Rejection Group Research Articles

Regulatory B Cells Profile in Kidney Transplant Recipients With Chronic-Active Antibody-Mediated Rejection

This study aims to reveal the relationship between regulatory B cell (Breg) subsets and chronic-active antibody-mediated rejection (c-aABMR) in renal transplant recipients. Our study involved 3 groups of participants: renal transplant recipients with biopsy-proven c-aABMR as the chronic rejection group (c-aABMR, n=23), recipients with stable graft functions as the patient control group (PC; n=11), and healthy volunteers (HV; n=11). Breg subsets, immature/transitional B cells, plasmablastic cells, B10 cells, and BR1 cells were isolated from venous blood samples by flow cytometry. The median values of Breg frequencies in the total lymphocyte population were analyzed. There were no significant differences between the study groups for immature and/or transitional B cell frequencies. Plasmablastic cell frequencies of the c-aABMR group (7.80 [2.10-27.40]) and the PC group (6.00 [1.80-55.50]) were similar, but both of these values were significantly higher than the HVs' (3.40 [1.20-8.50]), (respectively, P=.005 and P=.039). B10 cell frequencies were also similar, comparing the c-aABMR (4.20 [0.10-7.40]) and the PC groups (4.10 [0.10-5.90]), whereas the HVs (5.90 [2.90-8.50]) had the highest B10 cell frequency with an only statistical significance against the PC group (respectively, P=.09 and P=.028). The c-aABMR and the PC groups were similar regarding BR1 cell frequencies. However, the HV group significantly had the highest frequency of BR1 cells (5.50 [2.80-10.80]) than the other groups (P < .001 for both). We demonstrated that frequencies of B10 and BR1 cells were higher in HVs than in transplant recipients, regardless of rejection state. However, there was no significant relation between Breg frequencies and the c-aABMR state.

Changes and Significance of Interleukin 17 Expression in Patients After Renal Transplantation

The purpose of this study was to investigate the expression and significance of interleukin (IL)-17 in acute rejection after renal transplantation. From September 2019 to December 2021, patients and healthy volunteers who underwent renal allograft transplantation and renal biopsy in our hospital were selected and divided into 4 groups: the stable group (stable) showed no obvious abnormality in renal transplantation pathology; the pathologic diagnosis of acute rejection was the rejection group; the pathologic diagnosis of renal transplantation was immunosuppressive poisoning in the drug group; and the normal group (control) was healthy volunteers. The expression of IL-17 was detected by reverse transcription-polymerase chain reaction, quantitative enzyme-linked immunosorbent assay, and immunohistochemistry. Analysis of the area under the curve and sensitivity and specificity of IL-17 for the diagnosis of acute rejection after raw transplantation was done using receiver operating characteristic curves. Compared with those in the normal group and the stable group, the expression of IL-17 DNA in the blood, the value of IL-17 in the blood, and the integrated optical density value of IL-17 in the transplanted kidney were significantly higher in the rejection group (P < .05). There were no statistically significant differences in the expression of blood IL-17 DNA, the value of blood IL-17, or the integrated optical density value of transplanted kidney IL-17 between the normal group and the stable group (P > .05). IL-17 is involved in acute rejection after renal transplantation. Increased expression of IL-17 is seen in the blood and kidneys of patients with acute rejection after renal transplantation. The detection of IL-17 may provide a theoretical basis for diagnosing and treating acute rejection in human kidney transplantation.

Assessment of Cytokines as Potential Biomarkers to Predict Allograft Rejection in Kidney Transplant Recipients

Objectives: Allograft rejection is the most common event that occurs posttransplant and the current diagnostic tools like the measurement of serum creatinine or core kidney biopsy could not serve as a predictive tool and have certain drawbacks. Thus, our target was to find potential, non-invasive, and predictive biomarkers which could identify recipients at the risk of post-transplant allograft rejection. Methods: It is a prospective longitudinal cohort study comprising of 40 live-related kidney transplant recipients whose peripheral blood was collected at three time points viz pre, one and threemonths post-transplant to assess the plasma cytokine levels of FOXP3, IL- 6, IL-17 and TGF-b using Enzyme linked immunosorbent assay (ELISA). The patients were clinically followed for period of two years to determine their posttransplant outcome. Findings: 13 of our recipients had biopsy proven allograft rejection at the end of follow-up period. The pre-transplant concentration of FOXP3 were less in the rejection group whereas the concentration of IL-6, IL- 17 and TGF- b were more in the rejection group (p<0.05). The classification accuracy of the markers FOXP3, TGF- b , IL-17 and IL-6 were assessed and they had an AUC of 1.0, 1.0, 0.96 and 0.84 respectively with high sensitivity, specificity, and statistical significance. Novelty: Pre-transplant levels of plasma cytokines were able to predict recipients at the risk of allograft rejection. Thus, our study confirms the predictive power of our cytokine biomarkers FOXP3, IL- 6, IL-17 and TGF-b and further paves way for personalized immunosuppressive regimen. Keywords: Predictive Biomarkers; Allograft Rejection; FOXP3; IL6; IL17; TGFb

