Background. Highly HLA-sensitized (HS) patients are at increased risk for cell-mediated rejection (CMR)/antibody-mediated rejection (AMR) posttransplant and require intense immunosuppression to prevent these events. HS patients who experience calcineurin inhibitor (CNI) neuro/nephrotoxicity have few options as conversion to CNI-free regimens with cytotoxic T-lymphocyte-associated antigen (CTLA4)-Ig may risk donor specific antibody (DSA) rebound, CMR, and AMR. This remains an unmet medical need. Anti-interleukin (IL)-6/IL-6R treatment is useful in treating AMR and CMR and preventing rejection in HS patients after transplantation (tx). Here, we examined the utility of combining CTLA4-Ig with anti-IL-6/IL-6R treatment in 4 HS patients with CNI toxicity. Methods. Four HS patients (3 with calculated panel reactive antibodies >97% at tx and 1 who developed chronic-active AMR (c-aAMR) 14 y post-tx were evaluated. Two patients, 100% calculated panel reactive antibody and +flow crossmatch at transplantation, were desensitized with plasma exchange + clazakizumab (anti-IL-6) and maintained on clazakizumab post-tx and transitioned from CNI to CTLA4-Ig. Two patients treated with anti-IL-6R (tocilizumab) were transitioned to CTLA4-Ig at 2 and 11 y post-tx. Patients were monitored for renal function (RF), DSA, de novo DSA (dn-DSA), immune cell subsets, and patient and graft survival. Results. Clazakizumab and tocilizumab treated patients showed improvements in RF, with elimination of existing DSAs, except for 1 patient and no dn-DSAs (>6 y). We also saw reductions in CD8+ TEFF/MEM, TFH, BMEM, and NK cells post-CTLA4-Ig + Anti-IL-6/IL-6R treatment. TREG and BREG cell populations also increased, suggesting, but not confirming, possible immune modulation. Conclusions. Combining CTLA4-Ig + anti-IL-6/IL-6R appears to offer benefits in sustaining RF, DSA reduction, possibly immune cell modulation, and prevention of dn-DSA. Importantly, CNI toxicity abated in all patients.

Background.Highly HLA-sensitized (HS) patients are at increased risk for cell-mediated rejection (CMR)/antibody-mediated rejection (AMR) posttransplant and require intense immunosuppression to prevent these events. HS patients who experience calcineurin inhibitor (CNI) neuro/nephrotoxicity have few options as conversion to CNI-free regimens with cytotoxic T-lymphocyte-associated antigen (CTLA4)-Ig may risk donor specific antibody (DSA) rebound, CMR, and AMR. This remains an unmet medical need. Anti-interleukin (IL)-6/IL-6R treatment is useful in treating AMR and CMR and preventing rejection in HS patients after transplantation (tx). Here, we examined the utility of combining CTLA4-Ig with anti-IL-6/IL-6R treatment in 4 HS patients with CNI toxicity.Methods.Four HS patients (3 with calculated panel reactive antibodies >97% at tx and 1 who developed chronic-active AMR (c-aAMR) 14 y post-tx were evaluated. Two patients, 100% calculated panel reactive antibody and +flow crossmatch at transplantation, were desensitized with plasma exchange + clazakizumab (anti-IL-6) and maintained on clazakizumab post-tx and transitioned from CNI to CTLA4-Ig. Two patients treated with anti-IL-6R (tocilizumab) were transitioned to CTLA4-Ig at 2 and 11 y post-tx. Patients were monitored for renal function (RF), DSA, de novo DSA (dn-DSA), immune cell subsets, and patient and graft survival.Results.Clazakizumab and tocilizumab treated patients showed improvements in RF, with elimination of existing DSAs, except for 1 patient and no dn-DSAs (>6 y). We also saw reductions in CD8+ TEFF/MEM, TFH, BMEM, and NK cells post-CTLA4-Ig + Anti-IL-6/IL-6R treatment. T-REG and B-REG cell populations also increased, suggesting, but not confirming, possible immune modulation.Conclusions.Combining CTLA4-Ig + anti-IL-6/IL-6R appears to offer benefits in sustaining RF, DSA reduction, possibly immune cell modulation, and prevention of dn-DSA. Importantly, CNI toxicity abated in all patients.