Optimisation of a primary human CAR-NK cell manufacturing pipeline.

Objectives: Autologous chimeric antigen receptor (CAR) T-cell therapy of B-cell malignancies achieves long-term disease remission in a high fraction of patients and has triggered intense research into translating this successful approach into additional cancer types. However, the complex logistics involved in autologous CAR-T manufacturing, the compromised fitness of patient-derived T cells, the high rates of serious toxicities and the overall cost involved with product manufacturing and hospitalisation have driven innovation to overcome such hurdles. One alternative approach is the use of allogeneic natural killer (NK) cells as a source for CAR-NK cell therapy. However, this source has traditionally faced numerous manufacturing challenges. Methods: To address this, we have developed an optimised expansion and transduction protocol for primary human NK cells primed for manufacturing scaling and clinical evaluation. We have performed an in-depth comparison of primary human NK cell sources as a starting material by characterising their phenotype, functionality, expansion potential and transduction efficiency at crucial timepoints of our CAR-NK manufacturing pipeline. Results: We identified adult peripheral blood-derived NK cells to be the superior source for generating a CAR-NK cell product because of a higher maximum yield of CAR-expressing NK cells combined with potent natural, as well as CAR-mediated anti-tumor effector functions. Conclusions: Our optimised manufacturing pipeline dramatically improves lentiviral transduction efficiency of primary human NK cells. We conclude that the exponential expansion pre- and post-transduction and high on-target cytotoxicity make peripheral blood-derived NK cells a feasible and attractive CAR-NK cell product for clinical utility.

IKAROS and AIOLOS directly regulate AP-1 transcriptional complexes and are essential for NK cell development.

Ikaros transcription factors are essential for adaptive lymphocyte function, yet their role in innate lymphopoiesis is unknown. Using conditional genetic inactivation, we show that Ikzf1/Ikaros is essential for normal natural killer (NK) cell lymphopoiesis and IKZF1 directly represses Cish, a negative regulator of interleukin-15 receptor resulting in impaired interleukin-15 receptor signaling. Both Bcl2l11 and BIM levels, and intrinsic apoptosis were increased in Ikzf1-null NK cells, which in part accounts for NK lymphopenia as both were restored to normal levels when Ikzf1 and Bcl2l11 were co-deleted. Ikzf1-null NK cells presented extensive transcriptional alterations with reduced AP-1 transcriptional complex expression and increased expression of Ikzf2/Helios and Ikzf3/Aiolos. IKZF1 and IKZF3 directly bound AP-1 family members and deletion of both Ikzf1 and Ikzf3 in NK cells resulted in further reductions in Jun/Fos expression and complete loss of peripheral NK cells. Collectively, we show that Ikaros family members are important regulators of apoptosis, cytokine responsiveness and AP-1 transcriptional activity.

CD45 limits early Natural Killer cell development.

The clinical development of Natural Killer (NK) cell-mediated immunotherapy marks a milestone in the development of new cancer therapies and has gained traction due to the intrinsic ability of the NK cell to target and kill tumor cells. To fully harness the tumor killing ability of NK cells, we need to improve NK cell persistence and to overcome suppression of NK cell activation in the tumor microenvironment. The trans-membrane, protein tyrosine phosphatase CD45, regulates NK cell homeostasis, with the genetic loss of CD45 in mice resulting in increased numbers of mature NK cells. This suggests that CD45-deficient NK cells might display enhanced persistence following adoptive transfer. However, we demonstrate here that adoptive transfer of CD45-deficiency did not enhance NK cell persistence in mice, and instead, the homeostatic disturbance of NK cells in CD45-deficient mice stemmed from a developmental defect in the progenitor population. The enhanced maturation within the CD45-deficient NK cell compartment was intrinsic to the NK cell lineage, and independent of the developmental defect. CD45 is not a conventional immune checkpoint candidate, as systemic loss is detrimental to T and B cell development, compromising the adaptive immune system. Nonetheless, this study suggests that inhibition of CD45 in progenitor or stem cell populations may improve the yield of in vitro generated NK cells for adoptive therapy.