Neoadjuvant programmed cell death 1 inhibitor before liver transplantation for HCC is not associated with increased graft loss.

Immune checkpoint inhibitors (ICIs) may lead to rejection and even graft loss of solid organ transplant recipients, making them not widely used in transplant patients. There is insufficient clinical experience in using ICIs as a bridging or downstaging therapy before transplantation. We performed a retrospective review of patients receiving programmed cell death 1 inhibitor (PD1) before liver transplantation for HCC in our center and analyzed the data of these patients with the purpose of investigating the safety and feasibility of preoperative PD1 inhibitor among liver transplant recipients and exploring the preoperative correlation ICIs and the postoperative risk of rejection and immune-related graft loss. A total of 16 patients enrolled in this study. Acute rejection occurred in 9 patients, with an incidence of 56.3%. The median time of rejection was 7 days after surgery. The median FK506 concentration at the time of rejection was 7.1μg/L. All rejection reactions were reversed after adjusting the immunosuppression regimen. The interval between the last PD1 inhibitor and transplantation in the rejection group was shorter than that in the nonrejection group, and there was a statistical difference [21.0 (15.5-27.5) days vs. 60.0 (34.0-167.0) days, p =0.01]. In conclusion, PD1 inhibitor is a safe and feasible method for bridging or downstaging treatment before liver transplantation. Although preoperative PD1 inhibitor may increase the incidence of postoperative rejection, it is not associated with increased immune-related graft loss and patient death.

Assessment of renal allograft rejection with diffusion tensor imaging.

To investigate the value of DTI in differentiation of renal allograft rejection from well-functioning stable allograft, using fractional anisotropy (FA) and apparent diffusion coefficient (ADC) values. In this prospective study, 22 transplant recipients with well-functioning stable allograft (group A) and 20 patients with renal allograft rejection (group B + C) were recruited over a period of 19 months from January 2018 to July 2019. DTI-MRI was performed in all the patients, and FA and ADC values were measured in cortical and medullary regions of the transplanted kidney. On biopsy, graft rejection was classified as acute (group B) (n = 7) and chronic graft rejection (group C) (n = 13) based on the BANNF scoring system. Statistical analysis was performed using STATA v.14.0. Statistically significant difference between group A and group B + C was noted for cortical (p < 0.001), and medullary (p = 0.003) FA values, and cortical (p = 0.020), and medullary (p = 0.046) ADC values. Cortical(p < 0.001) and Medullary(p = 0.020) FA values showed statistically significant difference between group A and group C, and cortical FA value(p = 0.012) also showed statistically significant difference between group B and group C. AUC (to differentiate between renal allograft rejection and well-functioning stable allograft) for cortical, and medullary FA values and cortical and medullary ADC values were 0.853(p < 0.001), 0.757(p = 0.004), 0.709(p = 0.021) and 0.736(p = 0.009), respectively. DTI is a promising functional MRI technique for the non-invasive assessment of renal allograft function. Diffusion parameters, such as FA and ADC values, can be useful in the differentiation of renal allograft rejection from well-functioning stable allograft.

Artificial intelligence promotes shared decision-making through recommending tests to febrile pediatric outpatients.

