DNAX Activating Protein of 12 kDa/Triggering Receptor Expressed on Myeloid Cells 2 Expression by Mouse and Human Liver Dendritic Cells: Functional Implications and Regulation of Liver Ischemia-Reperfusion Injury.

Liver interstitial dendritic cells (DCs) have been implicated in the control of ischemia-reperfusion injury (IRI) and host immune responses following liver transplantation. Mechanisms underlying these regulatory functions of hepatic DCs remain unclear. We have shown recently that the transmembrane immunoadaptor DNAX-activating protein of 12 kDa (DAP12) negatively regulates mouse liver DC maturation and proinflammatory and immune stimulatory functions. Here, we used PCR analysis and flow cytometry to characterize expression of DAP12 and its associated triggering receptor, triggering receptor expressed on myeloid cells 2 (TREM2), by mouse and human liver DCs and other immune cells compared with DCs in other tissues. We also examined the roles of DAP12 and TREM2 and their expression by liver DCs in the regulation of liver IRI. Injury was induced in DAP12-/- , TREM2-/- , or wild-type (WT) mice by 1 hour of 70% clamping and quantified following 6 hours of reperfusion. Both DAP12 and TREM2 were coexpressed at comparatively high levels by liver DCs. Mouse liver DCs lacking DAP12 or TREM2 displayed enhanced levels of nuclear factor κB and costimulatory molecule expression. Unlike normal WT liver DCs, DAP12-/- liver DC failed to inhibit proliferative responses of activated T cells. In vivo, DAP12-/- and TREM2-/- mice exhibited enhanced IRI accompanied by augmented liver DC activation. Elevated alanine aminotransferase levels and tissue injury were markedly reduced by infusion of WT but not DAP12-/- DC. Conclusion: Our data reveal a close association between DAP12 and TREM2 expression by liver DC and suggest that, by negatively regulating liver DC stimulatory function, DAP12 promotes their control of hepatic inflammatory responses; the DAP12/TREM2 signaling complex may represent a therapeutic target for control of acute liver injury/liver inflammatory disorders.

The COVID-19 pandemic: A community approach.

An unprecedented global pandemic caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has quickly overwhelmed the health care systems worldwide. While there is an absence of consensus among the community in how to manage solid organ transplant recipients and donors, a platform provided by the American Society of Transplantation online community "Outstanding Questions in Transplantation," hosted a collaborative multicenter, multinational discussions to share knowledge in a rapidly evolving global situation. Here, we present a summary of the discussion in addition to the latest published literature.

Graft-infiltrating PD-L1hi cross-dressed dendritic cells regulate antidonor T cell responses in mouse liver transplant tolerance.

Although a key role of cross-dressing has been established in immunity to viral infection and more recently in the instigation of transplant rejection, its role in tolerance is unclear. We investigated the role of intragraft dendritic cells (DCs) and cross-dressing in mouse major histocompatibility complex (MHC)-mismatched liver transplant tolerance that occurs without therapeutic immunosuppression. Although donor interstitial DCs diminished rapidly after transplantation, they were replaced in the liver by host DCs that peaked on postoperative day (POD) 7 and persisted indefinitely. Approximately 60% of these recipient DCs displayed donor MHC class I, indicating cross-dressing. By contrast, only a very minor fraction (0%-2%) of cross-dressed DCs (CD-DCs) was evident in the spleen. CD-DCs sorted from liver grafts expressed much higher levels of T cell inhibitory programed death ligand 1 (PD-L1) and high levels of interleukin-10 compared with non-CD-DCs (nCD-DCs) isolated from the graft. Concomitantly, high incidences of programed death protein 1 (PD-1)hi T cell immunoglobulin and mucin domain containing 3 (TIM-3)+ exhausted graft-infiltrating CD8+ T cells were observed. Unlike nCD-DCs, the CD-DCs failed to stimulate proliferation of allogeneic T cells but markedly suppressed antidonor host T cell proliferation. CD-DCs were much less evident in allografts from DNAX-activating protein of 12 kDa (DAP12)-/- donors that were rejected acutely. CONCLUSION: These findings suggest that graft-infiltrating PD-L1hi CD-DCs may play a key role in the regulation of alloimmunity and in the induction of liver transplant tolerance. (Hepatology 2018;67:1499-1515).

