Cellular strategies to induce immune tolerance after liver transplantation: Clinical perspectives.

Liver transplantation (LT) has become the most efficient treatment for pediatric and adult end-stage liver disease and the survival time after transplantation is becoming longer due to the development of surgical techniques and perioperative management. However, long-term side-effects of immunosuppressants, like infection, metabolic disorders and malignant tumor are gaining more attention. Immune tolerance is the status in which LT recipients no longer need to take any immunosuppressants, but the liver function and intrahepatic histology maintain normal. The approaches to achieve immune tolerance after transplantation include spontaneous, operational and induced tolerance. The first two means require no specific intervention but withdrawing immunosuppressant gradually during follow-up. No clinical factors or biomarkers so far could accurately predict who are suitable for immunosuppressant withdraw after transplantation. With the understanding to the underlying mechanisms of immune tolerance, many strategies have been developed to induce tolerance in LT recipients. Cellular strategy is one of the most promising methods for immune tolerance induction, including chimerism induced by hematopoietic stem cells and adoptive transfer of regulatory immune cells. The safety and efficacy of various cell products have been evaluated by prospective preclinical and clinical trials, while obstacles still exist before translating into clinical practice. Here, we will summarize the latest perspectives and concerns on the clinical application of cellular strategies in LT recipients.

FOXP3 full length splice variant is associated with kidney allograft tolerance.

Background: Progressive decline of allograft function leads to premature graft loss. Forkhead box P3 (FOXP3), a characteristic gene of T-regulatory cells, is known to be essential for auto-antigen tolerance. We assessed the hypothesis that low FOXP3 mRNA splice variant levels in peripheral blood cells early after transplantation are associated with progressive allograft injury. Methods: Blood samples were prospectively collected from 333 incident kidney transplant recipients on the first and 29th postoperative day. We used quantitative polymerase chain reaction to determine transcripts of 3 isotypes of FOXP3 splice variants, including pre-mature FOXP3 and full length FOXP3 (FOXP3fl). We investigated the association between FOXP3 splice variant levels and the declines in estimated glomerular filtration rate (eGFR) of more than 5ml/min/1.73m2 within the first-year post-transplant using logistic regression. Results: We observed lower FOXP3fl levels in recipients with declining eGFR (N = 132) than in recipients with stable eGFR (N = 201), (logarithmic value -4.13 [IQR -4.50 to -3.84] vs -4.00 [4.32 to -3.74], p=0.02). In ad hoc analysis pre-transplant FOXP3fl levels were similar in both groups. The association between FOXP3fl and declining eGFR was confirmed by multivariable analysis adjusted for potential confounding factors (Odds Ratio 0.51, 95% confidence interval 0.28 to 0.91: p=0.02). When stratifying FOXP3fl levels into quartiles, recipients with lower day1 FOXP3fl had the highest rate of declining eGFR (p=0.04). Conclusion: Low FOXP3fl splice variant levels at the first postoperative day in kidney transplant recipients were associated with severe decline of eGFR, a well-known surrogate for hard endpoints.

Disulfiram, an Anti-alcoholic Drug, Targets Macrophages and Attenuates Acute Rejection in Rat Lung Allografts.

Macrophages contribute to post-transplant lung rejection. Disulfiram (DSF), an anti-alcoholic drug, has an anti-inflammatory effect and regulates macrophage chemotactic activity. Here, we investigated DSF efficacy in suppressing acute rejection post-lung transplantation. Male Lewis rats (280-300 g) received orthotopic left lung transplants from Fisher 344 rats (minor histocompatibility antigen-mismatched transplantation). DSF (0.75 mg/h) monotherapy or co-solvent only (50% hydroxypropyl-ß-cyclodextrin) as control was subcutaneously administered for 7 days (n = 10/group). No post-transplant immunosuppressant was administered. Grades of acute rejection, infiltration of immune cells positive for CD68, CD3, or CD79a, and gene expression of monocyte chemoattractant protein and pro-inflammatory cytokines in the grafts were assessed 7 days post-transplantation. The DSF-treated group had significantly milder lymphocytic bronchiolitis than the control group. The infiltration levels of CD68+ or CD3+ cells to the peribronchial area were significantly lower in the DSF than in the control groups. The normalized expression of chemokine ligand 2 and interleukin-6 mRNA in allografts was lower in the DSF than in the control groups. Validation assay revealed interleukin-6 expression to be significantly lower in the DSF than in the control groups. DSF can alleviate acute rejection post-lung transplantation by reducing macrophage accumulation around peripheral bronchi and suppressing pro-inflammatory cytokine expression.