To promote the shared decision-making (SDM) between patients and doctors in pediatric outpatient departments, this study was designed to validate artificial intelligence (AI) -initiated medical tests for children with fever. We designed an AI model, named Xiaoyi, to suggest necessary tests for a febrile child before visiting a pediatric outpatient clinic. We calculated the sensitivity, specificity, and F1 score to evaluate the efficacy of Xiaoyi's recommendations. The patients were divided into the rejection and acceptance groups. Then we analyzed the rejected examination items in order to obtain the corresponding reasons. We recruited a total of 11,867 children with fever who had used Xiaoyi in outpatient clinics. The recommended examinations given by Xiaoyi for 10,636 (89.6%) patients were qualified. The average F1 score reached 0.94. A total of 58.4% of the patients accepted Xiaoyi's suggestions (acceptance group), and 41.6% refused (rejection group). Imaging examinations were rejected by most patients (46.7%). The tests being time-consuming were rejected by 2,133 patients (43.2%), including rejecting pathogen studies in 1,347 patients (68.5%) and image studies in 732 patients (31.8%). The difficulty of sampling was the main reason for rejecting routine tests (41.9%). Our model has high accuracy and acceptability in recommending medical tests to febrile pediatric patients, and is worth promoting in facilitating SDM.

Implementation of the Management of Anticoagulation in the Periprocedural Period App into an Electronic Health Record: A Cost-Effectiveness Analysis.

The Management of Anticoagulation in the Periprocedural Period (MAPPP) app is a free tool providing up-to-date guidelines on the periprocedural management of patients on long-term anticoagulants. After validating its effectiveness in the post-procedural period, we aimed to study its overall cost-effectiveness. SF-12 surveys were sent to eligible patients, converted into SF-6D forms, and subsequently into quality-adjusted life years (QALYs) to calculate the incremental cost-effectiveness ratio (ICER). The number of 30-day readmissions was used to calculate hospitalization costs, utilizing publicly available data. From 1/1/2018 to 1/31/2019, 642 patients were screened for enrollment, with an overall response rate of 94% (164/175) among the consented and 49% (164/336) among all eligible patients. The average QALY score was 0.7134 (95% CI [0.6836, 0.7431]) for the patients whose treatment plan followed the MAPPP app recommendations (acceptance group) and 0.7104 (95% CI [0.6760, 0.7448]) for those who did not (rejection group), without statistically significant differences. The difference in ICER scores was -$429 866.67, with the negative sign demonstrating that acceptance was the dominant strategy. By utilizing QALYs and ICER scores we have shown that the acceptance of MAPPP app recommendations is the dominant strategy for the periprocedural management of patients on long-term anticoagulation.

Dendritic cells originating exosomal miR-193b-3p induces regulatory T cells to alleviate liver transplant rejection

Exosomes exert considerable influence in mediating regulatory T (Treg) cells differentiation, which attach great importance to attenuating acute cellular rejection after liver transplantation (LT). And, miRNAs are known to play essential roles in cell-cell communication delivered by exosomes. However, the function of exosomal miRNAs in regulating Treg cells after LT remains unknown. Here, we performed an expression profiling analysis of exosome-miRNAs from human plasma after LT and investigated their immunoregulatory effects on Treg cells. Fifty-eight LT patients and nine donors were included in this report. miRNA profiles in plasma exosomes were analyzed using next-generation sequencing. Flow cytometry, HE and multiplex immunofluorescent staining were used to identify Treg cells in the liver and peripheral blood. A lentiviral vector system was used to overexpress miR-193b-3p in dendritic cells (DCs), and exosomes isolated from these transfected cells were co-cultured with spleen lymphocytesin vitro. A quantitative Real-time PCR and enzyme-linked immunosorbent assay were used to detect the expression of cytokines. Treg cell infiltration was increased in the liver along with Th17 and CD8+ T cell, and it was down-regulated in peripheral blood in the acute rejection group. High-throughput sequencing revealed that miR-193b-3p was markedly up-regulated in plasma exosomes of non-rejection LT patients. The NLRP3 inflammasome was screened as a target for miR-193b-3p based on target prediction and functional enrichment analyses. Exosomal miR-193b-3p derived from DCs increased Treg cells as demonstrated in vitro. miR-193b-3p overexpression down-regulated NLRP3 as well as the inflammatory cytokines IL-1β and IL-17A while increasing levels of the cytokines IL-10 and TGF-β. DC derived exosomal miR-193b-3p promoted Treg cells by inhibiting NLRP3 expression. These findings not only provide a new perspective on the mechanisms, but also hold great promise for the treatment or prevention of liver allograft rejection.

