Lymphotoxin ß receptor and tertiary lymphoid organs shape acute and chronic allograft rejection.

Solid organ transplantation remains the life-saving treatment for end-stage organ failure, but chronic rejection remains a major obstacle to long-term allograft outcomes and has not improved substantially. Tertiary lymphoid organs (TLO) are ectopic lymphoid structures that form under conditions of chronic inflammation, and evidence from human transplantation suggests that TLO regularly form in allografts undergoing chronic rejection. In this study, we utilized a mouse renal transplantation model and manipulation of the lymphotoxin alpha (LTα) - lymphotoxin beta receptor (LTßR) pathway, which is essential for TLO formation, to define the role of TLO in transplantation. We showed that intragraft TLO are sufficient to activate the alloimmune response and mediate graft rejection in a model where the only lymphoid organs are TLO in the allograft. When transplanted to recipients with a normal set of secondary lymphoid organs, the presence of graft TLO or LTα overexpression accelerated rejection. If the LTßR pathway was disrupted in the donor graft, TLO formation was abrogated, and graft survival prolonged. Intravital microscopy of renal TLO demonstrated that local T and B cell activation in TLOs is similar to that observed in secondary lymphoid organs. In summary, we demonstrated that immune activation in TLO contributes to local immune responses, leading to earlier allograft failure. TLO and the LTαß-LTßR pathway are therefore prime targets to limit local immune responses and prevent allograft rejection. These findings are applicable to other diseases such as autoimmunity or tumors, where either limiting or boosting local immune responses is beneficial and improves disease outcomes.

Innate Allorecognition in Transplantation: Ancient Mechanisms With Modern Impact.

Through the effective targeting of the adaptive immune system, solid organ transplantation became a life-saving therapy for organ failure. However, beyond 1 y of transplantation, there is little improvement in transplant outcomes. The adaptive immune response requires the activation of the innate immune system. There are no modalities for the specific targeting of the innate immune system involvement in transplant rejection. However, the recent discovery of innate allorecognition and innate immune memory presents novel targets in transplantation that will increase our understanding of organ rejection and might aid in improving transplant outcomes. In this review, we look at the latest developments in the study of innate allorecognition and innate immune memory in transplantation.

Tissue-resident memory T cell maintenance during antigen persistence requires both cognate antigen and interleukin-15.

Our understanding of tissue-resident memory T (TRM) cell biology has been largely developed from acute infection models in which antigen is cleared and sterilizing immunity is achieved. Less is known about TRM cells in the context of chronic antigen persistence and inflammation. We investigated factors that underlie TRM maintenance in a kidney transplantation model in which TRM cells drive rejection. In contrast to acute infection, we found that TRM cells declined markedly in the absence of cognate antigen, antigen presentation, or antigen sensing by the T cells. Depletion of graft-infiltrating dendritic cells or interruption of antigen presentation after TRM cells were established was sufficient to disrupt TRM maintenance and reduce allograft pathology. Likewise, removal of IL-15 transpresentation or of the IL-15 receptor on T cells during TRM maintenance led to a decline in TRM cells, and IL-15 receptor blockade prevented chronic rejection. Therefore, antigen and IL-15 presented by dendritic cells play nonredundant key roles in CD8 TRM cell maintenance in settings of antigen persistence and inflammation. These findings provide insights that could lead to improved treatment of chronic transplant rejection and autoimmunity.

Transitional B cell cytokines risk stratify early borderline rejection after renal transplantation.

Borderline rejection (BL) in renal transplantation is associated with decreased allograft survival, yet many patients with BL maintain stable graft function. Identifying patients with early BL at risk for shortened allograft survival would allow for timely targeted therapeutic intervention aimed at improving outcomes. 851/1187 patients transplanted between 2013-18 underwent early biopsy (0-4 mos). 217/851 (25%) had BL and were compared to 387/851 without significant inflammation (NI). Serial surveillance and for-cause biopsies and seven-year follow-up were used to evaluate histological and clinical progression. To identify high-risk patients, we examined clinical/histological parameters using regression and non-linear dimensionality reduction (tSNE) and a biomarker based on peripheral blood transitional-1 B cell (T1B) IL-10/TNFα ratio. Compared to NI, early BL was associated with increased progression to late acute rejection (AR; 5-12 mos), premature interstitial fibrosis and tubular atrophy (IFTA) and decreased seven-year graft survival. However, decreased graft survival was limited to BL patients who progressed to late AR or IFTA, and was not influenced by treatment. Although tSNE clustered patients into groups based on clinical factors, the ability of these factors to risk stratify BL patients was modest. In contrast, a low T1B IL-10/TNFα ratio at 3 months identified BL patients at high risk for progression to AR (ROC AUC 0.87) and poor 7-yr graft survival (52% vs. 92%, p=0.003), while BL patients with a high ratio had similar graft survival to patients with NI (91%, p=NS). Thus, progressive early allograft inflammation manifested as BL that progresses to late AR in the first post-transplant year represents a high-risk clinical state for poor allograft outcomes. Such high-risk status can be predicted by the T1B IL-10/TNFα ratio before irreversible scarring sets in, thus allowing timely risk stratification.

