CD8 T cell is an essential component of tumor-infiltrating lymphocytes (TIL) and tumor immune microenvironment (TIME). Using the xCell CD8 T cell score of whole tumor gene expression data, we estimated these cells in total of 3837 breast cancer patients from TCGA, METABRIC and various GEO cohorts. The CD8 score correlated strongly with expression of CD8 genes. The score was highest for triple-negative breast cancer (TNBC), and a high score was associated with high tumor immune cytolytic activity and better survival in TNBC but not other breast cancer subtypes. In TNBC, tumors with a high CD8 score had enriched expression of interferon (IFN)-α and IFN-γ response and allograft rejection gene sets, and greater infiltration of anti-cancerous immune cells. The score strongly correlated with CD4 memory T cells in TNBC, and tumors with both a high CD8 score and high CD4 memory T cell abundance had significantly better survival. Finally, a high CD8 score was significantly associated with high expression of multiple immune checkpoint molecules. In conclusion, a high CD8 T cell score is associated with better survival in TNBC, particularly when tumor CD4 memory T cells were elevated. Our findings also suggest a possible use of the score as a predictive biomarker for response to immune checkpoint therapy.

Keywords: CD4 memory T cells; CD8 T cells; biomarker; cytolytic activity; immune cell; survival analysis; triple negative breast cancer; tumor immune microenvironment; tumor infiltrating lymphocyte; xCell.

The clinical importance of subclinical, early T-cell mediated rejection (Banff TCMR 1A and borderline lesions) remains unclear, due, in part to the fact that histologic lesions used to characterize early TCMR can be non-specific. Donor-derived cell-free DNA (dd-cfDNA) is an important molecular marker of active graft injury. Over a study period from June 2017 to May 2019, we assessed clinical outcomes in 79 patients diagnosed with TCMR 1A/borderline rejection across 11 United States transplant centers with a simultaneous measurement of dd-cfDNA. 42 patients had elevated dd-cfDNA (≥0.5%) while 37 patients had low levels (<0.5%). Elevated levels of dd-cfDNA predicted adverse clinical outcomes: among patients with elevated cfDNA, estimated glomerular filtration rate declined by 8.5% (IQR -16.22% to -1.39%) (-3.50 mL/min/1.73m² IQR -8.00 to -1.00) vs. 0% (-4.92%, 4.76%) in low dd-cfDNA patients (p=0.004), de novo donor-specific antibody formation was seen in 40% (17/42) vs. 2.7% (p<0.0001), and future or persistent rejection occurred in 9/42 patients (21.4%) vs. 0% (p=0.003). The use of dd-cfDNA may complement the Banff classification and to risk stratify patients with borderline/TCMR 1A identified on biopsy.

Limited data exist on safety and efficacy of immune checkpoint inhibitors (ICIs) among organ transplant recipients. The objective of this study was to report a case series of two patients with renal transplant who received treatment with an ICI and to conduct a pooled analysis of published cases to describe the safety and efficacy of ICIs in organ transplant patients. A systematic search in the Google Scholar and PubMed databases was carried out to include all the published cases of organ transplant patients who received treatment with ICIs including programmed cell death protein 1 (PD-1), programmed death-ligand 1, or cytotoxic lymphocyte antigen-4 inhibitors since their inscription to January 31, 2019. In the present series of two cases with renal allografts who received pembrolizumab, one patient with squamous cell carcinoma of the skin experienced complete response (CR), whereas another patient with melanoma had a mixed response. Both patients experienced allograft rejection, but graft was salvaged. The pooled analysis of 64 patients published in literature showed that overall allograft rejection rate is 41% in organ transplant recipients following ICI therapy. The graft rejection rate was 44% (17/39) for renal, 39% (7/19) for liver, and 20% (1/5) for cardiac allografts. The highest risk was seen among patients who were treated with PD-1 inhibitors, 20/42 (48%)-13/24 (54%) on nivolumab and 7/18 (39%) on pembrolizumab. The risk was lowest with ipilimumab, 23% (3/13). The overall response rate (CR + partial response [PR]) was 20% with ipilimumab, 26% with nivolumab, and 53% with pembrolizumab, whereas disease control rate (CR + PR + stable disease) was 35% with ipilimumab, 37% with nivolumab, and 53% with pembrolizumab. None of the variables including age, gender, type of cancer, type of allograft, type of immunosuppression, time since transplantation to initiation of ICI, and prior history of rejection were significantly associated with the transplant rejection on univariate analysis. The efficacy of ICI among patients with organ transplant appears promising, warranting testing in prospective clinical trials. The risk of rejection and allograft loss is considerable; therefore, the risk and alternative form of therapies should be thoroughly discussed with the transplant patients prior to initiating ICI therapy. IMPLICATIONS FOR PRACTICE: Transplant recipients are at higher risk of developing cancers. Although immune checkpoint inhibitors have been shown to improve the outcome in more than one cancer type, transplant recipients were excluded from these trials. Most of the data on the safety and efficacy of immune checkpoint inhibitors in transplant patients are based upon case series and case reports. The pooled data from these reports suggest that anti-programmed death-ligand 1 inhibitors have reasonable safety and efficacy among organ transplant patients, which warrants testing in clinical trials.

