Missing Self–Induced Microvascular Rejection of Kidney Allografts: A Population-Based Study

BackgroundCirculating anti-HLA donor-specific antibodies (HLA-DSA) are often absent in kidney transplant recipients with microvascular inflammation (MVI). Missing self, the inability of donor endothelial cells to provide HLA I–mediated signals to inhibitory killer cell Ig-like receptors (KIRs) on recipient natural killer cells, can cause endothelial damage in vitro, and has been associated with HLA-DSA–negative MVI. However, missing self’s clinical importance as a nonhumoral trigger of allograft rejection remains unclear.MethodsIn a population-based study of 924 consecutive kidney transplantations between March 2004 and February 2013, we performed high-resolution donor and recipient HLA typing and recipient KIR genotyping. Missing self was defined as the absence of A3/A11, Bw4, C1, or C2 donor genotype, with the presence of the corresponding educated recipient inhibitory KIR gene.ResultsWe identified missing self in 399 of 924 transplantations. Co-occurrence of missing self types had an additive effect in increasing MVI risk, with a threshold at two concurrent types (hazard ratio [HR], 1.78; 95% confidence interval [95% CI], 1.26 to 2.53), independent of HLA-DSA (HR, 5.65; 95% CI, 4.01 to 7.96). Missing self and lesions of cellular rejection were not associated. No HLA-DSAs were detectable in 146 of 222 recipients with MVI; 28 of the 146 had at least two missing self types. Missing self associated with transplant glomerulopathy after MVI (HR, 2.51; 95% CI, 1.12 to 5.62), although allograft survival was better than with HLA-DSA–associated MVI.ConclusionMissing self specifically and cumulatively increases MVI risk after kidney transplantation, independent of HLA-DSA. Systematic evaluation of missing self improves understanding of HLA-DSA–negative MVI and might be relevant for improved diagnostic classification and patient risk stratification.

Soluble and Exosome-Bound α-Galactosylceramide Mediate Preferential Proliferation of Educated NK Cells with Increased Anti-Tumor Capacity

Natural killer (NK) cells can kill target cells via the recognition of stress molecules and down-regulation of major histocompatibility complex class I (MHC-I). Some NK cells are educated to recognize and kill cells that have lost their MHC-I expression, e.g., tumor or virus-infected cells. A desired property of cancer immunotherapy is, therefore, to activate educated NK cells during anti-tumor responses in vivo. We here analyze NK cell responses to α-galactosylceramide (αGC), a potent activator of invariant NKT (iNKT) cells, or to exosomes loaded with αGC. In mouse strains which express different MHC-I alleles using an extended NK cell flow cytometry panel, we show that αGC induces a biased NK cell proliferation of educated NK cells. Importantly, iNKT cell-induced activation of NK cells selectively increased in vivo missing self-responses, leading to more effective rejection of tumor cells. Exosomes from antigen-presenting cells are attractive anti-cancer therapy tools as they may induce both innate and adaptive immune responses, thereby addressing the hurdle of tumor heterogeneity. Adding αGC to antigen-loaded dendritic-cell-derived exosomes also led to an increase in missing self-responses in addition to boosted T and B cell responses. This study manifests αGC as an attractive adjuvant in cancer immunotherapy, as it increases the functional capacity of educated NK cells and enhances the innate, missing self-based antitumor response.

Missing Self-Induced Activation of NK Cells Combines with Non-Complement-Fixing Donor-Specific Antibodies to Accelerate Kidney Transplant Loss in Chronic Antibody-Mediated Rejection

BackgroundBinding of donor-specific antibodies (DSAs) to kidney allograft endothelial cells that does not activate the classic complement cascade can trigger the recruitment of innate immune effectors, including NK cells. Activated NK cells contribute to microvascular inflammation leading to chronic antibody-mediated rejection (AMR). Recipient NK cells can also trigger antibody-independent microvascular inflammation by sensing the absence of self HLA class I molecules (“missing self”) on allograft endothelial cells. This translational study investigated whether the condition of missing self amplifies DSA-dependent NK cell activation to worsen chronic AMR.Methods and ResultsAmong 1682 kidney transplant recipients who underwent an allograft biopsy at Lyon University Hospital between 2004 and 2017, 135 fulfilled the diagnostic criteria for AMR and were enrolled in the study. Patients with complement-fixing DSAs identified by a positive C3d binding assay (n=73, 54%) had a higher risk of transplant failure (P=0.002). Among the remaining patients with complement-independent chronic AMR (n=62, 46%), those in whom missing self was identified through donor and recipient genotyping exhibited worse allograft survival (P=0.02). In multivariable analysis, only proteinuria (HR: 7.24; P=0.01) and the presence of missing self (HR: 3.57; P=0.04) were independent predictors for transplant failure following diagnosis of chronic AMR. Cocultures of human NK cells and endothelial cells confirmed that addition of missing self to DSA-induced NK cell activation increased endothelial damage.ConclusionsThe assessment of missing self at the time of diagnosis of chronic AMR identifies patients at higher risk for kidney transplant failure.