CIS controls the functional polarization of GM-CSF-derived macrophages.

The cytokine granulocyte-macrophage-colony stimulating factor (GM-CSF) possesses the capacity to differentiate monocytes into macrophages (MØs) with opposing functions, namely, proinflammatory M1-like MØs and immunosuppressive M2-like MØs. Despite the importance of these opposing biological outcomes, the intrinsic mechanism that regulates the functional polarization of MØs under GM-CSF signaling remains elusive. Here, we showed that GM-CSF-induced MØ polarization resulted in the expression of cytokine-inducible SH2-containing protein (CIS) and that CIS deficiency skewed the differentiation of monocytes toward immunosuppressive M2-like MØs. CIS deficiency resulted in hyperactivation of the JAK-STAT5 signaling pathway, consequently promoting downregulation of the transcription factor Interferon Regulatory Factor 8 (IRF8). Loss- and gain-of-function approaches highlighted IRF8 as a critical regulator of the M1-like polarization program. In vivo, CIS deficiency induced the differentiation of M2-like macrophages, which promoted strong Th2 immune responses characterized by the development of severe experimental asthma. Collectively, our results reveal a CIS-modulated mechanism that clarifies the opposing actions of GM-CSF in MØ differentiation and uncovers the role of GM-CSF in controlling allergic inflammation.

Therapeutic inhibition of the SRC-kinase HCK facilitates T cell tumor infiltration and improves response to immunotherapy.

Although immunotherapy has revolutionized cancer treatment, many immunogenic tumors remain refractory to treatment. This can be largely attributed to an immunologically "cold" tumor microenvironment characterized by an accumulation of immunosuppressive myeloid cells and exclusion of activated T cells. Here, we demonstrate that genetic ablation or therapeutic inhibition of the myeloid-specific hematopoietic cell kinase (HCK) enables activity of antagonistic anti-programmed cell death protein 1 (anti-PD1), anti-CTLA4, or agonistic anti-CD40 immunotherapies in otherwise refractory tumors and augments response in treatment-susceptible tumors. Mechanistically, HCK ablation reprograms tumor-associated macrophages and dendritic cells toward an inflammatory endotype and enhances CD8+ T cell recruitment and activation when combined with immunotherapy in mice. Meanwhile, therapeutic inhibition of HCK in humanized mice engrafted with patient-derived xenografts counteracts tumor immunosuppression, improves T cell recruitment, and impairs tumor growth. Collectively, our results suggest that therapeutic targeting of HCK activity enhances response to immunotherapy by simultaneously stimulating immune cell activation and inhibiting the immunosuppressive tumor microenvironment.

Mesenchymal stromal cell apoptosis is required for their therapeutic function.

Multipotent mesenchymal stromal cells (MSCs) ameliorate a wide range of diseases in preclinical models, but the lack of clarity around their mechanisms of action has impeded their clinical utility. The therapeutic effects of MSCs are often attributed to bioactive molecules secreted by viable MSCs. However, we found that MSCs underwent apoptosis in the lung after intravenous administration, even in the absence of host cytotoxic or alloreactive cells. Deletion of the apoptotic effectors BAK and BAX prevented MSC death and attenuated their immunosuppressive effects in disease models used to define MSC potency. Mechanistically, apoptosis of MSCs and their efferocytosis induced changes in metabolic and inflammatory pathways in alveolar macrophages to effect immunosuppression and reduce disease severity. Our data reveal a mode of action whereby the host response to dying MSCs is key to their therapeutic effects; findings that have broad implications for the effective translation of cell-based therapies.

Differential requirement for the Polycomb repressor complex 2 in dendritic cell and tissue-resident myeloid cell homeostasis.