Immune modulation in transplant medicine: a comprehensive review of cell therapy applications and future directions.

Balancing the immune response after solid organ transplantation (SOT) and vascularized composite allotransplantation (VCA) remains an ongoing clinical challenge. While immunosuppressants can effectively reduce acute rejection rates following transplant surgery, some patients still experience recurrent acute rejection episodes, which in turn may progress to chronic rejection. Furthermore, these immunosuppressive regimens are associated with an increased risk of malignancies and metabolic disorders. Despite significant advancements in the field, these IS related side effects persist as clinical hurdles, emphasizing the need for innovative therapeutic strategies to improve transplant survival and longevity. Cellular therapy, a novel therapeutic approach, has emerged as a potential pathway to promote immune tolerance while minimizing systemic side-effects of standard IS regiments. Various cell types, including chimeric antigen receptor T cells (CAR-T), mesenchymal stromal cells (MSCs), regulatory myeloid cells (RMCs) and regulatory T cells (Tregs), offer unique immunomodulatory properties that may help achieve improved outcomes in transplant patients. This review aims to elucidate the role of cellular therapies, particularly MSCs, T cells, Tregs, RMCs, macrophages, and dendritic cells in SOT and VCA. We explore the immunological features of each cell type, their capacity for immune regulation, and the prospective advantages and obstacles linked to their application in transplant patients. An in-depth outline of the current state of the technology may help SOT and VCA providers refine their perioperative treatment strategies while laying the foundation for further trials that investigate cellular therapeutics in transplantation surgery.

Memory B-cell derived donor-specific antibodies do not predict outcome in sensitized kidney transplant recipients: a retrospective single-center study.

Background: Repeated exposure to sensitizing events can activate HLA-specific memory B cells, leading to the production of donor-specific memory B cell antibodies (DSAm) that pose a risk for antibody-mediated rejection (ABMR) in kidney transplant recipients (KTRs). This single-center retrospective study aimed to identify DSAm and assess their association with outcomes in a cohort of KTRs with pretransplant serum donor-specific antibodies (DSA). Methods: We polyclonally activated pretransplant peripheral blood mononuclear cells (PBMCs) from 60 KTRs in vitro, isolated and quantified IgG from the culture supernatant using ELISA, and analyzed the HLA antibodies of eluates with single antigen bead (SAB) assays, comparing them to the donor HLA typing for potential DSAm. Biopsies from 41 KTRs were evaluated for rejection based on BANFF 2019 criteria. Results: At transplantation, a total of 37 DSAm were detected in 26 of 60 patients (43%), of which 13 (35%) were found to be undetectable in serum. No significant association was found between pretransplant DSAm and ABMR (P=0.53). Similar results were observed in a Kaplan-Meier analysis for ABMR within the first year posttransplant (P=0.29). Additionally, MFI levels of DSAm showed no significant association with ABMR (P=0.28). Conclusion: This study suggests no significant association between DSAm and biopsy-proven clinical ABMR. Further prospective research is needed to determine whether assessing DSAm could enhance existing immunological risk assessment methods for monitoring KTRs, particularly in non-sensitized KTRs.

Novel factors potentially initiating acute antibody-mediated rejection in pig kidney xenografts despite an efficient immunosuppressive regimen.

Antibody-mediated rejection (AMR) is a common cause of graft failure after pig-to-nonhuman primate organ transplantation, even when the graft is from a pig with multiple genetic modifications. The specific factors that initiate AMR are often uncertain. We report two cases of pig kidney transplantation into immunosuppressed baboons in which we identify novel factors associated with the initiation of AMR. In the first, membranous nephropathy was the initiating factor that was then associated with the apparent loss of the therapeutic anti-CD154 monoclonal antibody in the urine when severe proteinuria was present. This observation suggests that proteinuria may be associated with the loss of any therapeutic monoclonal antibody, for example, anti-CD154 or eculizumab, in the urine, resulting in xenograft rejection. In the second case, the sequence of events and histopathology tentatively suggested that pyelonephritis may have initiated acute-onset AMR. The association of a urinary infection with graft rejection has been well-documented in ABO-incompatible kidney allotransplantation based on the expression of an antigen on the invading microorganism shared with the kidney graft, generating an immune response to the graft. To our knowledge, these potential initiating factors of AMR in pig xenografts have not been highlighted previously.

MEK1/2-PKM2 Pathway Modulates the Immunometabolic Reprogramming of Proinflammatory Allograft-infiltrating Macrophages During Heart Transplant Rejection.