Assessing self-driving vehicle awareness in Hungarian rejecting groups

Autonomous vehicles can be seen as a radical innovation that develop innovative aspects in the mobility system, which can affect every part of our lives. Numerous studies have examined the attitudes of consumers toward autonomous vehicles with different models. It is striking that several models used questionnaires, and the empirical investigation of the social aspects of the issue of self-driving cars is complicated by the fact that people have little or no direct experience in Hungary. Research has demonstrated that there are certain groups that are more critical of technology. The aim of the research is to examine the attitudes of Hungarian people towards autonomous vehicles with a special focus and methodology: focusing on „rejection groups”, we perform a series of analyses. The results obtained draw attention to the impact of social processes on innovation. The study contributes to a deeper understanding of the Hungarian population's attitude toward autonomous vehicles and our goal is to enrich further research with our results.

Risk factors for acute rejection in liver transplantation and its impact on the outcomes of recipients

ObjectiveTo determine the risk factors for acute rejection in liver transplantation and its impact on the outcomes of the recipients. MethodsClinicopathological data of 290 patients who underwent liver transplantation from January 2012 to December 2021 at our center were retrospectively evaluated. Patients were grouped into an acute rejection (AR) group and a normal (NM) group based on the confirmed histopathological diagnosis of acute rejection. Univariate and multivariate logistic regression were used to determine the risk factors for acute rejection. Results244 patients were included in the study. Acute rejection occurred in 27 (11.1%) of the patients. Warm ischemia time (P = 0.137), cold ischemia time (P = 0.064) and chronic liver failure (P = 0.001) were potential risk factors for acute rejection. Chronic liver failure (P < 0.001, OR = 8.22, 95% CI = 2.47–27.32) was the independent risk factor. There was no significant difference in overall survival between recipients with acute rejection and those without it (P = 0.985). The 1-, 3- and 5-year overall survival in the NM group was 98.1%, 85.7% and 78.6% respectively vs 88.9%, 82.5% and 82.5% respectively in the AR group. ConclusionAcute rejection does not appear to affect the long-term survival of the recipients. Only chronic liver failure was an independent risk factor for acute rejection. Our findings further illustrate that contradictions still exist on which factors influence acute rejection in liver transplant recipients. SummaryClinicopathological data of 290 liver transplant recipients at our center between January 2012 and December 2021 were retrospectively evaluated to determine the risk factors for acute rejection and its impact on the outcomes of the recipients. 244 patients were included in the analysis. 27 of the 244 experienced acute rejection. Propensity score matching was performed to reduce the confounding effect. Patients were assigned to an acute rejection group (n = 27) and a normal group (n = 54). Chronic liver failure (P < 0.001, OR = 8.22, 95% CI = 2.47–27.32) was the determined to be independent risk factor for acute rejection. Acute rejection did not appear to affect the long-term survival of the recipients and there was no significant difference in overall survival between the patients with acute rejection and those without it.

Investigation of the Association Between the ITPA Gene 94C>A Gene Sequence Variant and Liver Transplant Rejection in Iranian Liver Transplant Recipients.

Inosine triphosphate pyrophosphatase is an enzyme encoded by the ITPA gene and functions to prevent the incorporation of thiopurine nucleotides into DNA and RNA. Thiopurine drug metabolites such as azathioprine and 6-mercaptopurine have been included in the lists of inosine triphosphate pyrophosphatase substrates. Inosine triphosphatase gene alterations are other pharmacogenetic sequence variants possibly involved in thiopurine metabolism. This study aimed to evaluate the possible association between ITPA 94C>A gene sequence variant (C-to-A substitution at nucleotide 94) in liver transplant recipients. The genotyping of ITPA 94C>A was evaluated by the polymerase chain reaction- restriction fragment length polymorphism method in 200 liver transplant recipients as well as 100 controls. Data were analyzed with SPSS statistical software. This study showed statistically significant associations between the CA genotype of the ITPA 94C>A sequence variant and liver transplant in the rejection and nonrejection groups. Moreover, the results reported in this study showed no significant differences between sex, age, and blood group in patients with liver transplant (with or without transplant rejection). Our results indicated that there were statistically significant associations of the CA genotype of ITPA 94C>A sequence variant with liver transplant in the rejection and nonrejection groups. Further studies are recommended.

Quantitative ultrasound for non-invasive evaluation of subclinical rejection in renal transplantation.