Background: Little is known about immune cell infiltrate type in the kidney allograft of patients with chronic-active antibody-mediated rejection (c-aABMR). Methods: In this study, multiplex immunofluorescent staining was performed on 20 cases of biopsy-proven c-aABMR. T-cell subsets (CD3, CD8, Foxp3, and granzyme B), macrophages (CD68 and CD163), B cells (CD20), and natural killer cells (CD57) were identified and counted in the glomeruli (cells/glomerulus) and the tubulointerstitial (TI) compartment [cells/high-power field (HPF)]. Results: In the glomerulus, T cells and macrophages were the dominant cell types with a mean of 5.5 CD3⁺ cells/glomerulus and 4 CD68⁺ cells/glomerulus. The majority of T cells was CD8⁺ (62%), and most macrophages were CD68⁺CD163⁺ (68%). The TI compartment showed a mean of 116 CD3⁺ cells/HPF, of which 54% were CD8⁺. Macrophage count was 21.5 cells/HPF with 39% CD68⁺CD163⁺. CD20⁺ cells were sporadically present in glomeruli, whereas B-cell aggregates in the TI compartment were frequently observed. Natural killer cells were rarely identified. Remarkably, increased numbers of CD3⁺FoxP3⁺ cells in the TI compartment were associated with decreased graft survival (p = 0.004). Conclusions: Renal allograft biopsies showing c-aABMR show a predominance of infiltrating CD8⁺ T cells, and increased numbers of interstitial FoxP3⁺ T cells are associated with inferior allograft survival.

Heme Oxygen-1 (HO-1)-modified bone marrow mesenchymal stem cells (BMMSCs) are effective to protect and repair transplanted small bowel and intestinal epithelial cells (IECs); however, the mechanism and the role of HO-1/BMMSCs-derived exosomes is unclear. In the present study, we aimed to verify that exosomes from a HO-1/BMMSCs and IEC-6 cells (IEC-6s) co-culture system could reduce the apoptosis of IEC-6s and decrease the expression of the tight junction protein, zona occludens 1, in the inflammatory environment. Using mass spectrometry, we revealed that high mobility group box 3 (HMGB3) and phosphorylated c-Jun NH2-terminal kinase (JNK), under the influence of differentially abundant proteins identified through proteomic analysis, play critical roles in the mechanism. Further studies indicated that microRNA miR-200b, which was upregulated in exosomes derived from the co-culture of HO-1/BMMSCs and IEC-6s, exerted its role by targeting the 3' untranslated region of Hmgb3 in this biological process. Functional experiments confirmed that miR-200b overexpression could reduce the inflammatory injury of IEC-6s, while intracellular miR-200b knockdown could significantly block the protective effect of HO-1/BMMSCs exosomes on the inflammatory injury of IEC-6s. In addition, the level of miR-200b in cells and exosomes derived from HO-1/BMMSCs stimulated by tumor necrosis factor alpha was significantly upregulated. In a rat small bowel transplantation model of allograft rejection treated with HO-1/BMMSCs, we confirmed that the level of miR-200b in the transplanted small bowel tissue was increased significantly, while the level of HMGB3/JNK was downregulated significantly. In conclusion, we identified that exosomes derived from HO-1/BMMSCs play an important role in alleviating the inflammatory injury of IECs. The mechanism is related to miR-200b targeting the abnormally increased expression of the Hmgb3 gene in IECs induced by inflammatory injury. The reduced level of HMGB3 then decreases the inflammatory injury.

