Natural Antibodies Are Associated With Rejection and Long-term Renal Allograft Loss in a Multicenter International Cohort.

BACKGROUND: Potentially harmful nonhuman leukocyte antigen antibodies have been identified in renal transplantation, including natural immunoglobulin G antibodies (Nabs) reactive to varied antigenic structures, including apoptotic cells. METHODS: In this retrospective, multicenter study, we assessed Nabs by reactivity to apoptotic cells in sera collected from 980 kidney transplant recipients across 4 centers to determine their association with graft outcomes. RESULTS: Elevated pretransplant Nabs were associated with graft loss (hazard ratio [HR] 2.71; 95% confidence interval [CI], 1.15-6.39; P = 0.0232), the composite endpoint of graft loss or severe graft dysfunction (HR 2.40; 95% CI, 1.13-5.10; P = 0.0232), and T cell-mediated rejection (odds ratio [OR] 1.77; 95% CI, 1.07-3.02; P = 0.0310). High pretransplant Nabs together with donor-specific antibodies (DSAs) were associated with increased risk of composite outcomes (HR 6.31; 95% CI, 1.81-22.0; P = 0.0039). In patients with high pretransplant Nabs, the subsequent development of posttransplant Nabs was associated with both T cell-mediated rejection (OR 3.64; 95% CI, 1.61-8.36; P = 0.0021) and mixed rejection (OR 3.10; 95% CI, 1.02-9.75; P = 0.0473). Finally, elevated pre- and posttransplant Nabs combined with DSAs were associated with increased risk of composite outcomes (HR 3.97; 95% CI, 1.51-10.43; P = 0.0052) and T cell-mediated rejection (OR 7.28; 95% CI, 2.16-25.96; P = 0.0016). CONCLUSIONS: The presence of pre- and posttransplant Nabs, together with DSAs, was associated with increased risk of poor graft outcomes and rejection after renal transplantation.

Weak antibody response to three doses of mRNA vaccine in kidney transplant recipients treated with belatacept.

Poor responses to mRNA COVID-19 vaccine have been reported after 2 vaccine injections in kidney transplant recipients (KTRs) treated with belatacept. We analyzed the humoral response in belatacept-treated KTRs without a history of SARS-CoV-2 infection who received three injections of BNT162b2-mRNA COVID-19 vaccine. We also investigated vaccine immunogenicity in belatacept-treated KTRs with prior COVID-19 and characterized symptomatic COVID-19 infections after the vaccine in belatacept-treated KTRs. Among the 62 belatacept-treated KTRs (36 [58%] males), the median age (63.5 years IQR [51-72]), without COVID-19 history, only four patients (6.4%) developed anti-SARS-CoV-2 IgG with low antibody titers (median 209, IQR [20-409] AU/ml). 71% were treated with mycophenolic acid and 100% with steroids in association with belatacept. In contrast, in all the 5 KTRs with prior COVID-19 history, mRNA vaccine induced a strong antibody response with high antibody titers (median 10 769 AU/ml, IQR [6410-20 069]) after two injections. Seroprevalence after three-vaccine doses in 35 non-belatacept-treated KTRs was 37.1%. Twelve KTRs developed symptomatic COVID-19 after vaccination, including severe forms (50% of mortality). Breakthrough COVID-19 occurred in 5% of fully vaccinated patients. Administration of a third dose of BNT162b2 mRNA COVID-19 vaccine did not improve immunogenicity in KTRs treated with belatacept without prior COVID-19. Other strategies aiming to improve patient protection are needed.

CRISPR/Cas9-Engineered HLA-Deleted Glomerular Endothelial Cells as a Tool to Predict Pathogenic Non-HLA Antibodies in Kidney Transplant Recipients.

BACKGROUND: After kidney transplantation, donor-specific antibodies against human leukocyte antigen donor-specific antibodies (HLA-DSAs) drive antibody-mediated rejection (ABMR) and are associated with poor transplant outcomes. However, ABMR histology (ABMRh) is increasingly reported in kidney transplant recipients (KTRs) without HLA-DSAs, highlighting the emerging role of non-HLA antibodies (Abs). METHODS: W e designed a non-HLA Ab detection immunoassay (NHADIA) using HLA class I and II-deficient glomerular endothelial cells (CiGEnCΔHLA) that had been previously generated through CRISPR/Cas9-induced B2M and CIITA gene disruption. Flow cytometry assessed the reactivity to non-HLA antigens of pretransplantation serum samples from 389 consecutive KTRs. The intensity of the signal observed with the NHADIA was associated with post-transplant graft histology assessed in 951 adequate biopsy specimens. RESULTS: W e sequentially applied CRISPR/Cas9 to delete the B2M and CIITA genes to obtain a CiGEnCΔHLA clone. CiGEnCΔHLA cells remained indistinguishable from the parental cell line, CiGEnC, in terms of morphology and phenotype. Previous transplantation was the main determinant of the pretransplantation NHADIA result (P<0.001). Stratification of 3-month allograft biopsy specimens (n=298) according to pretransplantation NHADIA tertiles demonstrated that higher levels of non-HLA Abs positively correlated with increased glomerulitis (P=0.002), microvascular inflammation (P=0.003), and ABMRh (P=0.03). A pretransplantation NHADIA threshold of 1.87 strongly discriminated the KTRs with the highest risk of ABMRh (P=0.005, log-rank test). A multivariate Cox model confirmed that NHADIA status and HLA-DSAs were independent, yet synergistic, predictors of ABMRh. CONCLUSION: The NHADIA identifies non-HLA Abs and strongly predicts graft endothelial injury independent of HLA-DSAs.

