Single-cell transcriptomic analysis of renal allograft rejection reveals insights into intragraft TCR clonality.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/ß sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8+ T cell clonotypes (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCR-α/ß cDNAs from CD8EXP into Jurkat 76 cells (TCR-/-) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection.

Single cell transcriptomic analysis of renal allograft rejection reveals novel insights into intragraft TCR clonality.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding. We performed combined single cell RNA transcriptomic and TCRα/ß sequencing on rBx from patients with ACR under differing immunosuppression (IS): tacrolimus, iscalimab, and belatacept. TCR analysis revealed a highly restricted CD8 + T cell clonal expansion (CD8 EXP ), independent of HLA mismatch or IS type. Subcloning of TCRα/ß cDNAs from CD8 EXP into Jurkat76 cells (TCR -/- ) conferred alloreactivity by mixed lymphocyte reaction. scRNAseq analysis of CD8 EXP revealed effector, memory, and exhausted phenotypes that were influenced by IS type. Successful anti-rejection treatment decreased, but did not eliminate, CD8 EXP , while CD8 EXP were maintained during treatment-refractory rejection. Finally, most rBx-derived CD8 EXP were also observed in matching urine samples. Overall, our data define the clonal CD8 + T cell response to ACR, providing novel insights to improve detection, assessment, and treatment of rejection.

Reducing Donor-specific Antibody During Acute Rejection Diminishes Long-term Renal Allograft Loss: Comparison of Early and Late Rejection.

BACKGROUND: Reduction in donor-specific antibody (DSA) has been associated with improved renal allograft survival after antibody-mediated rejection (AMR). These observations have not been separately analyzed for early and late AMR and mixed acute rejection (MAR). The purpose of this study was to evaluate long-term responses to proteasome inhibitor-based therapy for 4 rejection phenotypes and to determine factors that predict allograft survival. METHODS: Retrospective cohort study evaluating renal transplant recipients with first AMR episodes treated with proteasome inhibitor-based therapy from January 2005 to July 2015. RESULTS: A total of 108 patients were included in the analysis. Immunodominant DSA reduction at 14 days differed significantly (early AMR 79.6%, early MAR 54.7%, late AMR 23.4%, late MAR 21.1%, P < 0.001). Death-censored graft survival (DCGS) differed at 3 years postrejection (early AMR 88.3% versus early MAR 77.8% versus late AMR 56.7% versus late MAR 54.9%, P = 0.02). Multivariate analysis revealed that immunodominant DSA reduction > 50% at 14 days was associated with improved DCGS (odds ratio, 0.12, 95% CI, 0.02-0.52, P = 0.01). CONCLUSIONS: In summary, significant differences exist across rejection phenotypes with respect to histological and DSA responses. The data suggest that DSA reduction may be associated with improved DCGS in both early and late AMR.

Belatacept-based immunosuppression with simultaneous calcineurin inhibitor avoidance and early corticosteroid withdrawal: A prospective, randomized multicenter trial.

Simultaneous calcineurin inhibitor avoidance (CNIA) and early corticosteroid withdrawal (ESW) have not been achieved primarily due to excessive acute rejection. This trial compared 2 belatacept-based CNIA/ESW regimens with a tacrolimus-based ESW regimen. Kidney transplant recipients were randomized to receive alemtuzumab/belatacept, rabbit anti-thymocyte globulin (rATG)/belatacept, or rATG/tacrolimus. The combinatorial primary endpoint consisted of patient death, renal allograft loss, or a Modification of Diet in Renal Disease-calculated eGFR of <45 mL/min/1.73 m2 at 12 months. Results are reported by treatment group (alemtuzumab/belatacept, rATG/belatacept, and rATG/tacrolimus). Superiority was not observed at 1 year for the primary endpoint (9/107 [8.4%], 15/104 [14.4%], and 14/105 [13.3%], respectively; P = NS) for either belatacept-based regimen. Differences were not observed for secondary endpoints (death, death-censored graft loss, or estimated glomerular filtration rates < 45 mL/min/1.73 m2 ). Differences were observed in biopsy-proved acute cellular rejection (10.3%, 18.3%, and 1.9%, respectively) (P < .001), but not in antibody-mediated rejection, mixed acute rejection, or de novo donor-specific anti-HLA antibodies. Neurologic and electrolyte abnormality adverse events were less frequent under belatacept. Belatacept-based CNIA/ESW regimens did not prove to be superior for the primary or secondary endpoints. Belatacept-treated patients demonstrated an increase in biopsy-proved acute cellular rejection and reduced neurologic and metabolic adverse events. These results demonstrate that simultaneous CNIA/ESW is feasible without excessive acute rejection.