Regulatory T cells (Tregs) are a subpopulation of CD4⁺ T cells with a fundamental role in maintaining immune homeostasis and inhibiting unwanted immune responses using several different mechanisms. Recently, the intercellular transfer of molecules between Tregs and their target cells has been shown via trogocytosis and the release of small extracellular vesicles (sEVs). In this study, CD4⁺CD25⁺CD127^lo human Tregs were found to produce sEVs capable of inhibiting the proliferation of effector T cells (Teffs) in a dose dependent manner. These vesicles also modified the cytokine profile of Teffs leading to an increase in the production of IL-4 and IL-10 whilst simultaneously decreasing the levels of IL-6, IL-2, and IFNγ. MicroRNAs found enriched in the Treg EVs were indirectly linked to the changes in the cytokine profile observed. In a humanized mouse skin transplant model, human Treg derived EVs inhibited alloimmune-mediated skin tissue damage by limiting immune cell infiltration. Taken together, Treg sEVs may represent an exciting cell-free therapy to promote transplant survival.

Keywords: Regulatory T (Treg) cells; allograft rejection; cytokines; extracellular vesicles; miRNA – microRNA.

Post-transplantation cyclophosphamide (PTCy) has been highly successful at preventing severe acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). The clinical application of this approach was based on extensive studies in major histocompatibility complex (MHC)-matched murine skin allografting models, in which cyclophosphamide was believed to act via three main mechanisms: (1) selective elimination of alloreactive T cells; (2) intrathymic clonal deletion of alloreactive T-cell precursors; and (3) induction of suppressor T cells. In these models, cyclophosphamide was only effective in very specific contexts, requiring particular cell dose, cell source, PTCy dose, and recipient age. Achievement of transient mixed chimerism also was required. Furthermore, these studies showed differences in the impact of cyclophosphamide on transplanted cells (tumor) versus tissue (skin grafts), including the ability of cyclophosphamide to prevent rejection of the former but not the latter after MHC-mismatched transplants. Yet, clinically PTCy has demonstrated efficacy in MHC-matched or partially-MHC-mismatched HCT across a wide array of patients and HCT platforms. Importantly, clinically significant acute GVHD occurs frequently after PTCy, inconsistent with alloreactive T-cell elimination, whereas PTCy is most active against severe acute GVHD and chronic GVHD. These differences between murine skin allografting and clinical HCT suggest that the above-mentioned mechanisms may not be responsible for GVHD prevention by PTCy. Indeed, recent work by our group in murine HCT has shown that PTCy does not eliminate alloreactive T cells nor is the thymus necessary for PTCy's efficacy. Instead, other mechanisms appear to be playing important roles, including: (1) reduction of alloreactive CD4⁺ effector T-cell proliferation; (2) induced functional impairment of surviving alloreactive CD4⁺ and CD8⁺ effector T cells; and (3) preferential recovery of CD4⁺ regulatory T cells. Herein, we review the history of cyclophosphamide's use in preventing murine skin allograft rejection and our evolving new understanding of the mechanisms underlying its efficacy in preventing GVHD after HCT. Efforts are ongoing to more fully refine and elaborate this proposed new working model. The completion of this effort will provide critical insight relevant for the rational design of novel approaches to improve outcomes for PTCy-treated patients and for the induction of tolerance in other clinical contexts.

The proteasome is a multicatalytic protease in the cytosol and nucleus of all eukaryotic cells that controls numerous cellular processes through regulated protein degradation. Proteasome inhibitors have significantly improved the survival of multiple myeloma patients. However, clinically approved proteasome inhibitors have failed to show efficacy against solid tumors, neither alone nor in combination with other therapies. Targeting the immunoproteasome with selective inhibitors has been therapeutically effective in preclinical models for several autoimmune diseases and colon cancer. Moreover, immunoproteasome inhibitors prevented the chronic rejection of allogeneic organ transplants. In recent years, it has become apparent that inhibition of one single active center of the proteasome is insufficient to achieve therapeutic benefits. In this review we summarize the latest insights how targeting multiple catalytically active proteasome subunits can interfere with disease progression in autoimmunity, growth of solid tumors, and allograft rejection.