COVID-19 severity in kidney transplant recipients is similar to nontransplant patients with similar comorbidities.

Higher rates of severe COVID-19 have been reported in kidney transplant recipients (KTRs) compared to nontransplant patients. We aimed to determine if poorer outcomes were specifically related to chronic immunosuppression or underlying comorbidities. We used a 1:1 propensity score-matching method to compare survival and severe disease-free survival (defined as death and/or need for intensive care unit [ICU]) incidence in hospitalized KTRs and nontransplant control patients between February 26 and May 22, 2020. Patients were matched for risk factors of severe COVID-19: age, sex, body mass index, diabetes mellitus, preexisting cardiopathy, chronic lung disease, and basal renal function. We included 100 KTRs (median age [interquartile range (IQR)]) 64.7 years (55.3-73.1) in three French transplant centers. After a median follow-up of 13 days (7-30), transfer to ICU was required for 34 patients (34%) and death occurred in 26 patients (26%). Overall, 43 patients (43%) developed a severe disease during a median follow-up of 8.5 days (2-14). Propensity score matching to a large French cohort of 2017 patients hospitalized in 24 centers, revealed that survival was similar between KTRs and matched nontransplant patients with respective 30-day survival of 62.9% and 71% (p = .38) and severe disease-free 30-day survival of 50.6% and 47.5% (p = .91). These findings suggest that severity of COVID-19 in KTRs is related to their associated comorbidities and not to chronic immunosuppression.

In situ multiplex immunofluorescence analysis of the inflammatory burden in kidney allograft rejection: A new tool to characterize the alloimmune response.

The exact composition of leukocyte infiltration during kidney allograft rejection is difficult to comprehend and visualize on the same biopsy slide. Using an innovative technology of multiplex immunofluorescence (mIF), we were able to detect simultaneously NK cells, macrophages, and T cells and to determine their intra- or extravascular localization using an endothelial marker. Twenty antibody-mediated rejection (ABMR), 20 T cell-mediated rejection (TCMR), and five normal biopsies were labeled, with automatic leukocyte quantification and localization. This method was compared to a classic NKp46 immunohistochemistry (IHC) with manual quantification and to mRNA quantification. mIF automatic quantification was strongly correlated to IHC (r = .91, P < .001) and to mRNA expression levels (r > .46, P < .021). T cells and macrophages were the 2 predominant populations involved in rejection (48.0 ± 4.4% and 49.3 ± 4.4%, respectively, in ABMR; 51.8 ± 6.0% and 45.3 ± 5.8% in TCMR). NK cells constituted a rare population in both ABMR (2.7 ± 0.7%) and TCMR (2.9 ± 0.6%). The intravascular compartment was mainly composed of T cells, including during ABMR, in peritubular and glomerular capillaries. However, NK cell and macrophage densities were significantly higher during ABMR in glomerular and peritubular capillaries. To conclude, this study demonstrates the feasibility and utility of mIF imaging to study and better understand the kidney allograft rejection process.

Early Acute Microvascular Kidney Transplant Rejection in the Absence of Anti-HLA Antibodies Is Associated with Preformed IgG Antibodies against Diverse Glomerular Endothelial Cell Antigens.

BACKGROUND: Although anti-HLA antibodies (Abs) cause most antibody-mediated rejections of renal allografts, non-anti-HLA Abs have also been postulated to contribute. A better understanding of such Abs in rejection is needed. METHODS: We conducted a nationwide study to identify kidney transplant recipients without anti-HLA donor-specific Abs who experienced acute graft dysfunction within 3 months after transplantation and showed evidence of microvascular injury, called acute microvascular rejection (AMVR). We developed a crossmatch assay to assess serum reactivity to human microvascular endothelial cells, and used a combination of transcriptomic and proteomic approaches to identify non-HLA Abs. RESULTS: We identified a highly selected cohort of 38 patients with early acute AMVR. Biopsy specimens revealed intense microvascular inflammation and the presence of vasculitis (in 60.5%), interstitial hemorrhages (31.6%), or thrombotic microangiopathy (15.8%). Serum samples collected at the time of transplant showed that previously proposed anti-endothelial cell Abs-angiotensin type 1 receptor (AT1R), endothelin-1 type A and natural polyreactive Abs-did not increase significantly among patients with AMVR compared with a control group of stable kidney transplant recipients. However, 26% of the tested AMVR samples were positive for AT1R Abs when a threshold of 10 IU/ml was used. The crossmatch assay identified a common IgG response that was specifically directed against constitutively expressed antigens of microvascular glomerular cells in patients with AMVR. Transcriptomic and proteomic analyses identified new targets of non-HLA Abs, with little redundancy among individuals. CONCLUSIONS: Our findings indicate that preformed IgG Abs targeting non-HLA antigens expressed on glomerular endothelial cells are associated with early AMVR, and that in vitro cell-based assays are needed to improve risk assessments before transplant.