Allelic variation of KIR and HLA tunes the cytolytic payload and determines functional hierarchy of NK cell repertoires.

The functionality of natural killer (NK) cells is tuned during education and is associated with remodeling of the lysosomal compartment. We hypothesized that genetic variation in killer cell immunoglobulin-like receptor (KIR) and HLA, which is known to influence the functional strength of NK cells, fine-tunes the payload of effector molecules stored in secretory lysosomes. To address this possibility, we performed a high-resolution analysis of KIR and HLA class I genes in 365 blood donors and linked genotypes to granzyme B loading and functional phenotypes. We found that granzyme B levels varied across individuals but were stable over time in each individual and genetically determined by allelic variation in HLA class I genes. A broad mapping of surface receptors and lysosomal effector molecules revealed that DNAM-1 and granzyme B levels served as robust metric of the functional state in NK cells. Variation in granzyme B levels at rest was tightly linked to the lytic hit and downstream killing of major histocompatibility complex-deficient target cells. Together, these data provide insights into how variation in genetically hardwired receptor pairs tunes the releasable granzyme B pool in NK cells, resulting in predictable hierarchies in global NK cell function.

Effect of mTOR Inhibition with Sirolimus on Natural Killer Cell Reconstitution in Allogeneic Stem Cell Transplantation.

Sirolimus is an inhibitor of the mammalian target of rapamycin (mTOR) and is emerging as a promising component of graft-versus-host disease (GVHD) prophylaxis regimens in the context of allogeneic hematopoietic stem cell transplantation (HSCT). Multiple studies have explored the clinical benefits of adding sirolimus to GVHD prophylaxis; however, detailed immunologic studies have not yet been carried out in this context. Mechanistically, mTOR is at the center of metabolic regulation in T cells and natural killer (NK) cells and is critical for their differentiation to mature effector cells. Therefore, close evaluation of the inhibition of mTOR in the context of immune reconstitution post-HSCT is warranted. In this work, we studied the effect of sirolimus on immune reconstitution using a biobank of longitudinal samples from patients receiving either tacrolimus/sirolimus (TAC/SIR) or cyclosporin A/methotrexate (CSA/MTX) as conventional GVHD prophylaxis. Healthy donor controls, donor graft material, and samples from 28 patients (14 with TAC/SIR, 14 with CSA/MTX) at 3 to 4 weeks and 34 to 39 weeks post- HSCT were collected. Multicolor flow cytometry was used to perform broad immune cell mapping, with a focus on NK cells. NK cell proliferation was evaluated over a 6-day in vitro homeostatic proliferation protocol. Furthermore, in vitro NK cell responses to cytokine stimulation or tumor cells were evaluated. Systems-level assessment of the immune repertoire revealed a deep and prolonged suppression (weeks 34 to 39 post-HSCT) of the naïve CD4 T cell compartment with relative sparing of regulatory T cells and enrichment of CD69+Ki-67+HLA-DR+ CD8 T cells, independent of the type of GVHD prophylaxis. Early after transplantation (weeks 3 to 4), while patients were still on TAC/SIR or CSA/MTX, we found a relative increase in less-differentiated CD56bright NK cells and NKG2A+CD57-KIR- CD56dim NK cells and a distinct loss of CD16 and DNAM-1 expression. Both regimens led to suppressed proliferative responses ex vivo and functional impairment with preferential loss of cytokine responsiveness and IFN-γ production. Patients who received TAC/SIR as GVHD prophylaxis showed delayed NK cell reconstitution with lower overall NK cell counts and fewer CD56bright and NKG2A+ CD56dim NK cells. Treatment with sirolimus-containing regimens generated similar immune cell profiles as conventional prophylaxis; however, the NK cell compartment seemed to be composed of slightly more mature NK cells. These effects were also present after the completion of GVHD prophylaxis, suggesting that mTOR inhibition with sirolimus leaves a lasting imprint on homeostatic proliferation and NK cell reconstitution following HSCT.

Adaptive single-KIR+NKG2C+ NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia.

BACKGROUND: Natural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)+NKG2C+ adaptive NK cells to maximize missing-self reactivity. METHODS: We developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML. RESULTS: ADAPT-NK cells were >90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45dim blast subtypes. CONCLUSIONS: These preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.