CD8+ Effector T Cell Migration to Pancreatic Islet Grafts Is Dependent on Cognate Antigen Presentation by Donor Graft Cells.

Pancreatic islet transplantation is a promising therapy for diabetes, but acute rejection of the islets by host effector T cells has hindered clinical application. In this study, we addressed the mechanisms of CD8(+) effector T cell migration to islet grafts because interrupting this step is key to preventing rejection. We found that effector T cell migration to revascularized islet transplants in mice is dependent on non-self Ag recognition rather than signaling via Gαi-coupled chemokine receptors. Presentation of non-self Ag by donor cells was necessary for migration, whereas Ag presentation by recipient cells was dispensable. We also observed that deficiency of SKAP1, an immune cell adaptor downstream of the TCR and important for integrin activation, prolongs allograft survival but does not reduce effector T cell migration to the graft. Therefore, effector T cell migration to transplanted islets is Ag driven, not chemokine driven, but SKAP1 does not play a critical role in this process.

New insights into the mechanisms of Treg function.

PURPOSE OF REVIEW: CD4Foxp3 regulatory T cells (Tregs) are crucial in controlling immunity and self-tolerance. Consequently, in transplantation, Tregs play a central role in inhibiting acute rejection and promoting allograft tolerance. A more complete understanding of Treg biology may lead to novel therapeutic approaches to enhance Treg numbers and function. RECENT FINDINGS: The maintenance of self-tolerance in nonlymphoid tissues requires the differentiation of Tregs in secondary lymphoid organs from naïve-like central Tregs into effector Tregs. Antigen and environmental cues guide this Treg differentiation, which parallels the types of adaptive immune responses taking place, allowing them to enter and function within specific nonlymphoid tissues. In addition to controlling inflammation, tissue-infiltrating Tregs unexpectedly regulate nonimmune processes, including metabolic homeostasis and tissue repair. Finally, Tregs can be directly and specifically targeted in vivo to augment their numbers or enhance their function in both secondary lymphoid organs and nonlymphoid tissues. SUMMARY: Tregs exhibit a previously unrecognized breadth of function, which includes tissue-specific specialization and the regulation of both immune and nonimmune processes. This is of particular importance in transplantation since allo-reactive memory T cells can act directly within the allograft. Thus, therapeutic approaches may need to promote Treg function in transplanted tissue, as well as in secondary lymphoid organs. Such therapy would not only prevent inflammation and acute rejection, but may also promote nonimmune processes within the allograft such as tissue homeostasis and repair.

Tipping the balance toward transplantation tolerance: in vivo therapy using a mutated IL-2.

Immune tolerance to allogenic transplanted tissues remains elusive, and therapeutics promoting CD4+FOXP3+ Tregs are required to achieve this ultimate goal. In this issue of the JCI, Efe and colleagues engineered an Fc domain fused to a human mutein IL-2 (mIL-2-Fc) bearing mutations that confer preferential binding to the high-affinity IL-2 receptor expressed on Tregs. In vivo mIL-2-Fc therapy effectively heightened mouse, monkey, and human Treg numbers, promoted tolerance to minor antigen mismatched skin grafts in mice, and synergized with immunosuppressive drugs used in the clinic. These findings warrant clinical trials that assess the efficacy of mIL-2-Fc in transplantation.

Regulatory T cells require mammalian target of rapamycin signaling to maintain both homeostasis and alloantigen-driven proliferation in lymphocyte-replete mice.