CAR19 iPSC-Derived NK Cells Utilize the Innate Functional Potential Mediated through NKG2A-Driven Education and Override the HLA-E Check Point to Effectively Target B Cell Lymphoma

Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells offer a promising platform for off-the-shelf immunotherapy against cancer. A unique benefit of iPSC-derived immune effector cells is the possibility to perform multiple precision editing steps at the single cell level to achieve a homogenous effector cell population tailored to target a desired cancer type and equipped with selected functional properties. These functional edits are superimposed on the innate reactivity of NK cells to stress ligands and MHC downregulation (missing self). The ability of NK cells to sense missing self is based on a functional calibration to self MHC during a process termed NK cell education, the latter being critically dependent on signaling through inhibitory receptors, including CD94/NKG2A and killer cell immunoglobulin-like receptors (KIR). Whereas the process of NK cell differentiation into mature effector cells from iPSCs has been well characterized, the role of natural variation in inhibitory receptor expression and NK cell education remains poorly defined in iNK cells. We used mass cytometry to map the receptor repertoire in series of iNK cell lines and genetic edits thereof during differentiation and in vitro expansion (Figure 1A and B). Similar to peripheral blood NK cells, the receptor repertoire was diversified but genetically hardwired showing consistent patterns within each iNK cell line but with slight variation between genetically distinct lines. NKG2A was the dominantly expressed inhibitory receptor ranging from 13% to 87% with the highest expression in multi-edited iNK cell lines engineered to express a chimeric antigen receptor against CD19, a high affinity, non-cleavable FcγRIIIa receptor (CD16) and a recombinant IL15 signaling complex (CAR19-iNK cells). KIR expression was generally low in all tested iNK cell lines but increased gradually during culture and was further increased by genetic silencing of NKG2A receptors. Interestingly, silencing of NKG2A lead to increased levels of the activating receptor NKG2C. We monitored degranulation by iNK cell variants against K562 engineered to express varying levels of HLA-E as well as CD19+ Nalm-6 cells. Genetic silencing of ß2microglobulin (ß2m), associated with reduced levels of HLA-class I and HLA-E, led to dampened global functional responses in iNK cells, suggesting a positive impact of education during iNK cell differentiation and expansion (Figure 1C). Subset stratification revealed that NKG2A+ iNK cells showed superior functionality compared to NKG2A- iNK cells across all iNK cell lines tested, albeit less striking in CAR19-iNK cells that showed the highest overall natural cytotoxicity (Figure 1D). Knockdown of NKG2A led to a general reduction in functional capacity of NK92 cells (Figure 1E-F) and CAR19-iNK cells (Figure 1H), supporting a critical role for NKG2A-driven education in iNK cells. Given the superior functionality of NKG2A+ iNK cells, we next addressed whether this advantage was countered by expression of the check point ligand HLA-E during target cell interactions. Although we noted a slight inhibitory impact on natural cytotoxicity in NK cells isolated and expanded from peripheral blood (PB-NK) against K562 cells expressing physiological levels of HLA-E, this effect was completely overridden in iNK cells and did not interfere with NKG2A+ CAR-iNK cell recognition of HLA-E expressing CD19+ target cells (Figure 1G-H). Indeed, NKG2A+ CAR19-iNK showed superior degranulation against HLA-E expressing CD19+ Nalm-6 targets compared to CRISPR-edited NKG2A-/- CAR19-iNK cells (Figure 1I). Our results shed light on the regulatory gene circuits and cellular programs that determine functional potential in iPSC-derived NK cells products. Specifically, our results point to a crucial role for NKG2A-driven acquisition of a mature effector cell phenotype in combination with functional education through cognate ligands. Importantly, iNK cell education is operational during iNK cell differentiation and expansion without interfering with recognition of tumor targets expressing HLA-E. Figure 1 Disclosures Cichocki: Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding. Mahmood:Fate Therapeutics, Inc: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Chu:Fate Therapeutics, Inc: Current Employment. Groff:Fate Therapeutics, Inc: Current Employment. Denholtz:Fate Therapeutics, Inc: Current Employment. Miller:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; GT Biopharma: Consultancy, Patents & Royalties, Research Funding. Lee:Fate Therapeutics, Inc.: Current Employment. Kaufman:Fate Therapeutics: Consultancy. Goodridge:Fate Therapeutics, Inc: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Malmberg:Fate Therapeutics: Consultancy, Patents & Royalties; Vycellix: Membership on an entity's Board of Directors or advisory committees.

Missing self triggers NK cell-mediated chronic vascular rejection of solid organ transplants

Current doctrine is that microvascular inflammation (MVI) triggered by a transplant -recipient antibody response against alloantigens (antibody-mediated rejection) is the main cause of graft failure. Here, we show that histological lesions are not mediated by antibodies in approximately half the participants in a cohort of 129 renal recipients with MVI on graft biopsy. Genetic analysis of these patients shows a higher prevalence of mismatches between donor HLA I and recipient inhibitory killer cell immunoglobulin-like receptors (KIRs). Human in vitro models and transplantation of β2-microglobulin-deficient hearts into wild-type mice demonstrates that the inability of graft endothelial cells to provide HLA I-mediated inhibitory signals to recipient circulating NK cells triggers their activation, which in turn promotes endothelial damage. Missing self-induced NK cell activation is mTORC1-dependent and the mTOR inhibitor rapamycin can prevent the development of this type of chronic vascular rejection.