BACKGROUND: Emerging evidence has highlighted the role of macrophages in heart transplant rejection (HTR). However, the molecular signals modulating the immunometabolic phenotype of allograft-infiltrating macrophages (AIMs) during HTR remain unknown. METHODS: We analyzed single-cell RNA sequencing data from cardiac graft-infiltrating immunocytes to characterize the activation patterns and metabolic features of AIMs. We used flow cytometry to determine iNOS and PKM2 expression and MEK/ERK signaling activation levels in AIMs. We then generated macrophage-specific Mek1/2 knockout mice to determine the role of the MEK1/2-PKM2 pathway in the proinflammatory phenotype and glycolytic capacity of AIMs during HTR. RESULTS: Single-cell RNA sequencing analysis showed that AIMs had a significantly elevated proinflammatory and glycolytic phenotype. Flow cytometry analysis verified that iNOS and PKM2 expressions were significantly upregulated in AIMs. Moreover, MEK/ERK signaling was activated in AIMs and positively correlated with proinflammatory and glycolytic signatures. Macrophage-specific Mek1/2 deletion significantly protected chronic cardiac allograft rejection and inhibited the proinflammatory phenotype and glycolytic capacity of AIMs. Mek1/2 ablation also reduced the proinflammatory phenotype and glycolytic capacity of lipopolysaccharides + interferon-γ-stimulated macrophages. Mek1/2 ablation impaired nuclear translocation and PKM2 expression in macrophages. PKM2 overexpression partially restored the proinflammatory phenotype and glycolytic capacity of Mek1/2 -deficient macrophages. Moreover, trametinib, an Food and Drug Administration-approved MEK1/2 inhibitor, ameliorated chronic cardiac allograft rejection. CONCLUSIONS: These findings suggest that the MEK1/2-PKM2 pathway is essential for immunometabolic reprogramming of proinflammatory AIMs, implying that it may be a promising therapeutic target in clinical heart transplantation.

Design and testing of a humanized porcine donor for xenotransplantation.

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

Luteolin attenuates acute liver allograft rejection in rats by inhibiting T cell proliferation and regulating T cell subsets.

Allograft rejection continues to be a significant cause of morbidity and graft failure for liver transplant recipients. Existing immunosuppressive regimens have many drawbacks, thus safe and effective long-term immunosuppressive regimens are still required. Luteolin (LUT), a natural component found in many plants, has a variety of biological and pharmacological effects and shows good anti-inflammatory activity in inflammatory and autoimmune diseases. Nevertheless, it remains unclear how it affects acute organ rejection after allogeneic transplantation. In this study, a rat liver transplantation model was constructed to investigate the effect of LUT on acute rejection of organ allografts. We found that LUT significantly protected the structure and function of liver grafts, prolonged recipient rat survival, ameliorated T cell infiltration, and downregulated proinflammatory cytokines. Moreover, LUT inhibited the proliferation of CD4+ T cells and Th cell differentiation but increased the proportion of Tregs, which is the key to its immunosuppressive effect. In vitro, LUT also significantly inhibited CD4+ T cell proliferation and Th1 differentiation. There may be important implications for improving immunosuppressive regimens for organ transplantation as a result of this discovery.

Clinical application of immune repertoire sequencing in solid organ transplant.

Background: Measurement of T cell receptor (TCR) or B cell receptor (BCR) gene utilization may be valuable in monitoring the dynamic changes in donor-reactive clonal populations following transplantation and enabling adjustment in therapy to avoid the consequences of excess immune suppression or to prevent rejection with contingent graft damage and to indicate the development of tolerance. Objective: We performed a review of current literature to examine research in immune repertoire sequencing in organ transplantation and to assess the feasibility of this technology for clinical application in immune monitoring. Methods: We searched MEDLINE and PubMed Central for English-language studies published between 2010 and 2021 that examined T cell/B cell repertoire dynamics upon immune activation. Manual filtering of the search results was performed based on relevancy and predefined inclusion criteria. Data were extracted based on study and methodology characteristics. Results: Our initial search yielded 1933 articles of which 37 met the inclusion criteria; 16 of these were kidney transplant studies (43%) and 21 were other or general transplantation studies (57%). The predominant method for repertoire characterization was sequencing the CDR3 region of the TCR ß chain. Repertoires of transplant recipients were found to have decreased diversity in both rejectors and non-rejectors when compared to healthy controls. Rejectors and those with opportunistic infections were more likely to have clonal expansion in T or B cell populations. Mixed lymphocyte culture followed by TCR sequencing was used in 6 studies to define an alloreactive repertoire and in specialized transplant settings to track tolerance. Conclusion: Methodological approaches to immune repertoire sequencing are becoming established and offer considerable potential as a novel clinical tool for pre- and post-transplant immune monitoring.