Contrast-Induced Nephropathy in Renal Transplant Recipients: A Single Center Experience.

BACKGROUND: Contrast-induced nephropathy (CIN) in native kidneys is associated with a significant increase in mortality and morbidity. Data regarding CIN in renal allografts are limited, however. We retrospectively studied CIN in renal allografts at our institution: its incidence, risk factors, and effect on long-term outcomes including allograft loss and death. METHODS: One hundred thirty-five renal transplant recipients undergoing 161 contrast-enhanced computed tomography (CT) scans or coronary angiograms (Cath) between years 2000 and 2014 were identified. Contrast agents were iso- or low osmolar. CIN was defined as a rise in serum creatinine (SCr) by >0.3 mg/dl or 25% from baseline within 4 days of contrast exposure. After excluding 85 contrast exposures where patients had no SCr within 4 days of contrast administration, 76 exposures (CT: n = 45; Cath: n = 31) in 50 eligible patients were analyzed. Risk factors assessed included demographics, comorbid conditions, type/volume of contrast agent used, IV fluids, N-acetylcysteine administration, and calcineurin inhibitor use. Bivariate and multivariable analyses were used to assess the risk of CIN. RESULTS: Incidence of CIN was 13% following both, CT (6 out of 45) and Cath (4 out of 31). Significant bivariate predictors of CIN were IV fluid administration (p = 0.05), lower hemoglobin (p = 0.03), and lower albumin (p = 0.02). In a multivariable model, CIN was predicted by N-acetylcysteine (p = 0.03) and lower hemoglobin (p = 0.01). Calcineurin inhibitor use was not associated with CIN. At last follow-up, CIN did not affect allograft or patient survival. CONCLUSION: CIN is common in kidney transplant recipients, and there is room for quality improvement with regards to careful renal function monitoring post-contrast exposure. In our study, N-acetylcysteine exposure and lower hemoglobin were associated with CIN. Calcineurin inhibitor use was not associated with CIN. Our sample size is small, however, and larger prospective studies of CIN in renal allografts are needed.

Pharmacokinetic and pharmacogenetic analysis of immunosuppressive agents after laparoscopic sleeve gastrectomy.

BACKGROUND: Severe obesity has been shown to limit access to renal transplantation in patients with end-stage renal disease (ESRD). Laparoscopic sleeve gastrectomy (LSG) has been performed in the ESRD population to assist in achieving waitlist and transplant eligibility. Little is known about how LSG impacts the bioequivalence of tacrolimus products and immunosuppression pharmacokinetics. METHODS: This was a prospective, open-label, single-dose, crossover, two-period pharmacokinetic (PK) study. The purpose of this study was to assess single-dose PK of immediate-release tacrolimus (IR-TAC), extended-release tacrolimus (ER-TAC), and mycophenolic acid (MPA) in adult ESRD patients post-LSG. RESULTS: Twenty-three subjects were included in the 24-hour PK assessments. The ratio of geometric means between ER-TAC and IR-TAC was 103.5% (90% CI; 89.6%-119.6%) for AUC0-24 and 92.5% (90% CI; 80.4%-106.4%) for Cmax . PK parameters were similar between ER-TAC and IR-TAC, except for Cmin (P=.004) and Cmax (P=.04). MPA AUC0-24 was similar when given with either ER-TAC or IR-TAC (P=.32). Patients expressing CYP3A5*1 genotypes had lower tacrolimus AUC0-24 values vs those with CYP3A5*3/*3 (IR-TACP<.001; ER-TACP=.008). Genotype did not impact MPA PK. CONCLUSION: Dose modification of immunosuppressants post-LSG may not be necessary aside from standard therapeutic drug monitoring.

A Banff Component Scoring-based Histologic Assessment of Bortezomib-based Antibody-mediated Rejection Therapy.