Inflammation has been linked with cancer, but whether it is part of the problem or part of the solution remains to be a matter of debate in breast cancer. Our group and others have demonstrated that inflammation aggravates cancer progression; however, some claim that inflammation may support immune cell infiltration and suppress cancer. We defined the gene set variation analysis of the Molecular Signatures Database Hallmark inflammatory response gene set as the inflammatory pathway score and analyzed 3632 tumors in total from 4 breast cancer cohorts (METABRIC, TCGA, GSE25066, and GSE21094). In the whole breast cancer cohort, high-score tumors were associated with aggressive clinical characteristics, such as worse disease specific survival, higher Nottingham histological grade, and younger age. Inflammatory score was significantly higher in triple-negative (TNBC) as well as basal and normal subtypes compared with the other subtypes, which suggest that the detrimental effect of high level of inflammation may be because it includes a more aggressive subtype. On the contrary, high score within TNBC was significantly associated with better survival. TNBC with high score enriched not only IFN-α, IFN-γ response, IL-2/STAT5 signaling, Allograft rejection, Complement, p53 pathway, Reactive Oxygen, and Apoptosis but also TNF-α signaling, IL6-JAK-STAT signaling, TGF-β signaling, Coagulation, Angiogenesis, EMT, KRAS signaling, and PI3K-AKT-MTOR signaling gene sets. High score was associated with mainly favorable anticancerous immune cell infiltration as well as Leukocyte fraction, TIL regional fraction, Lymphocyte infiltration, IFN-γ response, TGF-β response, and cytolytic activity scores. Although the inflammatory pathway score was not associated with neoadjuvant treatment response, it associated with expressions of immune checkpoint molecules. In conclusion, inflammation was associated with worse outcome in the whole breast cancer cohort, but with better outcome in TNBC, which was associated with favorable anticancerous immune response and immune cell infiltrations.

Extracellular vesicles (EVs), including exosomes, ectosomes and apoptotic vesicles, play an essential role in communication between cells of the innate and adaptive immune systems. Recent studies showed that EVs released after transplantation of allogeneic tissues and organs are involved in the immune recognition and response leading to rejection or tolerance in mice. After skin, pancreatic islet, and solid organ transplantation, donor-derived EVs were shown to initiate direct inflammatory alloresponses by T cells leading to acute rejection. This occurred through presentation of intact allogeneic MHC molecules on recipient antigen presenting cells (MHC cross-dressing) and subsequent activation of T cells via semi-direct allorecognition. On the other hand, some studies have documented the role of EVs in maternal tolerance of fetal alloantigens during pregnancy and immune privilege associated with spontaneous tolerance of liver allografts in laboratory rodents. The precise nature of the EVs, which are involved in rejection or tolerance, and the cells which produce them, is still unclear. Nevertheless, several reports showed that EVs released in the blood and urine by allografts can be used as biomarkers of rejection. This article reviews current knowledge on the contribution of EVs in allorecognition by T cells and discusses some mechanisms underlying their influence on T cell alloimmunity in allograft rejection or tolerance.

Mesenchymal stem cells (MSC) are used in various clinical and preclinical models for immunomodulation. However, it remains unclear how the immunomodulatory effect of MSC is communicated. MSC-induced immunomodulation is known to be mediated through both MSC-secreted cytokines and direct cell-cell interactions. Recently, it has been demonstrated that metabolically inactive, heat-inactivated MSC (HI-MSC) have similar anti-inflammatory capacities in LPS-induced sepsis compared to viable MSC. To further investigate the immunomodulatory effects of MSC, we introduced MSC and HI-MSC in two animal models with different immunological causes. In the first model, allogeneic hearts were transplanted from C57BL/6 mice to BALB/c recipients. MSC in combination with mycophenolate mofetil (MMF) significantly improved graft survival compared to MMF alone, whereas the application of HI-MSC had no effect on graft survival. We revealed that control MSC dose-dependently inhibited CD3⁺ and CD8⁺ T-cell proliferation in vitro, whereas HI-MSC had no effect. In the second model, sepsis was induced in mice via cecal ligation and puncture (CLP). HI-MSC treatment significantly improved the overall survival whereas control MSC had no effect. In vitro studies demonstrated that HI-MSC are more effectively phagocytosed by monocytes than control MSC and induced cell death in particular of activated CD16⁺ monocytes, which may explain the immune protective effect of HI-MSC in the sepsis model. The results of our study demonstrate that MSC-mediated immunomodulation in sepsis is dependent on a passive recognition of MSC by monocytes, whereas fully functional MSC are required for inhibition of T-cell mediated allograft rejection. © AlphaMed Press 2020 SIGNIFICANCE STATEMENT: Although MSC offer great potential in the treatment of diseases associated with an aberrant immune system, the development of efficacious and reproducible immune therapies is hampered by the paucity of information on MSC's precise mechanism of action. The present study clearly demonstrates that MSC carry out their therapeutic functions through two different mechanisms, which differentially contribute to resolution of disease. An intact MSC cell metabolism contributes to T-cell suppression, whereas MSC structures passively enable monocyte recognition, phagocytosis, and selective apoptosis. It further indicates that viable MSC target different host immune cell populations than metabolically inactive, secretome-deficient MSC. This study highlights the importance of tailoring MSC treatments to different disease types, rather than utilizing MSC therapy as a "one size fit all" solution. This knowledge not only provides insights into MSC's mechanisms of action but also contributes to the development of efficacious MSC therapy through the identification of disease types that can benefit from MSC treatment.