CD16+ natural killer cells in bronchoalveolar lavage are associated with antibody-mediated rejection and chronic lung allograft dysfunction.

Acute and chronic rejections limit the long-term survival after lung transplant. Pulmonary antibody-mediated rejection (AMR) is an incompletely understood driver of long-term outcomes characterized by donor-specific antibodies (DSAs), innate immune infiltration, and evidence of complement activation. Natural killer (NK) cells may recognize DSAs via the CD16 receptor, but this complement-independent mechanism of injury has not been explored in pulmonary AMR. CD16+ NK cells were quantified in 508 prospectively collected bronchoalveolar lavage fluid samples from 195 lung transplant recipients. Associations between CD16+ NK cells and human leukocyte antigen mismatches, DSAs, and AMR grade were assessed by linear models adjusted for participant characteristics and repeat measures. Cox proportional hazards models were used to assess CD16+ NK cell association with chronic lung allograft dysfunction and survival. Bronchoalveolar lavage fluid CD16+ NK cell frequency was associated with increasing human leukocyte antigens mismatches and increased AMR grade. Although NK frequencies were similar between DSA+ and DSA- recipients, CD16+ NK cell frequencies were greater in recipients with AMR and those with concomitant allograft dysfunction. CD16+ NK cells were associated with long-term graft dysfunction after AMR and decreased chronic lung allograft dysfunction-free survival. These data support the role of CD16+ NK cells in pulmonary AMR.

Effects of periodontal pathogen-induced intestinal dysbiosis on transplant immunity in an allogenic skin graft model.

Periodontal disease can induce dysbiosis, a compositional and functional alteration in the microbiota. Dysbiosis induced by periodontal disease is known to cause systemic inflammation and may affect transplant immunity. Here, we examined the effects of periodontal disease-related intestinal dysbiosis on transplant immunity using a mouse model of allogenic skin graft in which the mice were orally administered the periodontal pathogen Porphyromonas gingivalis (Pg). For 6 weeks, the Pg group orally received Pg while the control group orally received phosphate-buffered saline solution. After that, both groups received allogenic skin grafts. 16 s rRNA analysis of feces revealed that oral administration of Pg significantly increased three short chain fatty acids (SCFAs) producing genera. SCFA (acetate and propionate) levels were significantly higher in the Pg group (p = 0.040 and p = 0.005). The ratio of regulatory T cells, which are positively correlated with SCFAs, to total CD4+ T cells in the peripheral blood and spleen was significantly greater (p = 0.002 and p < 0.001) in the Pg group by flowcytometry. Finally, oral administration of Pg significantly prolonged skin graft survival (p < 0.001) and reduced pathological inflammation in transplanted skin grafts. In conclusion, periodontal pathogen-induced intestinal dysbiosis may affect transplant immunity through increased levels of SCFAs and regulatory T cells. (198 words).

Rejection and graft outcomes in kidney transplant recipients with and without angiotensin II receptor type 1 antibodies.

AIM: Angiotensin II type 1 receptor antibody (AT1R Ab) is a non-Human Leucocyte Antigen (HLA) antibody that is maybe associated with early severe kidney transplant rejection and worse graft outcomes. This study aimed to assess the association between AT1R Ab and kidney transplant rejection and graft outcomes. METHODS: We performed a retrospective analysis of all adult kidney transplant recipients in an Australian centre who had an AT1R Ab test between 1 January 2015 to 30 June 2020. AT1R Ab positive patients were compared to AT1R Ab negative patients. Primary outcomes were rejection risk, type and histopathological severity scores. Secondary outcomes were 8-week graft function and graft loss. RESULTS: Of 965 kidney transplants that were performed during the study period, 73 patients had AT1R Ab tested; 16 (22%) were positive and 57(78%) were negative. Positive patients were on average younger and had higher level of donor-specific HLA antibodies. Rejection occurred in 13 (81%) positive patients and 41 (72%) negative patients (P = 0.45). No significant differences in rejection type or severity were found. HLA mismatch and peak panel reactive antibody ≥80%, but not AT1R Ab, independently predicted rejection. Average (132 vs. 177 mmol/L, P = 0.302) and graft loss were not significantly different between groups. CONCLUSION: The study found no evidence that AT1R Ab is associated with rejection type, severity or worse graft function. Future studies should assess its relationship with graft outcomes to help complement immunological risk assessment and potentially provide therapeutic options to alter outcomes.