BACKGROUND: Histology remains a cornerstone for antibody-mediated rejection (AMR) diagnosis. Little data exist supporting histology for assessing therapeutic responses. This study evaluates histologic components in assessing AMR therapeutic responses. METHODS: Antibody-mediated rejection was diagnosed using Antibody Working Group criteria and Banff component scoring, and C4d staining data were analyzed. Statistics included independent and paired samples t test, χ(2), Fisher exact, or the Wilcoxon-signed rank test. Fifty-five AMR patients were analyzed. Early AMR was defined as occurring within 6 months after transplantation and treated with a single rituximab dose and 4 bortezomib doses preceded by plasmapheresis. Allograft biopsies were performed within 48 hours of treatment; repeat biopsy was performed 14 to 21 days later. RESULTS: Early AMR demonstrated histologic improvement in mean scores for acute Banff components glomerulitis (g), C4d, g+ peritubular capillaritis (ptc) and acute composite score, but showed deterioration in chronic Banff components tubular atrophy and interstitial fibrosis. Late AMR showed improved mean scores for acute Banff components tubulitis, interstitial inflammation, g, ptc, g + ptc, C4d, and acute composite score, but chronic scores did not change. Significant changes in distribution of Banff scores after treatment were observed for g, C4d, tubular atrophy, and interstitial fibrosis scores in early AMR patients and tubulitis, interstitial inflammation, g, ptc, and C4d in late AMR. CONCLUSIONS: These results show that: (1) Banff component scoring provides insights into histologic responses to AMR therapy and may provide a potential endpoint for clinical AMR trials. (2) Early and late AMR demonstrate differences in acute and chronic Banff components at the time of the AMR diagnostic biopsy, as well as differential responses to AMR therapy.

Prospective evaluation of the toxicity profile of proteasome inhibitor-based therapy in renal transplant candidates and recipients.

BACKGROUND: A prospective intermediate-term evaluation of toxicities associated with bortezomib therapy for antibody-mediated rejection (AMR) and desensitization was conducted. METHODS: Patients were graded for bortezomib-related toxicities: hematologic and gastrointestinal toxicities by Common Terminology Criteria for Adverse Events and peripheral neuropathy by modified Functional Assessment of Cancer Therapy questionnaire and Common Terminology Criteria for Adverse Events. RESULTS: Fifty-one patients treated for AMR and 19 patients treated for desensitization received 96 bortezomib cycles (1.3 mg/m(2) ×4 doses); mean (SD) follow-up was 16.3 (9.0) months. Patients treated for AMR and patients treated for desensitization were similar in age, gender, ethnicity, and baseline peripheral neuropathy. Patients treated for AMR received a mean (SD) of 4.9 (2.0) bortezomib doses in 1.3 (0.5) cycles; and patients treated for desensitization, a mean of 7.3 (1.6) doses in 1.8 (0.4) cycles. Prevalence of diabetes and anemia were higher at baseline in patients treated for AMR. In the AMR cohort, two cases of cytomegalovirus infection, two cases of BK virus infection, and one case of Epstein-Barr virus infection were observed. No cases of viral infection were observed in the desensitization cohort. Malignancies were not observed. Significant bortezomib toxicities included anemia and peripheral neuropathy, which were manageable. Anemia was more common in patients treated for AMR; and peripheral neuropathy, more common in patients treated for desensitization. CONCLUSIONS: Bortezomib-related toxicities in kidney transplant candidates and recipients are low in incidence and severity and vary based on treatment population.

Proteasome inhibition for antibody-mediated allograft rejection.

Antibody-mediated rejection (AMR) is a major risk factor for graft loss following kidney transplantation. Traditional anti-humoral therapies provide suboptimal therapy and they do not deplete plasma cells, which are the source of antibody production. Proteasome inhibitors (PI) have been shown to deplete both transformed and nontransformed plasma cells in human transplant recipients and animal models; and therefore, offer a new paradigm for AMR, ie, plasma cell-targeted therapy. Bortezomib, a first in class proteasome inhibitor approved by the US Food and Drug Administration for treatment of multiple myeloma, has been used to treat AMR in several solid organ transplant recipients. The greatest experience with PI therapy for treating AMR is in kidney transplant recipients. Experiences to date with PI therapy have demonstrated that: (1) early AMR (within the first 6 months post-transplant) responds better than late AMR, and (2) the nature of the plasma cell clonal population influences sensitivity to PI therapy with plasma subsets greater than long-lived bone marrow niche-resident plasma cells. In conclusion, plasma cell-targeted therapy with PIs is a method for targeting plasma cells (the source of antibody production) with a well-elucidated mechanism of action and subsequent points of synergy, thereby providing an exciting new potential means for enhancing anti-humoral therapies.