Activated-memory T cells influence naïve T cell fate: a noncytotoxic function of human CD8 T cells.

T cells are endowed with the capacity to sense their environment including other T cells around them. They do so to set their numbers and activation thresholds. This form of regulation has been well-studied within a given T cell population - i.e., within the naïve or memory pool; however, less is known about the cross-talk between T cell subsets. Here, we tested whether memory T cells interact with and influence surrounding naïve T cells. We report that human naïve CD8 T cells (TN) undergo phenotypic and transcriptional changes in the presence of autologous activated-memory CD8 T cells (TMem). Following in vitro co-culture with activated central memory cells (TCM), ~3% of the TN acquired activation/memory canonical markers (CD45RO and CD95) in an MHC-I dependent-fashion. Using scRNA-seq, we also observed that ~3% of the TN acquired an activated/memory signature, while ~84% developed a unique activated transcriptional profile hybrid between naïve and activated memory. Pseudotime trajectory analysis provided further evidence that TN with an activated/memory or hybrid phenotype were derived from TN. Our data reveal a non-cytotoxic function of TMem with potential to activate autologous TN into the activated/memory pool. These findings may have implications for host-protection and autoimmunity that arises after vaccination, infection or transplantation.

Innate allorecognition in transplantation.

Successful allogeneic transplantation has been made possible by suppressing activation of the adaptive immune system. Current immunosuppressive therapy prevents rejection by targeting T and B cells. Despite this effective treatment, it is the innate immune system, which includes dendritic cells, monocytes, natural killer cells, that is responsible for the initiation of the adaptive immune response. Recent work has described that the innate immune system is capable of recognizing allogeneic nonself and some of the mechanisms of innate allorecognition have been uncovered. Better understanding of the role of the innate immune system in initiation and maintenance of the allo-immune response has potential to lead to better treatment strategies for transplant patients, prolonging allograft survival. Here, we review advances in our understanding of innate allorecognition in transplantation.

Resident memory T cells form during persistent antigen exposure leading to allograft rejection.

Tissue-resident memory T cells (TRM) contained at sites of previous infection provide local protection against reinfection. Whether they form and function in organ transplants where cognate antigen persists is unclear. This is a key question in transplantation as T cells are detected long term in allografts, but it is not known whether they are exhausted or are functional memory T cells. Using a mouse model of kidney transplantation, we showed that antigen-specific and polyclonal effector T cells differentiated in the graft into TRM and subsequently caused allograft rejection. TRM identity was established by surface phenotype, transcriptional profile, and inability to recirculate in parabiosis and retransplantation experiments. Graft TRM proliferated locally, produced interferon-γ upon restimulation, and their in vivo depletion attenuated rejection. The vast majority of antigen-specific and polyclonal TRM lacked phenotypic and transcriptional exhaustion markers. Single-cell analysis of graft T cells early and late after transplantation identified a transcriptional program associated with transition to the tissue-resident state that could serve as a platform for the discovery of therapeutic targets. Thus, recipient effector T cells differentiate into functional graft TRM that maintain rejection locally. Targeting these TRM could improve renal transplant outcomes.

Innate Allorecognition and Memory in Transplantation.

Over the past few decades, we have witnessed a decline in the rates of acute rejection without significant improvement in chronic rejection. Current treatment strategies principally target the adaptive immune response and not the innate response. Therefore, better understanding of innate immunity in transplantation and how to target it is highly desirable. Here, we review the latest advances in innate immunity in transplantation focusing on the roles and mechanisms of innate allorecognition and memory in myeloid cells. These novel concepts could explain why alloimmune response do not abate over time and shed light on new molecular pathways that can be interrupted to prevent or treat chronic rejection.

PIRs mediate innate myeloid cell memory to nonself MHC molecules.