The coronavirus disease 2019 (COVID-19) pandemic required transplant nephrologists, surgeons, and care teams to make decisions about the full spectrum of transplant program operations and clinical practices, in the absence of experience or data. Initially, across the country, there was a reduction in kidney transplant procedures, and a striking pause in the conduct of living donation and living donor transplant surgeries. Aspects of candidate evaluation and follow-up rapidly converted to telehealth. Months into the pandemic, much has been learned from experiences world-wide, yet many questions remain. In this Perspective, we reflect on some of the practice decisions made by the transplant community in the initial response to the pandemic, and consider lessons learned, including related to the risks, benefits, and logistical considerations of proceeding with versus delaying deceased donor transplantation, living donation, and living donor transplantation during the pandemic. We review the evolution of therapeutic strategies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and their use in transplant recipients, current consensus related to immunosuppression management in infected transplant recipients, and emerging information on vaccination against SARS-CoV-2. We share our thoughts on research priorities, discuss the areas in which we are still practicing with uncertainty, and look ahead to the next phase of the pandemic response.

Keywords: Evaluation; Follow-up; Kidney Transplantation; Living Kidney Donation; Pandemic; Screening; Telehealth; Therapeutics; Vaccines; allograft rejection; coronavirus disease 2019 (COVID-19); immunosuppression management; severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

There is growing emphasis on the potential significance of the placental microbiome and microbiome-metabolite interactions in immune responses and subsequent pregnancy outcome, especially in relation to preterm birth (PTB). This review discusses in detail the pathomechanisms of placental inflammatory responses and the resultant maternal-fetal allograft rejection in both microbial-induced and sterile conditions. It also highlights some potential placental-associated predictive markers of PTB for future investigation. The existence of a placental microbiome remains debatable. Therefore, an overview of our current understanding of the state and role of the placental microbiome (if it exists) and metabolome in human pregnancy is also provided. We critical evaluate the evidence for a placental microbiome, discuss its functional capacity through the elaborated metabolic products and also describe the consequent and more established fetomaternal inflammatory responses that stimulate the pathway to preterm premature rupture of membranes, preterm labour and spontaneous PTB.

Keywords: immunopathology; maternal-fetal; microbial pathogenic; placenta; pregnancy.

Background: In kidney transplantation, evaluating mismatches of HLA eplets-small patches of surface-exposed amino acids of the HLA molecule-instead of antigen mismatches might offer a better approach to assessing donor-recipient HLA incompatibility and improve risk assessment and prediction of transplant outcomes.

Methods: To evaluate the effect of number of eplet mismatches (mismatch load) on de novo formation of donor-specific HLA antibodies (DSAs) and transplant outcomes, we conducted a cohort study that included consecutive adult kidney recipients transplanted at a single center from March 2004 to February 2013. We performed retrospective high-resolution genotyping of HLA loci of 926 transplant pairs and used the HLAMatchmaker computer algorithm to count HLA eplet mismatches.

Results: De novo DSAs occurred in 43 (4.6%) patients. Multivariable analysis showed a significant independent association between antibody-verified eplet mismatch load and de novo DSA occurrence and graft failure, mainly explained by DQ antibody-verified eplet effects. The association with DQ antibody-verified eplet mismatches was linear, without a safe threshold at which de novo DSA did not occur. Odds for T cell- or antibody-mediated rejection increased by 5% and 12%, respectively, per antibody-verified DQ eplet mismatch.

Conclusions: Eplet mismatches in HLA-DQ confer substantial risk for de novo DSA formation, graft rejection, and graft failure after kidney transplantation. Mismatches in other loci seem to have less effect. The results suggest that antibody-verified HLA-DQ eplet mismatch load could be used to guide personalized post-transplant immunosuppression. Adoption of molecular matching for DQA₁ and DQB₁ alleles could also help to minimize de novo DSA formation and potentially improve transplant outcomes.