Rapamycin (Rapa), an immunosuppressive drug that acts through mammalian target of Rapa inhibition, broadly synergizes with tolerogenic agents in animal models of transplantation and autoimmunity. Rapa preferentially inhibits conventional CD4(+) Foxp3(-) T cells (Tconv) and promotes outgrowth of CD4(+)Foxp3(+) regulatory T cells (Treg) during in vitro expansion. Moreover, Rapa is widely perceived as augmenting both expansion and conversion of Treg in vivo. However, most quantitative studies were performed in lymphopenic hosts or in graft-versus-host disease models. We show in this study that in replete wild-type mice, Rapa significantly inhibits both homeostatic and alloantigen-induced proliferation of Treg, and promotes their apoptosis. Together, these lead to significant Treg depletion. Tconv undergo depletion to a similar degree, resulting in no change in the percent of Treg among CD4 cells. Moreover, in this setting, there was no evidence of conversion of Tconv into Treg. However, after withdrawal of Rapa, Treg recover Ag-induced proliferation more quickly than Tconv, leading to recovery to baseline numbers and an increase in the percent of Treg compared with Tconv. These findings suggest that the effects of Rapa on Treg survival, homeostasis, and induction, depend heavily on the cellular milieu and degree of activation. In vivo, the resistance of Treg to mammalian target of Rapa inhibition is relative and results from lymphopenic and graft-versus-host disease models cannot be directly extrapolated to settings more typical of solid organ transplantation or autoimmunity. Moreover, these results have important implications for the timing of Rapa therapy with tolerogenic agents designed to increase the number of Treg in vivo.

New perspectives in transplantation through intravital microscopy imaging.

PURPOSE OF REVIEW: Rejection of transplanted organs is a complex and highly dynamic immune process. Two-photon laser-scanning intravital microscopy (LSIM) allows for real-time, deep tissue, high-resolution imaging in physiological conditions. The recent application of this technology to study organ rejection started to provide a clearer picture of the spatiotemporal immunological dynamics of organ rejection. RECENT FINDINGS: To date, LSIM has been applied to transplanted skin, islet, and kidney in mice, as well to constantly moving organs such as transplanted lung and heart. To characterize the dynamics of innate and adaptive immune cell infiltration, time-lapse imaging of various fluorescent-reporter mice was performed. Overall, these studies revealed differences between the anatomical location of infiltrating neutrophils and monocytes in various transplanted organs. In addition, the dynamics of lymphocytic infiltration revealed different transendothelial migration routes in vascularized versus nonvascularized transplanted tissues. SUMMARY: LSIM is a very powerful tool that can be used to carefully dissect the immune cells dynamics in rejection and in tolerance induction in transplantation. Many dynamic biological processes can only be investigated using LSIM. Thus, LSIM promises to greatly enhance our knowledge in transplantation immunobiology and will help tailoring specific therapeutics in relation to the transplanted tissue.

Treg suppression of immunity within inflamed allogeneic grafts.

CD4+Foxp3+ regulatory T cells (Tregs) restrain inflammation and immunity. However, the mechanisms underlying Treg suppressor function in inflamed nonlymphoid tissues remain largely unexplored. Here, we restricted immune responses to nonlymphoid tissues and used intravital microscopy to visualize Treg suppression of rejection by effector T cells (Teffs) within inflamed allogeneic islet transplants. Despite their elevated motility, Tregs preferentially contacted antigen-presenting cells (APCs) over Teffs. Interestingly, Tregs specifically targeted APCs that were extensively and simultaneously contacted by Teffs. In turn, Tregs decreased MHC-II expression on APCs and hindered Teff function. Last, we demonstrate that Treg suppressive function within inflamed allografts required ectonucleotidase CD73 activity, which generated the antiinflammatory adenosine. Consequently, CD73-/- Tregs exhibited fewer contacts with APCs within inflamed allografts compared with WT Tregs, but not in spleen. Overall, our findings demonstrate that Tregs suppress immunity within inflamed grafts through CD73 activity and suggest that Treg-APC direct contacts are central to this process.

Graft-derived extracellular vesicles transported across subcapsular sinus macrophages elicit B cell alloimmunity after transplantation.

Despite the role of donor-specific antibodies (DSAs) in recognizing major histocompatibility complex (MHC) antigens and mediating transplant rejection, how and where recipient B cells in lymphoid tissues encounter donor MHC antigens remains unclear. Contrary to the dogma, we demonstrated here that migration of donor leukocytes out of skin or heart allografts is not necessary for B or T cell allosensitization in mice. We found that mouse skin and cardiac allografts and human skin grafts release cell-free donor MHC antigens via extracellular vesicles (EVs) that are captured by subcapsular sinus (SCS) macrophages in lymph nodes or analog macrophages in the spleen. Donor EVs were transported across the SCS macrophages, and donor MHC molecules on the EVs were recognized by alloreactive B cells. This triggered B cell activation and DSA production, which were both prevented by SCS macrophage depletion. These results reveal an unexpected role for graft-derived EVs and open venues to interfere with EV biogenesis, trafficking, or function to restrain priming or reactivation of alloreactive B cells.