This study aimed to investigate the predictive efficacy of shear-wave elastography, superb microvascular imaging (SMI), and CEUS for allograft rejection in kidney transplants without graft dysfunction. From January 2021 to November 2021, 72 consecutive patients who underwent both allograft biopsy and ultrasound were evaluated. Blood test results were obtained within a week of the ultrasound examinations, which were performed before the protocol biopsy. Resistive index (RI), tissue viscoelasticity, vascular index, and quantitative CEUS parameters were measured. Patients were divided based on biopsy results into the rejection and non-rejection groups. Among the 72 patients, 21 patients had pathological characteristics of acute rejection. RI of allograft was significantly higher in the rejection group (p = 0.007), compared to the non-rejection group. There were no significant between-group differences in vascular indices of SMI, mean elasticity, and mean viscosity. Meanwhile, among the parameters obtained by the time-intensity curve on CEUS, the cortical and medullary ratios of average contrast signal intensity, peak enhancement, wash-in area AUC, wash-in perfusion index, wash-out AUC, and wash-in and wash-out AUC were significantly different between the two groups (p < 0.05). In the receiver operating characteristic curve analysis for predicting allograft rejection, the AUC was 0.853 for the combination of six CEUS parameters, RI, and blood urea nitrogen. Among non-invasive quantitative ultrasound measurements, CEUS parameters are the most useful for diagnosing subclinical allograft rejection. Furthermore, the combination of CEUS parameters, RI, and blood urea nitrogen may be helpful for the early detection of renal allograft rejection. • Among non-invasive quantitative ultrasound measurements, CEUS parameters are the most useful for the diagnosis of subclinical allograft rejection. • On CEUS, the C/M ratios of MeanLin, PE, WiAUC, WiPI, WoAUC, and WiWoAUC are significantly lower in the rejection group; the combination of these showed reliable predictive performance for rejection. • The combination of CEUS parameters, RI, and BUN has a high predictive capability for subclinical allograft rejection.

Metabolomics profiling in acute liver transplant rejection in a pediatric population

Pediatric liver transplantation rejection affects 20% of children. Currently, liver biopsy, expensive and invasive, is the best method of diagnosis. Discovery and validation of clinical biomarkers from blood or other biospecimens would improve clinical care. For this study, stored plasma samples were utilized from two cross-sectional cohorts of liver transplant patients at Children’s Healthcare of Atlanta. High resolution metabolic profiling was completed using established methods. Children with (n = 18) or without (n = 25) acute cellular rejection were included in the analysis (n = 43 total). The mean age of these racially diverse cohorts ranged from 12.6 years in the rejection group and 13.6 years in the no rejection group. Linear regression provided 510 significantly differentiating metabolites between groups, and OPLS-DA showed 145 metabolites with VIP > 2. A total of 95 overlapping significant metabolites between OPLS-DA and linear regression analyses were detected. Pathway analysis (p < 0.05) showed bile acid biosynthesis and tryptophan metabolism as the top two differentiating pathways. Network analysis also identified tryptophan and clustered with liver enzymes and steroid use. We conclude metabolic profiling of plasma from children with acute liver transplant rejection demonstrates > 500 significant metabolites. This result suggests that development of a non-invasive biomarker-based test is possible for rejection screening.

Association Between the Intracellular Tacrolimus Concentration in CD3 + T Lymphocytes and CD14 + Monocytes and Acute Kidney Transplant Rejection.

Intracellular tacrolimus concentration in peripheral blood mononuclear cells (PBMCs) (TAC [PBMC] ) has been proposed to better represent its active concentration than its whole blood concentration. As tacrolimus acts on T lymphocytes and other white blood cells, including monocytes, we investigated the association of tacrolimus concentration in CD3 + T lymphocytes (TAC [CD3] ) and CD14 + monocytes (TAC [CD14] ) with acute rejection after kidney transplantation. From a total of 61 samples in this case-control study, 28 samples were obtained during biopsy-proven acute rejection (rejection group), and 33 samples were obtained in the absence of rejection (control group). PBMCs were collected from both cryopreserved (retrospectively) and freshly obtained (prospectively) samples. CD3 + T lymphocytes and CD14 + monocytes were isolated from PBMCs, and their intracellular tacrolimus concentrations were measured. The correlation between tacrolimus whole-blood and intracellular concentrations was poor. TAC [CD3] was significantly lower than TAC [CD14] (median 12.8 versus 81.6 pg/million cells; P < 0.001). No difference in TAC [PBMC] (48.5 versus 44.4 pg/million cells; P = 0.82), TAC [CD3] (13.4 versus 12.5 pg/million cells; P = 0.28), and TAC [CD14] (90.0 versus 72.8 pg/million cells; P = 0.27) was found between the rejection and control groups. However, freshly isolated PBMCs showed significantly higher TAC [PBMC] than PBMCs from cryopreserved samples. Subgroup analysis of intracellular tacrolimus concentrations from freshly isolated cells did not show a difference between rejectors and nonrejectors. Differences in TAC [CD3] and TAC [CD14] between patients with and without rejection could not be demonstrated. However, further optimization of the cell isolation process is required because a difference in TAC [PBMC] between fresh and cryopreserved cells was observed. These results need to be confirmed in a study with a larger number of patients.