Immunological memory specific to previously encountered antigens is a cardinal feature of adaptive lymphoid cells. However, it is unknown whether innate myeloid cells retain memory of prior antigenic stimulation and respond to it more vigorously on subsequent encounters. In this work, we show that murine monocytes and macrophages acquire memory specific to major histocompatibility complex I (MHC-I) antigens, and we identify A-type paired immunoglobulin-like receptors (PIR-As) as the MHC-I receptors necessary for the memory response. We demonstrate that deleting PIR-A in the recipient or blocking PIR-A binding to donor MHC-I molecules blocks memory and attenuates kidney and heart allograft rejection. Thus, innate myeloid cells acquire alloantigen-specific memory that can be targeted to improve transplant outcomes.

Cross-dressed dendritic cells sustain effector T cell responses in islet and kidney allografts.

Activation of host T cells that mediate allograft rejection is a 2-step process. The first occurs in secondary lymphoid organs where T cells encounter alloantigens presented by host DCs and differentiate to effectors. Antigen presentation at these sites occurs principally via transfer of intact, donor MHC-peptide complexes from graft cells to host DCs (cross-dressing) or by uptake and processing of donor antigens into allopeptides bound to self-MHC molecules (indirect presentation). The second step takes place in the graft, where effector T cells reengage with host DCs before causing rejection. How host DCs present alloantigens to T cells in the graft is not known. Using mouse islet and kidney transplantation models, imaging cytometry, and 2-photon intravital microscopy, we demonstrate extensive cross-dressing of intragraft host DCs with donor MHC-peptide complexes that occurred early after transplantation, whereas host DCs presenting donor antigen via the indirect pathway were rare. Cross-dressed DCs stably engaged TCR-transgenic effector CD8+ T cells that recognized donor antigen and were sufficient for sustaining acute rejection. In the chronic kidney rejection model, cross-dressing declined over time but was still conspicuous 8 weeks after transplantation. We conclude that cross-dressing of host DCs with donor MHC molecules is a major antigen presentation pathway driving effector T cell responses within allografts.

Donor SIRPα polymorphism modulates the innate immune response to allogeneic grafts.

Mice devoid of T, B, and natural killer (NK) cells distinguish between self and allogeneic nonself despite the absence of an adaptive immune system. When challenged with an allograft, they mount an innate response characterized by accumulation of mature, monocyte-derived dendritic cells (DCs) that produce interleukin-12 and present antigen to T cells. However, the molecular mechanisms by which the innate immune system detects allogeneic nonself to generate these DCs are not known. To address this question, we studied the innate response of Rag2-/- γc-/- mice, which lack T, B, and NK cells, to grafts from allogeneic donors. By positional cloning, we identified that donor polymorphism in the gene encoding signal regulatory protein α (SIRPα) is a key modulator of the recipient's innate allorecognition response. Donors that differed from the recipient in one or both Sirpa alleles elicited an innate alloresponse. The response was mediated by binding of donor SIRPα to recipient CD47 and was modulated by the strength of the SIRPα-CD47 interaction. Therefore, sensing SIRPα polymorphism by CD47 provides a molecular mechanism by which the innate immune system distinguishes between self and allogeneic nonself independently of T, B, and NK cells.

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.

Effects of rosiglitazone (PPAR†γ agonist) on the myocardium in non-hypertensive diabetic rats (PPAR γ).

BACKGROUND: There is ongoing controversy regarding the safety of rosiglitazone and its effects on the myocardium, in some cases causing severe cardiac pathology and even in some instances mortality. In this study we aimed at examining the effects of pharmacologic doses of rosiglitazone on cardiomyocytes in diabetic non-cardiac rats receiving sub-optimal doses of insulin. METHODS: Animals were distributed into six groups: normal, diabetic, and diabetic receiving insulin, each subdivided into a control group and an experimental group receiving pharmacologic doses of rosiglitazone. Cardiomyocyte hypertrophy was assessed using heart to body weight index and microscopic examination using the number of cardiomyocytes per quadrant of high power field and intercalated disks in a sector of 100 × field. Fibrosis was assessed using Masson Trichrome staining. A number of sections of each group were stained with periodic acid Shiff and others with Sudan III for glycogen and fat accumulation, respectively. One way ANOVA was used for statistical analysis as appropriate. RESULTS: Diffuse cardiomyopathic changes in diabetic control animals were observed consisting of cardiomyocyte hypertrophy, loss of striations and widespread vacuolation. These changes were reduced and even prevented by treatment with insulin and rosiglitazone. Masson staining showed that all rat groups had no more than +1 fibrosis that is equal to what was present in the normal controls. CONCLUSION: Rosiglitazone, in combination with even sub-optimal doses of insulin therapy, has protective effects on cardiac muscle in diabetic animals especially those expressed as muscle hypertrophy, muscle cell death, and fibrosis.