Identification of a putative pathway for the muscle homing of stem cells in a muscular dystrophy model.

Attempts to repair muscle damage in Duchenne muscular dystrophy (DMD) by transplanting skeletal myoblasts directly into muscles are faced with the problem of the limited migration of these cells in the muscles. The delivery of myogenic stem cells to the sites of muscle lesions via the systemic circulation is a potential alternative approach to treat this disease. Muscle-derived stem cells (MDSCs) were obtained by a MACS(R) multisort method. Clones of MDSCs, which were Sca-1+/CD34-/L-selectin+, were found to adhere firmly to the endothelium of mdx dystrophic muscles after i.v. or i.m. injections. The subpopulation of Sca-1+/CD34- MDSCs expressing L-selectin was called homing MDSCs (HMDSCs). Treatment of HMDSCs with antibodies against L-selectin prevented adhesion to the muscle endothelium. Importantly, we found that vascular endothelium from striate muscle of young mdx mice expresses mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a ligand for L-selectin. Our results showed for the first time that the expression of the adhesion molecule L-selectin is important for muscle homing of MDSCs. This discovery will aid in the improvement of a potential therapy for muscular dystrophy based on the systemic delivery of MDSCs.

Central tolerance to myogenic cell transplants does not include muscle neoantigens.

BACKGROUND: Duchenne muscular dystrophy is a fatal genetic disease caused by lack of dystrophin. Myogenic cell transplantation (MT), a potential therapy for Duchenne muscular dystrophy, can restore dystrophin expression in muscles. Because allogeneic MT is highly resistant to peripheral tolerance, we proposed to induce central tolerance. However, given its immunogenicity, we asked whether central tolerance to donor major histocompatibility complex would allow long-term expression of dystrophin, a tissue-specific neoantigen in dystrophic recipients. METHODS: Central tolerance was induced in C57BL/10J mdx (dystrophic) mice by allogeneic bone marrow transplantation (BMT) after conditioning with either lethal total body irradiation (TBI) or an established nonmyeloablative protocol (anti-CD154, anti-CD8 mAbs, and low-dose TBI). Recipients subsequently received donor-strain MT or skin grafts. RESULTS: Long-term hematopoietic chimeras generated using either lethal TBI or the nonmyeloablative regimen were tolerant to donor skin grafts and both primary and secondary donor MT (>90 days). Myogenic cell transplantation survival was decreased when chimerism was transient, which was most common with nonmyeloablative conditioning and fully rather than haplo-mismatched donors. Interestingly, regardless of conditioning, MT was associated with localized muscle infiltration with Foxp3CD4, CD25CD4, and PerforinCD8 cells, whereas skin grafts lacked infiltration. CONCLUSIONS: Central tolerance achieved using regimens that eliminate nearly all endogenous peripheral lymphocytes (i.e., lethal irradiation) or a nonmyeloablative protocol that depleted peripheral CD8 cells, results in lymphocytic infiltration in muscles that received MT but not in skin allografts. This suggests that muscle-specific infiltration may result from lack of negative selection for peripheral neoantigens in the thymus after BMT and that tolerance after MT may rely on peripheral regulatory mechanisms.

Induction of tolerance across fully mismatched barriers by a nonmyeloablative treatment excluding antibodies or irradiation use.