Urinary Exosomal Cystatin C and Lipopolysaccharide Binding Protein as Biomarkers for Antibody-Mediated Rejection after Kidney Transplantation.

We aimed to discover and validate urinary exosomal proteins as biomarkers for antibody−mediated rejection (ABMR) after kidney transplantation. Urine and for-cause biopsy samples from kidney transplant recipients were collected and categorized into the discovery cohort (n = 36) and a validation cohort (n = 65). Exosomes were isolated by stepwise ultra-centrifugation for proteomic analysis to discover biomarker candidates for ABMR (n = 12). Of 1820 exosomal proteins in the discovery cohort, four proteins were specifically associated with ABMR: cystatin C (CST3), serum paraoxonase/arylesterase 1, retinol-binding protein 4, and lipopolysaccharide−binding protein (LBP). In the validation cohort, the level of urinary exosomal LBP was significantly higher in the ABMR group (n = 25) compared with the T-cell-mediated rejection (TCMR) group and the no major abnormality (NOMOA) group. Urinary exosomal CST3 level was significantly higher in the ABMR group compared with the control and NOMOA groups. Immunohistochemical staining showed that LBP and CST3 in the glomerulus were more abundant in the ABMR group compared with other groups. The combined prediction probability of urinary exosomal LBP and CST3 was significantly correlated with summed LBP and CST3 intensity scores in the glomerulus and peritubular capillary as well as Banff g + ptc scores. Urinary exosomal CST3 and LBP could be potent biomarkers for ABMR after kidney transplantation.

Vitamin D deficiency may predispose patients to increased risk of kidney transplant rejection.

BACKGROUNDVitamin D deficiency occurs in more than 80% of kidney transplant recipients. Its immunomodulatory effects can predispose transplant recipients to rejection and chronic allograft nephropathy (CAN). This study determined the association between serum 25 (OH) vitamin D, biopsy-proven allograft rejection, and CAN rates.AIMTo determine the relationship between serum 25 (OH) vitamin D level and biopsy-proven allograft rejection and CAN rate in renal transplant recipients.METHODSAdult renal transplant recipients followed at the clinic between January 2013 and 2018 were included. Recipients requiring graft biopsy due to declined function, hematuria, and proteinuria were reviewed. The two groups were compared regarding collected data, including the biopsy results, immunologic parameters, vitamin D, parathyroid hormone (PTH), phosphorus, albumin levels, and graft function tests.RESULTSFifty-two recipients who underwent graft biopsy met the inclusion criteria. In all, 14 recipients had a vitamin D level > 15 ng/mL (group 1) vs ≤ 15 ng/mL (group 2) in 38. In total, 27 patients had biopsy-proven rejection, and 19 had CAN. There was only 1 recipient with biopsy-proven rejection in group 1, whereas there were 24 patients with rejection in group 2. The rejection rate was significantly higher in group 2 than in group 1 (P < 0.001). Four patients were diagnosed with CAN in group 1 vs fifteen in group 2. There was no significant difference in the CAN rate between the two groups. PTH was higher at the time of graft biopsy (P = 0.009, P = 0.022) in group 1 with a mean of 268 pg/mL. Donor-specific antibodies were detected in 14 (56.0%) of the recipients with rejection. Vitamin D level was 9.7 ± 3.4 ng/mL in the rejection group vs 14.7 ± 7.2 in the non-rejection group; this difference was statistically significant (P = 0.003). The albumin levels were significantly lower in patients with rejection than in those without rejection (P = 0.001). In univariate regression analysis of risk factors affecting rejection, sex, serum vitamin D, phosphorus and albumin were found to have an impact (P = 0.027, P = 0.007, P = 0.023, P = 0.008). In multivariate regression analysis, the same factors did not affect rejection.CONCLUSIONThe serum 25 (OH) vitamin D level in kidney transplant recipients remained low. Although low serum vitamin D level emerged as a risk factor for rejection in univariate analysis, this finding was not confirmed by multivariate analysis. Prospective studies are required to determine the effect of serum vitamin D levels on allograft rejection.