Keywords: HLA; acute allograft rejection; histocompatibility; kidney transplantation; organ allocation.

Type 2 innate lymphoid cells (ILC2s) are a subset of ILCs with critical roles in immunoregulation. However, the possible role of ILC2s as immunotherapy against allograft rejection remains unclear. Here, we show that IL-33 significantly prolonged islet allograft survival. IL-33-treated mice had elevated numbers of ILC2s and regulatory T cells (Tregs). Depletion of Tregs partially abolished the protective effect of IL-33 on allograft survival, and additional ILC2 depletion in Treg-depleted DEREG mice completely abolished the protective effects of IL-33, indicating that ILC2s play critical roles in IL-33-mediated islet graft protection. Two subsets of ILC2s were identified in islet allografts of IL-33-treated mice: IL-10 producing ILC2s (ILC2¹⁰ ) and non-IL-10 producing ILC2s (non-ILC¹⁰ ). Intravenous transfer of ILC2¹⁰ cells, but not non-ILC¹⁰ , prolonged islet allograft survival in an IL-10-dependent manner. Locally transferred ILC2¹⁰ cells led to long-term islet graft survival, suggesting that ILC2¹⁰ cells are required within the allograft for maximal suppressive effect and graft protection. This study has uncovered a major protective role of ILC2¹⁰ in islet transplantation which could be potentiated as a therapeutic strategy.

Keywords: IL-10; IL-33; innate lymphoid cells; islet transplantation; type 1 diabetes.

Background: Circulating extracellular vesicles (EVs) are raising considerable interest as a non-invasive diagnostic tool, as they are easily detectable in biologic fluids and contain a specific set of nucleic acids, proteins, and lipids reflecting pathophysiologic conditions. We aimed to investigate differences in plasma-derived EV surface protein profiles as a biomarker to be used in combination with endomyocardial biopsies (EMBs) for the diagnosis of allograft rejection.

Methods: Plasma was collected from 90 patients (53 training cohort, 37 validation cohort) before EMB. EV concentration was assessed by nanoparticle tracking analysis. EV surface antigens were measured using a multiplex flow cytometry assay composed of 37 fluorescently labeled capture bead populations coated with specific antibodies directed against respective EV surface epitopes.

Results: The concentration of EVs was significantly increased and their diameter decreased in patients undergoing rejection as compared with negative ones. The trend was highly significant for both antibody-mediated rejection and acute cellular rejection (p < 0.001). Among EV surface markers, CD3, CD2, ROR1, SSEA-4, human leukocyte antigen (HLA)-I, and CD41b were identified as discriminants between controls and acute cellular rejection, whereas HLA-II, CD326, CD19, CD25, CD20, ROR1, SSEA-4, HLA-I, and CD41b discriminated controls from patients with antibody-mediated rejection. Receiver operating characteristics curves confirmed a reliable diagnostic performance for each single marker (area under the curve range, 0.727-0.939). According to differential EV-marker expression, a diagnostic model was built and validated in an external cohort of patients. Our model was able to distinguish patients undergoing rejection from those without rejection. The accuracy at validation in an independent external cohort reached 86.5%. Its application for patient management has the potential to reduce the number of EMBs. Further studies in a higher number of patients are required to validate this approach for clinical purposes.

Conclusions: Circulating EVs are highly promising as a new tool to characterize cardiac allograft rejection and to be complementary to EMB monitoring.

Keywords: allograft rejection; biomarker; extracellular vesicles; heart transplant; machine learning.

Endocrine therapy is the gold-standard treatment for ER-positive/HER2-negative breast cancer. Although its clear benefit, patient compliance is poor (50-80%) due to its long administration period and adverse effects. Therefore, a predictive biomarker that can predict whether endocrine therapy is truly beneficial may improve patient compliance. In this study, we use estrogen response early gene sets of gene set enrichment assay algorithm as the score. We hypothesize that the score could predict the response to endocrine therapy and survival of breast cancer patients. A total of 6549 breast cancer from multiple patient cohorts were analyzed. The score was highest in ER-positive/HER2-negative compared to the other subtypes. Earlier AJCC stage, as well as lower Nottingham pathological grade, were associated with a high score. Low score tumors enriched only allograft rejection gene set, and was significantly infiltrated with immune cells, and high cytolytic activity score. A low score was significantly associated with a worse response to endocrine therapy and worse survival in both primary and metastatic breast cancer patients. The hazard ratio was double that of ESR1 expression. In conclusion, the estrogen response early score predicts response to endocrine therapy and is associated with survival in primary and metastatic breast cancer.