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system with essential roles in immune surveillance, tissue homeostasis and inflammation. In the kidney, NK cells comprise a heterogeneous population that includes both circulating and tissue-resident subsets, each shaped by environmental cues and genetic factors through interactions with major histocompatibility complex class I molecules. The latest research data highlight multifaceted NK cell contributions to kidney physiology and pathology. In steady state, NK cells support kidney immune surveillance and crosstalk with epithelial, myeloid and lymphoid cells. In disease, NK cells can promote injury through direct cytotoxicity and pro-inflammatory cytokine release. Experimental models demonstrate pathogenic roles for NK cells in ischaemia–reperfusion injury and chronic kidney disease, with emerging evidence implicating NK cell-derived mediators in fibrogenesis. In kidney transplantation, NK cells are effectors of antibody-dependent and antibody-independent allograft injury. Educated NK cells expressing CD16a (also known as FcγRIIIa) mediate antibody-dependent cellular cytotoxicity, whereas loss of inhibitory receptor–ligand interactions (for example, due to killer immunoglobulin-like receptor–HLA mismatch) can trigger NK cell activation independently of donor-specific antibodies. Advances in high-resolution profiling have deepened mechanistic insights and uncovered novel therapeutic targets. Here, we provide a comprehensive overview of NK cell biology in the kidney, highlighting roles in health, disease and transplantation, and we consider its translational implications for diagnosis and therapy. This Review discusses the latest advances in natural killer cell biology, including insights into population heterogeneity, and examines the roles of these innate lymphocytes in kidney health and disease, as well as their contribution to kidney allograft rejection and its therapeutic implications.

Extracellular vesicles (EVs) contain proteins, lipids, and nucleic acids from their cells of origin. By delivering these cargos to distant acceptor cells, EVs modulate many biologic processes, including adaptive immunity. Following transplantation, EV's expressing donor major histocompatibility complexes bind to host dendritic cells (DCs), permitting donor major histocompatibility complexes expression by recipient DCs and priming antidonor T cell responses. The mechanisms through which circulating EVs bind DCs are poorly understood. The complement system opsonizes pathogens and damaged cells and enhances subsequent recognition by antigen-presenting cells through surface-expressed complement receptors. Here, we newly show that complement opsonization of graft-released EVs augments their binding to recipient DCs in a CD11c-dependent manner. Enhanced delivery of donor antigen by EVs induces antidonor T cell responses and graft rejection, which can be mitigated by pharmacologic inhibition of complement activation. Our findings reveal a previously unrecognized mechanism linking complement activation to EV function with important implications for T cell immunity.

INTRODUCTIONMaladaptive hypertrophy, podocyte stress, and depletion contribute to kidney function decline. Although insulin-like growth factor 1 (IGF-1) plays a key role in early hypertrophic responses in the single kidney state, its impact on kidney transplant (KTx) outcomes remains uncertain. This report tests the hypothesis that early IGF-1 exposure reduces KTx survival. METHODSPopulation datasets compared incident death-censored graft failure (DCGF) rates by age at KTx (n = 366,404) with IGF-1 levels by age (n = 15,014). A clinical study of 216 KTx recipients evaluated the association of IGF-1 exposure with DCGF and secondary outcomes of proteinuria and biopsy-proven acute rejection. IGF-1 exposure was modeled using pre-KTx IGF-1 levels and donor kidney dose estimated from the donor/recipient body surface area ratio reflecting allograft hyperfiltration. The association of DCGF with an IGF1 SNP linked to high IGF-1 levels was assessed in 724 genotyped allograft recipients. Single-cell transcriptomic data from first-year post-KTx patients and binephric donors were compared to assess intrarenal cellular expression of IGF1, IGF1R, and growth hormone receptor (GHR) transcripts. RESULTSDCGF risk by age at KTx paralleled IGF-1 levels by age. Higher IGF-1 exposure was associated with significantly increased risks of DCGF, proteinuria, and T cell-mediated rejection. Genotypic analysis showed a 50% increase in DCGF risk per risk allele at IGF1 expression quantitative trait locus rs35767. First-year biopsy results revealed no increase in intrarenal IGF1 transcripts, while GHR and IGF1R transcripts were suppressed, consistent with circulating IGF-1 (vs. graft-derived IGF-1) being the primary source of IGF-1 exposure. CONCLUSIONWe identify a role for the growth hormone/IGF-1 axis in reducing KTx survival.