SARS-CoV-2 Nsp13 encodes for an HLA-E-stabilizing peptide that abrogates inhibition of NKG2A-expressing NK cells.

Natural killer (NK) cells are innate immune cells that contribute to host defense against virus infections. NK cells respond to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and are activated in patients with acute coronavirus disease 2019 (COVID-19). However, by which mechanisms NK cells detect SARS-CoV-2-infected cells remains largely unknown. Here, we show that the Non-structural protein 13 of SARS-CoV-2 encodes for a peptide that is presented by human leukocyte antigen E (HLA-E). In contrast with self-peptides, the viral peptide prevents binding of HLA-E to the inhibitory receptor NKG2A, thereby rendering target cells susceptible to NK cell attack. In line with these observations, NKG2A-expressing NK cells are particularly activated in patients with COVID-19 and proficiently limit SARS-CoV-2 replication in infected lung epithelial cells in vitro. Thus, these data suggest that a viral peptide presented by HLA-E abrogates inhibition of NKG2A+ NK cells, resulting in missing self-recognition.

PRDX-1 Supports the Survival and Antitumor Activity of Primary and CAR-Modified NK Cells under Oxidative Stress.

Oxidative stress, caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses, is one of the characteristic features of cancer. Here, we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo, when compared with blood or normal subcutaneous fluid. Therefore, we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells, and by comparing T, B, and NK cells' sensitivities to redox stress and their antioxidant capacities, we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However, the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore, we genetically modified NK cells to stably overexpress PRDX1, which led to increased survival and NK cell activity in redox stress conditions. Finally, we generated PD-L1-CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide, at concentrations detected in the TME, suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors.

Perturbed NK-cell homeostasis associated with disease severity in chronic neutropenia.

Neutrophils have been thought to play a critical role in terminal differentiation of NK cells. Whether this effect is direct or a consequence of global immune changes with effects on NK-cell homeostasis remains unknown. In this study, we used high-resolution flow and mass cytometry to examine NK-cell repertoires in 64 patients with neutropenia and 27 healthy age- and sex-matched donors. A subgroup of patients with chronic neutropenia showed severely disrupted NK-cell homeostasis manifesting as increased frequencies of CD56bright NK cells and a lack of mature CD56dim NK cells. These immature NK-cell repertoires were characterized by expression of the proliferation/exhaustion markers Ki-67, Tim-3, and TIGIT and displayed blunted tumor target cell responses. Systems-level immune mapping revealed that the changes in immunophenotypes were confined to NK cells, leaving T-cell differentiation intact. RNA sequencing of NK cells from these patients showed upregulation of a network of genes, including TNFSF9, CENPF, MKI67, and TOP2A, associated with apoptosis and the cell cycle, but different from the conventional CD56bright signatures. Profiling of 249 plasma proteins showed a coordinated enrichment of pathways related to apoptosis and cell turnover, which correlated with immature NK-cell repertoires. Notably, most of these patients exhibited severe-grade neutropenia, suggesting that the profoundly altered NK-cell homeostasis was connected to the severity of their underlying etiology. Hence, although our data suggest that neutrophils are dispensable for NK-cell development and differentiation, some patients displayed a specific gap in the NK repertoire, associated with poor cytotoxic function and more severe disease manifestations.

Intra-lineage Plasticity and Functional Reprogramming Maintain Natural Killer Cell Repertoire Diversity.

Natural killer (NK) cell repertoires are made up of phenotypically distinct subsets with different functional properties. The molecular programs involved in maintaining NK cell repertoire diversity under homeostatic conditions remain elusive. Here, we show that subset-specific NK cell proliferation kinetics correlate with mTOR activation, and global repertoire diversity is maintained through a high degree of intra-lineage subset plasticity during interleukin (IL)-15-driven homeostatic proliferation in vitro. Slowly cycling sorted KIR+CD56dim NK cells with an induced CD57 phenotype display increased functional potential associated with increased transcription of genes involved in adhesion and immune synapse formation. Rapidly cycling cells upregulate NKG2A, display a general loss of functionality, and a transcriptional signature associated with increased apoptosis/cellular stress, actin-remodeling, and nuclear factor κB (NF-κB) activation. These results shed light on the role of intra-lineage plasticity during NK cell homeostasis and suggest that the functional fate of the cell is tightly linked to the acquired phenotype and transcriptional reprogramming.