Keywords: ER; GSEA; GSVA; biomarker; endocrine; estrogen response; survival; treatment response.

Current immunosuppressive therapy has led to excellent short-term survival rates in organ transplantation. However, long-term graft survival rates are suboptimal, and a vast number of allografts are gradually lost in the clinic. An increasing number of animal and clinical studies have demonstrated that monocytes and macrophages play a pivotal role in graft rejection, as these mononuclear phagocytic cells recognize alloantigens and trigger an inflammatory cascade that activate the adaptive immune response. Moreover, recent studies suggest that monocytes acquire a feature of memory recall response that is associated with a potent immune response. This form of memory is called "trained immunity," and it is retained by mechanisms of epigenetic and metabolic changes in innate immune cells after exposure to particular ligands, which have a direct impact in allograft rejection. In this review article, we highlight the role of monocytes and macrophages in organ transplantation and summarize therapeutic approaches to promote tolerance through manipulation of monocytes and macrophages. These strategies may open new therapeutic opportunities to increase long-term transplant survival rates in the clinic.

Keywords: immune tolerance; macrophages; nanotherapy; organ transplantation; trained immunity.

Cell therapy with autologous donor-specific regulatory T cells (Tregs) is a promising strategy to minimize immunosuppression in transplant recipients. Chimeric antigen receptor (CAR) technology has recently been used successfully to generate donor-specific Tregs and overcome the limitations of enrichment protocols based on repetitive stimulations with alloantigens. However, the ability of CAR-Treg therapy to control alloreactivity in immunocompetent recipients is unknown. We first analyzed the effect of donor-specific CAR Tregs on alloreactivity in naive, immunocompetent mice receiving skin allografts. Tregs expressing an irrelevant or anti-HLA-A2-specific CAR were administered to Bl/6 mice at the time of transplanting an HLA-A2⁺ Bl/6 skin graft. Donor-specific CAR-Tregs, but not irrelevant-CAR Tregs, significantly delayed skin rejection, and diminished donor-specific antibodies (DSAs) and frequencies of DSA-secreting B cells. Donor-specific CAR Treg-treated mice also had a weaker recall DSA response, but normal responses to an irrelevant antigen, demonstrating antigen-specific suppression. When donor-specific CAR Tregs were tested in HLA-A2 sensitized mice, they were unable to delay allograft rejection or diminish DSAs. The finding that donor-specific CAR-Tregs restrain de novo but not memory alloreactivity has important implications for their use as an adoptive cell therapy in transplantation.

Regulatory T cells (T_regs) are important mediators of immunological self-tolerance and homeostasis. Being cluster of differentiation 4⁺Forkhead box protein3⁺ (CD4⁺FOXP3⁺), these cells are a subset of CD4⁺ T lymphocytes and can originate from the thymus (tT_regs) or from the periphery (pT_regs). The malfunction of CD4⁺ T_regs is associated with autoimmune responses such as rheumatoid arthritis (RA), multiple sclerosis (MS), type 1 diabetes (T1D), inflammatory bowel diseases (IBD), psoriasis, systemic lupus erythematosus (SLE), and transplant rejection. Recent evidence supports an opposed role in sepsis. Therefore, maintaining functional T_regs is considered as a therapy regimen to prevent autoimmunity and allograft rejection, whereas blocking T_reg differentiation might be favorable in sepsis patients. It has been shown that T_regs can be generated from conventional naïve T cells, called iT_regs, due to their induced differentiation. Moreover, T_regs can be effectively expanded in vitro based on blood-derived tT_regs. Taking into consideration that the suppressive role of T_regs has been mainly attributed to the expression and function of the transcription factor Foxp3, modulating its expression and binding to the promoter regions of target genes by altering the chromatin histone acetylation state may turn out beneficial. Hence, we discuss the role of histone deacetylation inhibitors as epigenetic modulators of T_regs in this review in detail.