African Americans have an increased risk of kidney disease due to exonic variants in Apolipoprotein-L1 (G1 and G2). These prevalent variants have also been linked with kidney rejection, but outside of association with African ancestry, underpinning causal mechanisms are unknown. We investigated T-cell function using transgenic mice with physiologic expression of wild type (G0-), G1-, or G2-APOL1. Mice with variant APOL1 showed greater CD8+T-cell activation with expansion of a central memory (TCM) subset. Stimulated G1-CD8+T-cells showed enhanced proliferation and cytokine production, which reversed with APOL1 inhibition. In MHC-mismatched cardiac transplants, G1-mice demonstrated greater CD8+T-cell infiltration and reduced survival. Bulk transcriptome of G1-CD8+T-cells, and single-cell transcriptome of graft infiltrating TCMs, showed enrichment of canonical T-cell receptor (TCR) pathways including Ca 2+ -signaling. G1-CD8+T-cells demonstrated baseline ER-Ca 2+ depletion followed by sustained increases in cytosolic-Ca 2+ upon TCR stimulation. G1-CD8+T-cells were more sensitive to Ca 2+ chelation, or store-operated Ca 2+ entry inhibition, and relatively resistant to calcineurin antagonism vs. G0-CD8+T-cells. Analogously, in a kidney transplant cohort, APOL1-variant recipients developed rejection when they had elevated peripheral TCMs before transplantation and despite significantly higher tacrolimus levels vs G0/G0-AAs with rejection. In summary, we unravel an excitatory T-cell intrinsic mechanism for APOL1 exonic variants, causally linking them with kidney rejection.

AbstractBackgroundLong-term kidney allograft survival remains suboptimal. Emerging evidence indicates donor-recipient (D-R) mismatches outside of human leukocyte antigens (HLA) contribute to graft survival but mechanisms remain unclear, specifically for those mismatches within intronic regions.MethodsWe analyzed genome-wide SNP data of D-R pairs from two well-phenotyped kidney transplant cohorts (median follow-up ~1800 days), Genomics of Chronic Allograft Rejection (GoCAR; n=385) and Clinical Trials in Organ Transplantation 1/17 (CTOT1/17; n=146), quantifying genetic mismatches outside of HLA for every D-R pair at variant, gene, and genome-wide scales.ResultsUnbiased genome-wide screen of GoCAR D-R pairs uncovered the LIMS1 locus as the topranked candidate where D-R mismatches associated with death censored graft loss (DCGL). Independent of HLA, a previously unreported relationship between mismatches at a highly linked, intronic haplotype of 30 SNPs was seen as associated with DCGL, with confirmatory association in intra-ancestry D-Rs. Validation testing within the CTOT-01/17 showed similar associations with DCGL. Haplotype D-R mismatches showed a dosage effect, and the introduction of minor alleles in the donor to major allele-carrying recipients showed a greater risk of DCGL. Both the new LIMS1 haplotype and the previously reported LIMS1 SNP rs893403 are expression quantitative trait loci (eQTL) for the gene GCC2 in recipient immune cells, without alterations in GCC2 or LIMS1 coding sequences. Transcriptome enrichment analyses performed on whole blood and within multiple T cell subsets demonstrated significant associations of GCC2 gene, and of either allelic locus, with regulation of TGF-beta-SMAD signaling, implying a role in Treg function and association with rejection.ConclusionsOur analysis unravels intronic non-HLA SNP mismatches within LIMS1 that do not induce protein sequence variation but associate with DCGL. By acting as cis-eQTLs for GCC2 expression, these SNPs modulates TGF-beta signaling and T cell function, associating with immune events and graft outcomes. The findings have clinical implications for risk assessment and individualized therapy in kidney transplant recipients.