Pre-transplant angiotensin II receptor type I antibodies in pediatric renal transplant recipients: An observational cohort study.

BACKGROUND: The role of angiotensin II type 1 receptor antibodies (AT1R-Ab) in pediatric renal transplantation is unclear. Here, we evaluated pre-transplant AT1R-Ab on transplant outcomes in the first 5 years. Secondary analysis compared pre-transplant AT1R-Ab levels by age. METHODS: Thirty-six patients, 2-20 years old, were divided into two groups: pre-transplant AT1R-Ab- (<17 U/ml; n = 18) and pre-transplant AT1R-Ab+ (≥17 U/ml; n = 18). eGFR was determined at 6-month, 1-, 2-, and 4-year post-transplant. Allograft biopsies were performed in the setting of strong HLA-DSA (MFI > 10 000), AT1R-Ab ≥17 U/ml, and/or elevated creatinine. RESULTS: Mean age in pre-transplant AT1R-Ab- was 13.3 years vs. 11.0 in pre-transplant AT1R-Ab+ (p = 0.16). At 6 months, mean eGFR was 111.3 ml/min/1.73 m2 in pre-transplant AT1R-Ab- vs. 100.2 in pre-transplant AT1R-Ab + at 1 year, 103.6 ml/min/1.73 m2 vs. 100.5; at 2 years, 98.9 ml/min/1.73 m2 vs. and 93.7; at 4 years, 72.6 ml/min/1.73 m2 vs. 80.9. 11/36 patients had acute rejection (6 in pre-transplant AT1R-Ab-, 5 in pre-transplant AT1R-Ab + ). There was no difference in rejection rates. All 6 subjects with de novo HLA-DSA and AT1R-Ab ≥17 U/ml at the time of biopsy experienced rejection. Mean age in those with the AT1R-Ab ≥40 U/ml was 10.0 years vs. 13.2 in those <40 U/ml (p = 0.07). CONCLUSION: In our small cohort, pre-transplant AT1R-Ab ≥17 U/ml was not associated with reduced graft function or rejection. The pathogenicity of pre-transplant AT1R-Ab in pediatric kidney transplantation requires further investigation.

TFNR2 in Ischemia-Reperfusion Injury, Rejection, and Tolerance in Transplantation.

Tumor necrosis factor receptor 2 (TNFR2) has been shown to play a crucial role in CD4+ T regulatory cells (CD4+Tregs) expansion and suppressive function. Increasing evidence has also demonstrated its role in a variety of immune regulatory cell subtypes such as CD8+ T regulatory cells (CD8+ Tregs), B regulatory cells (Bregs), and myeloid-derived suppressor cells (MDSCs). In solid organ transplantation, regulatory immune cells have been associated with decreased ischemia-reperfusion injury (IRI), improved graft survival, and improved overall outcomes. However, despite TNFR2 being studied in the context of autoimmune diseases, cancer, and hematopoietic stem cell transplantation, there remains paucity of data in the context of solid organ transplantation and islet cell transplantation. Interestingly, TNFR2 signaling has found a clinical application in islet transplantation which could guide its wider use. This article reviews the current literature on TNFR2 expression in immune modulatory cells as well as IRI, cell, and solid organ transplantation. Our results highlighted the positive impact of TNFR2 signaling especially in kidney and islet transplantation. However, further investigation of TNFR2 in all types of solid organ transplantation are required as well as dedicated studies on its therapeutic use during induction therapy or treatment of rejection.

T Cells With Activated STAT4 Drive the High-Risk Rejection State to Renal Allograft Failure After Kidney Transplantation.

In kidney transplantation, deteriorated progression of rejection is considered to be a leading course of postoperative mortality. However, the conventional histologic diagnosis is limited in reading the rejection status at the molecular level, thereby triggering mismatched pathogenesis with clinical phenotypes. Here, by applying uniform manifold approximation and projection and Leiden algorithms to 2,611 publicly available microarray datasets of renal transplantation, we uncovered six rejection states with corresponding signature genes and revealed a high-risk (HR) state that was essential in promoting allograft loss. By identifying cell populations from single-cell RNA sequencing data that were associated with the six rejection states, we identified a T-cell population to be the pathogenesis-triggering cells associated with the HR rejection state. Additionally, by constructing gene regulatory networks, we identified that activated STAT4, as a core transcription factor that was regulated by PTPN6 in T cells, was closely linked to poor allograft function and prognosis. Taken together, our study provides a novel strategy to help with the precise diagnosis of kidney allograft rejection progression, which is powerful in investigating the underlying molecular pathogenesis, and therefore, for further clinical intervention.