A mixed-chimerism approach is a major goal to circumvent sustained immunosuppression, but most of the proposed protocols need antibody treatment or host irradiation. Another promising experience involves busulfan combined with cyclophosphamide treatment. Additionally, recent publications demonstrated that, differing from busulfan, treosulfan administration does not present severe organ or hemato toxicities. Currently, Duchenne muscular dystrophy (DMD) patients are treated with chronic immunosuppression for muscle precursor cell transplantation (MT). We have developed a safe tolerance approach within this cellular allotransplantation therapy background. Thus, we have conditioned, prior to a donor BALB/c MT, the dystrophic mouse model C57Bl10J mdx/mdx, with our treatment based on a donor-specific transfusion, then a treosulfan treatment combined with single cyclophosphamide dose, and finally a donor bone marrow transplantation (TTCB). A first MT was performed in all mixed chimeric mice resulting from the TTCB treatment in the left tibialis anterior (TA) muscles. A second MT from the same donor strain was performed 100 days later in the right TA without any additional therapy. Results show that all treated mice developed permanent mixed chimerism. Long-lasting donor-positive fibers were present in both TAs of the mice, which received MT after the TTCB treatment. Only a basal level of infiltration was observed around donor fibers and mixed chimeric mice rejected third-party haplotype skin grafts. Thus, mixed chimerism development with this TTCB conditioning regimen promotes donor-specific stable tolerance, avoiding costimulatory blockade antibodies or irradiation use and side effects of sustained immunosuppressive treatments. This protocol could be eventually applied for MT to DMD patients or others tissue transplantations.

Treatment with anti-CD154 antibody and donor-specific transfusion prevents acute rejection of myoblast transplantation.

BACKGROUND: Achieving immunological tolerance to transplanted myoblasts would reduce the adverse effects associated with the sustained immunosuppression required for this experimental therapeutic approach in Duchenne muscular dystrophic patients. METHODS: Mdx mice were transplanted with fully allogeneic BALB/c myoblasts in the tibialis anterior muscles. Seven days before transplantation (-7), host mice received 107 total donor spleen cells i.v. (donor-specific transfusion, DST) with 500 microg of anti-CD154 mAb i.p. on days -7, -4, 0, +4. RESULTS: Results showed a high level of dystrophin expression in 83, 60, and 20% of the mice 1, 3, and 6 months, respectively, after transplantation of myoblasts. No antibodies against the donor cells were produced up to 3 months after transplantation. However, abundant activated cytotoxic cells were present in muscles still expressing high percentage of dystrophin positive fibers. CONCLUSIONS: In conclusion, the DST + anti-CD154 mAb treatments effectively prolonged myoblast survival, but this treatment could not develop tolerance to complete allogeneic myoblast transplantation.

CD45 ligation expands Tregs by promoting interactions with DCs.

Regulatory T cells (Tregs), which express CD4 and FOXP3, are critical for modulating the immune response and promoting immune tolerance. Consequently, methods to expand Tregs for therapeutic use are of great interest. While transfer of Tregs after massive ex vivo expansion can be achieved, in vivo expansion of Tregs would be more practical. Here, we demonstrate that targeting the CD45 tyrosine phosphatase with a tolerogenic anti-CD45RB mAb acutely increases Treg numbers in WT mice, even in absence of exogenous antigen. Treg expansion occurred through substantial augmentation of homeostatic proliferation in the preexisting Treg population. Moreover, anti-CD45RB specifically increased Treg proliferation in response to cognate antigen. Compared with conventional T cells, Tregs differentially regulate their conjugation with DCs. Therefore, we determined whether CD45 ligation could alter interactions between Tregs and DCs. Live imaging showed that CD45 ligation specifically reduced Treg motility in an integrin-dependent manner, resulting in enhanced interactions between Tregs and DCs in vivo. Increased conjugate formation, in turn, augmented nuclear translocation of nuclear factor of activated T cells (NFAT) and Treg proliferation. Together, these results demonstrate that Treg peripheral homeostasis can be specifically modulated in vivo to promote Treg expansion and tolerance by increasing conjugation between Tregs and DCs.

Cognate antigen directs CD8+ T cell migration to vascularized transplants.

The migration of effector or memory T cells to the graft is a critical event in the rejection of transplanted organs. The prevailing view is that the key steps involved in T cell migration - integrin-mediated firm adhesion followed by transendothelial migration - are dependent on the activation of Gαi-coupled chemokine receptors on T cells. In contrast to this view, we demonstrated in vivo that cognate antigen was necessary for the firm adhesion and transendothelial migration of CD8+ effector T cells specific to graft antigens and that both steps occurred independent of Gαi signaling. Presentation of cognate antigen by either graft endothelial cells or bone marrow-derived APCs that extend into the capillary lumen was sufficient for T cell migration. The adhesion and transmigration of antigen-nonspecific (bystander) effector T cells, on the other hand, remained dependent on Gαi, but required the presence of antigen-specific effector T cells. These findings underscore the primary role of cognate antigen presented by either endothelial cells or bone marrow-derived APCs in the migration of T cells across endothelial barriers and have important implications for the prevention and treatment of graft rejection.