216.15: Natural History of CXCR5+ t Follicular-like Helper (TFH) Cells in Kidney Transplant Recipients With Rejection

Introduction: T follicular-like helper cells (Tfh) have been associated with donor-specific antibody (DSA) and antibody-mediated rejection (ABMR) in kidney transplant recipients. However, changes in Tfh according to rejection type are less clear. We sought to describe the natural history of Tfh by rejection type. Methods: A pilot study of 4 recipients with ABMR were age-sex matched to recipients with TCMR, and recipients with no rejection. Prospectively collected blood samples were analysed retrospectively by cytometry by time of flight and manually gated to identify CD3+CD4+CXCR5+ Tfh (Figure 1). Pre-transplant Tfh frequency of CD3+ T cells was compared between rejection groups, pre-transplant DSA and de-novo DSA using student’s T-test. Change in Tfh frequency post-transplant was tested between rejection groups using linear mixed regression. Results: Pre-transplant Tfh frequency was highly variable between individuals (median=3.3%, IQR=1.1-5.9) and not significantly different between rejection groups (p=0.7), those with and without pre-transplant DSA (p=0.4) or those that developed and did not develop de-novo DSA (p=0.7). Early post-transplant Tfh frequency decreased in recipients with subsequent TCMR or stable function but increased in those who developed ABMR (Figure 2). Of the four recipients with AMBR, three had a Tfh frequency increase within 60 days prior to ABMR. Change in Tfh frequency over time was 2.7% (95%CI=0.3-5.2) higher in ABMR than stable recipients, adjusting for pre-transplant DSA and de-novo DSA (p<0.001). There was no difference in the change of Tfh frequency between TCMR and stable recipients. Conclusion: In the early post-transplant period and immediately before rejection, Tfh frequency increased above pre-transplant levels in patients who subsequently developed ABMR. This was not observed in stable patients or those with TCMR. These results will inform timepoints appropriate for screening in larger cohort studies. Kidney Health Australia. Ramaciotti Facility for Human Systems Biology. RPAH Transplant Insititute.

216.13: Spatial Protein Profiling of Leukocyte Infiltrates in Renal Biopsies After Cellular Therapy

Background: Regulatory T cells (Tregs) are powerful modulators of immune function, and are of increasing interest as an adoptive cellular therapy for the treatment of transplant rejection. Elucidate the impact of Treg therapeutics in vivo is crucial to identify the therapeutic efficacy. This study aimed to examine the infiltrated leukocytes to provide a clearer picture of the dynamic process that occurs in the transplanted tissue after cellular therapy compared to rejection. Methods: Cellular infiltrates were assessed by GeoMx digital spatial profiling (DSP) platform in tissue biopsies from three transplant patients who received Treg therapy and two control patients with confirmed rejection, with biopsies taken at similar time points after transplantation (6-12 months). Various regions of interest (ROIs) were selected (9 -12 regions) from each patient with diverse infiltration types (Figure 1). In addition, FOXP3hi CD4+ and FOXP3low/neg CD4+ regions were selected from both groups to assess the protein signatures of these cellular infiltrates. Results: The FOXP3 signal was highly enriched in regions from cell therapy compared to rejection patients, suggesting that infused Tregs are likely homing to the allograft. The B cell marker CD20 was highly expressed in cell therapy ROIs than in rejection ROIs, suggesting a link between infused Tregs and allograft infiltration by B cells. Conversely, the rejection ROIs have a high expression of Ki67, indicating the presence of highly proliferative cells. The monocyte/macrophage markers CD14, CD68, and CD163 were also enriched in the rejection ROIs, suggesting that macrophages may be associated with graft rejection in these patients. Further analysis revealed interesting differences between the cellular infiltrates in FOXP3hi segments from cell therapy patients compared with rejection group such as enrichment in proteins related to signalling, including STAT3 and p-STAT3, and coinciding with enrichment in regulatory molecules, including CTLA-4 and PD-1. VISTA protein was found to be highly enriched in FOXP3hi segments from cell therapy patients, suggesting that VISTA might play a regulatory role in suppressing the alloimmune response. Our analysis suggests that FOXP3hi cells that are present in regions collected from rejecting patients are likely suppressive Tregs, since FOXP3hi cells coincide with enrichment of Tregs-associated markers. Nevertheless, these Tregs failed to control the alloresponse, as demonstrated by the enrichment of CD8 T cells, which were notably represented in FOXP3hi regions in rejection tissues. Conclusion: The GeoMx DSP allowed for an in-depth analysis of leukocyte infiltrates and provided comprehensive information about the molecular processes associated with stable renal allograft in patients receiving Treg therapy, which in turn may assist in understanding the mechanisms of therapeutic immune regulation and optimal timing for immunosuppression minimisation.