Background: Therapeutic intervention targeted to restrict apoptosis could reduce left ventricle dysfunction.Ergo, noninvasive assessment of apoptosis could be of seminal clinical importance.However, imaging myocardial apoptosis may have differences in feasibility and efficacy when apoptosis is focal (as in the infarct zone) vs. diffuse (as in cardiac allograft rejection).Focal apoptosis imaging using Duramycin can avoid some limitations of Annexin A5 imaging and may be feasible after a major insult like MI, but its comparative efficacy in diffusely distributed apoptosis is unknown.Purpose: To evaluate the feasibility and effectiveness of imaging apoptosis with a novel method [(99mTc) Duramycin], in experimental models of both focal as well as diffuse myocardial insults that cause apoptosis. Methods: 2 models were studied:Protocol A [regional injury after ischemia-reperfusion (IR)] -In an IR injury model in 13 rabbits, 7mCi of 99mTc-labeled Duramycin (n = 10; 4 animals treated with an anti-apoptotic agent-minocycline) or 99mTc-Linear Duramycin (a negative tracer control, n = 3) were intravenously administered 30 minutes after reperfusion.In-vivo mSPECT-mCT imaging was performed 3 hours after reperfusion followed by ex-vivo imaging of the explanted heart, quantitative assessment of tracer uptake and pathological characterization.Protocol B [diffuse apoptosis in cardiac allograft rejection (CAR)] -In a CAR model, 16 mice received abdominal heterotopic cardiac allografts (n= 4 each).Group 1: Balb/c donor to B6 recipient (allogeneic transplant, ALO).Group 2: B6 donor to B6 recipient (syngeneic control transplant, CON).Group 3: Balb/c donor to B6 recipient treated with abatacept (allogeneic transplant and immunosuppressed, IMS).Group 4: Bm12 donor to B6 recipient (sub-allogeneic transplant to test for chronic CAR, CHR).Animals were sacrificed at fixed points (ALO and CON 6-7 days, IMS animals 14-15 days and CHR animals 21 days following transplant).MicroSPECT/CT imaging of the heterotopic transplant was performed in vivo and ex vivo after intravenous Duramycin administration for quantitative uptake.Results: Protocol A -Intense uptake ((%ID/g) of Duramycin was observed in the infarct area (0.751 ± 0.262%) compared to remote area (0.045 ± 0.029%; p < 0.01).Minocycline attenuated apoptosis (0.354 ± 0.0624%; p < 0.01).Protocol B -CAR animals showed intense uptake in ALO (5.8 ± 2.2), followed by CHR (1.8 ± 1.5), then IMS (1.2 ± 0.4), and CON (0.90 ± 0.04) [p< 0.05 for ALO vs. both CON and IMS groups].Rejection was ISHLT Grade 3R in ALO, and Grade 1-2R in IMS / CHR groups.Conclusions Duramycin is similarly effective in imaging apoptotic cell death in localized as well as diffuse myocardial damage and tracks changing levels of apoptosis.Clinical feasibility of apoptosis imaging with a safe PE-seeking antibiotic if proven, might reduce the need for frequent endomyocardial biopsies in patients after cardiac transplant.