Memory T cells migrate to and reject vascularized cardiac allografts independent of the chemokine receptor CXCR3.

BACKGROUND: Memory T cells migrate to and reject transplanted organs without the need for priming in secondary lymphoid tissues, but the mechanisms by which they do so are not known. Here, we tested whether CXCR3, implicated in the homing of effector T cells to sites of infection, is critical for memory T-cell migration to vascularized allografts. METHODS: CD4 and CD8 memory T cells were sorted from alloimmunized CXCR3 and wildtype B6 mice and cotransferred to congenic B6 recipients of BALB/c heart allografts. Graft-infiltrating T cells were quantitated 20 and 72 hr later by flow cytometry. Migration and allograft survival were also studied in splenectomized alymphoplastic (aly/aly) recipients, which lack secondary lymphoid tissues. RESULTS: We found that polyclonal and antigen-specific memory T cells express high levels of CXCR3. No difference in migration of wildtype versus CXCR3 CD4 and CD8 memory T cells to allografts could be detected in wildtype or aly/aly hosts. In the latter, wildtype and CXCR3 memory T cells precipitated acute rejection at similar rates. Blocking CCR5, a chemokine receptor also upregulated on memory T cells, did not delay graft rejection mediated by CXCR3 memory T cells. CONCLUSIONS: CXCR3 is not critical for the migration of memory T cells to vascularized organ allografts. Blocking CXCR3 or CXCR3 and CCR5 does not delay acute rejection mediated by memory T cells. These findings suggest that the mechanisms of memory T cell-homing to transplanted organs may be distinct from those required for their migration to sites of infection.

Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice.

T cell Ig domain and mucin domain protein 1 (TIM-1) is a costimulatory molecule that regulates immune responses by modulating CD4+ T cell effector differentiation. However, the function of TIM-1 on other immune cell populations is unknown. Here, we show that in vivo in mice, TIM-1 is predominantly expressed on B rather than T cells. Importantly, TIM-1 was expressed by a large majority of IL-10-expressing regulatory B cells in all major B cell subpopulations, including transitional, marginal zone, and follicular B cells, as well as the B cell population characterized as CD1d(hi)CD5+. A low-affinity TIM-1-specific antibody that normally promotes tolerance in mice, actually accelerated (T cell-mediated) immune responsiveness in the absence of B cells. TIM-1+ B cells were highly enriched for IL-4 and IL-10 expression, promoted Th2 responses, and could directly transfer allograft tolerance. Both cytokine expression and number of TIM-1+ regulatory B cells (Bregs) were induced by TIM-1-specific antibody, and this was dependent on IL-4 signaling. Thus, TIM-1 is an inclusive marker for IL-10+ Bregs that can be induced by TIM-1 ligation. These findings suggest that TIM-1 may be a novel therapeutic target for modulating the immune response and provide insight into the signals involved in the generation and induction of Bregs.

Absence of innate MyD88 signaling promotes inducible allograft acceptance.

Prior experimental strategies to induce transplantation tolerance have focused largely on modifying adaptive immunity. However, less is known concerning the role of innate immune signaling in the induction of transplantation tolerance. Using a highly immunogenic murine skin transplant model that resists transplantation tolerance induction when innate immunity is preserved, we show that absence of MyD88, a key innate Toll like receptor signal adaptor, abrogates this resistance and facilitates inducible allograft acceptance. In our model, absence of MyD88 impairs inflammatory dendritic cell responses that reduce T cell activation. This effect increases T cell susceptibility to suppression mediated by CD4+ CD25+ regulatory T cells. Therefore, this study provides evidence that absence of MyD88 promotes inducible allograft acceptance and implies that inhibiting innate immunity may be a potential, clinically relevant strategy to facilitate transplantation tolerance.