerated costimulation blockade-resistant rejection of skin allografts compared to non-infected animals (MST 13.5 d in infected animals vs 22 d in non-infected animals, p<.0001). In contrast, the duration of graft acceptance was equivalent between non-infected and infected animals when treated with combined anti-LFA-1/anti-VLA-4 adhesion blockade (MST 24 d for non-infected and 27 d for infected, p = n.s.). The combination of CTLA-4-Ig/anti-CD154-based costimulation blockade+anti-LFA-1/anti-VLA-4-based adhesion blockade led to prolonged graft acceptance in both non-infected and infected cohorts (MST>100 d for both, p<.0001 versus costimulation blockade for either). While in the non-infected cohort, either CTLA-4-Ig or anti-CD154 alone could effectively pair with adhesion blockade to prolong allograft acceptance, in infected animals, the prolonged acceptance of skin grafts could only be recapitulated when anti-LFA-1 and anti-VLA-4 antibodies were combined with anti-CD154 (without CTLA-4-Ig, MST>100 d). Graft acceptance was significantly impaired when CTLA-4-Ig alone (no anti-CD154) was combined with adhesion blockade (MST 41 d). These results suggest that in the setting of MHV68 infection, synergy occurs predominantly between adhesion pathways and CD154-based costimulation, and that combined targeting of both pathways may be required to overcome the increased risk of rejection that occurs in the setting of latent-virus-mediated immune deviation.