Gynaecologic and breast cancers share some similarities at the molecular level. The aims of our study are to highlight the similarities and differences about IDO1, an important immune-related gene in female cancers. The NGS data from TCGA of cervical squamous cell carcinoma (CESC), ovarian serous cystadenocarcinoma (OV), uterine corpus endometrial carcinoma (UCEC), uterine carcinosarcoma (UCS) and breast invasive carcinoma (BRCA) were analysed to identify molecular features, and clinically significant and potential therapeutic targets of IDO1. We found IDO1 was significantly up-regulated in four gynaecologic cancers and breast cancer. According to breast cancer PAM50 classification scheme, IDO1 expression was higher in tumours of basal than other subtypes and showed better survival prognosis in BRCA and OV. Through immune infiltration analysis, we found a strong correlation between IDO1 and immune cell populations especially for dendritic cells and T cells. In addition, we investigated the association between IDO1 and tumour mutation burden (TMB) and found that IDO1 was significantly correlated with TMB in BRCA and CESC. GSVA revealed that hallmarks significantly correlated with IDO1 were involved in interferon gamma response, allograft rejection and inflammatory response. We also found PD-L1 and LAG3 were highly positive related to IDO1 in gynaecologic cancers when comparing with their corresponding normal tissues. Our results indicated that IDO1 participated in anti-tumour immune process and is correlated with mutation burden. These findings may expand our outlook of potential anti-IDO1 treatments.

Immune suppressive donor regulatory T cells (Tregs) can prevent graft-versus-host disease (GVHD) or solid organ allograft rejection. We previously demonstrated inhibiting STAT3 phosphorylation (pSTAT3) augments FOXP3 expression, stabilizing induced Tregs (iTregs). Here we report human pSTAT3-inhibited iTregs prevent human skin graft rejection and xenogeneic GVHD yet spare donor anti-leukemia immunity. pSTAT3-inhibited iTregs express increased levels of skin-homing CLA antigen, immune suppressive GARP and PD-1, and IL-9 that supports tolerizing mast cells. Further, pSTAT3-inhibited iTregs significantly reduce alloreactive conventional T cells, Th1, and Th17 cells implicated in GVHD and tissue rejection, and impair infiltration by pathogenic Th2 cells. Mechanistically, pSTAT3 inhibition of iTregs provokes a shift in metabolism from oxidative phosphorylation (OxPhos) to glycolysis and reduced electron transport chain activity. Strikingly, co-treatment with coenzyme Q10 (coQ10) restores OxPhos in pSTAT3-inhibited iTregs and augments their suppressive potency. These findings support the rationale for clinically testing the safety and efficacy of metabolically tuned, human pSTAT3-inhibited iTregs to control alloreactive T cells.

Background: Short-chain fatty acids derived from gut microbial fermentation of dietary fiber have been shown to suppress autoimmunity through mechanisms that include enhanced regulation by T regulatory cells (Tregs).

Methods: Using a murine kidney transplantation model, we examined the effects on alloimmunity of a high-fiber diet or supplementation with the short-chain fatty acid acetate. Kidney transplants were performed from BALB/c(H2^d) to B6(H2^b) mice as allografts in wild-type and recipient mice lacking the G protein-coupled receptor GPR43 (the metabolite-sensing receptor of acetate). Allograft mice received normal chow, a high-fiber diet, or normal chow supplemented with sodium acetate. We assessed rejection at days 14 (acute) and 100 (chronic), and used 16S rRNA sequencing to determine gut microbiota composition pretransplantation and post-transplantation.

Results: Wild-type mice fed normal chow exhibited dysbiosis after receiving a kidney allograft but not an isograft, despite the avoidance of antibiotics and immunosuppression for the latter. A high-fiber diet prevented dysbiosis in allograft recipients, who demonstrated prolonged survival and reduced evidence of rejection compared with mice fed normal chow. Allograft mice receiving supplemental sodium acetate exhibited similar protection from rejection, and subsequently demonstrated donor-specific tolerance. Depletion of CD25⁺ Tregs or absence of the short-chain fatty acid receptor GPR43 abolished this survival advantage.

Conclusions: Manipulation of the microbiome by a high-fiber diet or supplementation with sodium acetate modified alloimmunity in a kidney transplant model, generating tolerance dependent on Tregs and GPR43. Diet-based therapy to induce changes in the gut microbiome can alter systemic alloimmunity in mice, in part through the production of short-chain fatty acids leading to Treg cell development, and merits study as a potential clinical strategy to facilitate transplant acceptance.

Keywords: acute allograft rejection; chronic allograft rejection; immunology; tolerance; transplantation.