Prognostic Value of Left and right ventricular deformation strain analysis on Acute Cellular rejection in Heart Transplant recipients: A 6-year outcome study

PurposeTwo-dimensional (2D) strain analysis is a sensitive method for detecting myocardial dysfunction in acute cellular rejection (ACR) from post-transplant complications. This study aims to evaluate the utility of novel left (LV) and right ventricular (RV) strain parameters for prognostic risk stratification associated with ACR burden at 1-year post transplantation.Methods128 Heart transplant patients, assessed between 2012 and 2018, underwent transthoracic echocardiography and endomyocardial biopsy. 2D strain analysis was performed and history of rejection burden was assessed and grouped according to ACR burden at 1-year post transplantation. The primary endpoint was all-cause mortality at 6-years follow up.Results21 patients met primary the endpoint. Multivariate analysis of 6-year all-cause mortality showed LV global longitudinal strain (LV GLS) (Hazard Ratio [HR] = 1.21, CI = 1.06–1.49), LV early diastolic strain rate (LV ESr) (HR = 1.31, CI = 1.12–1.54), RV GLS (HR = 1.12, CI = 1.02–1.25) and RV ESr (HR = 1.26, CI = 1.12–1.47) were significant predictors of outcome. Univariate analysis also showed LV GLS, LV ESr, RV GLS and RV ESr were significant predictors of outcome. Optimal cut-off for predicting 6-year mortality for LV GLS by receive operator characteristic was 15.5% (sensitivity: 92%, specificity: 79%). Significant reductions (p < 0.05) in LV GLS, RV GLS and LV and RV ESr between rejection groups were seen.ConclusionsNon-invasive LV and RV strain parameters are predictors of mortality in post-transplant patient with ACR. LV GLS and LV ESr are superior to other strain and conventional echo parameters.

Potential correlation of allograft infiltrating group 2 innate lymphoid cells with acute rejection after liver transplantation.

Accumulating evidence indicates the critical roles of group 2 innate lymphoid cells (ILC2s) in immunoregulation. However, the role of ILC2s in acute rejection after liver transplantation (LT) remains elusive. In this study, we analyzed the frequency, counts, and signature cytokines of ILC2s in liver transplant recipients by flow cytometric analysis and multiplex immunofluorescence assay. We also assessed the spatial distribution and correlation between hepatic ILC2s and Treg cells. The changes of ILC2s were dynamically monitored in the mouse LT model. We found that the frequencies of circulating ILC2s were comparable in liver transplant recipients with either rejection or non-rejection compared with the control group. The hepatic ILC2s counts were significantly increased in the rejection group than in the non-rejection and control groups, and a similar trend was observed for Treg cells. In the mouse LT model, allograft infiltrating ILC2s dramatically increased within 14 days post-transplant. The frequency of ILC2s in bone marrow significantly increased at 7 days post-transplant and rapidly decreased at 14 days after LT. Similarly, there was a significant increase in the frequency of splenic ILC2s within two weeks post-transplant. Multiplex immunofluorescence assay showed a close correlation between hepatic ILC2s and Treg cells by analyzing their spatial distribution and distance. In conclusion, the number of allograft infiltrating ILC2s was closely related to rejection after LT. Allograft infiltrating ILC2s may play inhibitory roles in posttransplant immune homeostasis, favoring resolution of liver allograft rejection by interacting with Treg cells or promoting the migration of Tregs cells into the liver allograft.