Dendritic cells (DCs) and macrophages are at the forefront of immune responses, modifying their transcriptional programs in response to their tissue environment or immunological challenge. Posttranslational modifications of histones, such as histone H3 lysine-27 trimethylation (H3K27me3) by the Polycomb repressive complex 2 (PRC2), are tightly associated with epigenetic regulation of gene expression. To explore whether H3K27me3 is involved in either the establishment or function of the mononuclear phagocyte system, we selectively deleted core components of PRC2, either EZH2 or SUZ12, in CD11c-expressing myeloid cells. Unexpectedly, EZH2 deficiency neither prevented the deposition and maintenance of H3K27me3 in DCs nor hindered DC/macrophage homeostasis. In contrast, SUZ12 deficiency markedly impaired the capacity of DCs and macrophages to maintain H3K27me3. SUZ12 ablation induced a rapid loss of the alveolar macrophage and Langerhans cell networks under both steady state and inflammatory conditions because these cells could no longer proliferate to facilitate their self-renewal. Despite the reduced H3K27me3, DC development and function were unaffected by SUZ12 ablation, suggesting that PRC2-mediated gene repression was dispensable for DC homeostasis. Thus, the role of SUZ12 highlights the fundamentally different homeostatic mechanisms used by tissue-resident myeloid cells versus DCs.

BCL-XL antagonism selectively reduces neutrophil life span within inflamed tissues without causing neutropenia.

Neutrophils help to clear pathogens and cellular debris, but can also cause collateral damage within inflamed tissues. Prolonged neutrophil residency within an inflammatory niche can exacerbate tissue pathology. Using both genetic and pharmacological approaches, we show that BCL-XL is required for the persistence of neutrophils within inflammatory sites in mice. We demonstrate that a selective BCL-XL inhibitor (A-1331852) has therapeutic potential by causing apoptosis in inflammatory human neutrophils ex vivo. Moreover, in murine models of acute and chronic inflammatory disease, it reduced inflammatory neutrophil numbers and ameliorated tissue pathology. In contrast, there was minimal effect on circulating neutrophils. Thus, we show a differential survival requirement in activated neutrophils for BCL-XL and reveal a new therapeutic approach to neutrophil-mediated diseases.

Single-cell analyses reveal the clonal and molecular aetiology of Flt3L-induced emergency dendritic cell development.

Regulation of haematopoietic stem and progenitor cell (HSPC) fate is crucial during homeostasis and under stress conditions. Here we examine the aetiology of the Flt3 ligand (Flt3L)-mediated increase of type 1 conventional dendritic cells (cDC1s). Using cellular barcoding we demonstrate this occurs through selective clonal expansion of HSPCs that are primed to produce cDC1s and not through activation of cDC1 fate by other HSPCs. In particular, multi/oligo-potent clones selectively amplify their cDC1 output, without compromising the production of other lineages, via a process we term tuning. We then develop Divi-Seq to simultaneously profile the division history, surface phenotype and transcriptome of individual HSPCs. We discover that Flt3L-responsive HSPCs maintain a proliferative 'early progenitor'-like state, leading to the selective expansion of multiple transitional cDC1-primed progenitor stages that are marked by Irf8 expression. These findings define the mechanistic action of Flt3L through clonal tuning, which has important implications for other models of 'emergency' haematopoiesis.

Hhex Directly Represses BIM-Dependent Apoptosis to Promote NK Cell Development and Maintenance.

Hhex encodes a homeobox transcriptional regulator important for embryonic development and hematopoiesis. Hhex is highly expressed in NK cells, and its germline deletion results in significant defects in lymphoid development, including NK cells. To determine if Hhex is intrinsically required throughout NK cell development or for NK cell function, we generate mice that specifically lack Hhex in NK cells. NK cell frequency is dramatically reduced, while NK cell differentiation, IL-15 responsiveness, and function at the cellular level remain largely normal in the absence of Hhex. Increased IL-15 availability fails to fully reverse NK lymphopenia following conditional Hhex deletion, suggesting that Hhex regulates developmental pathways extrinsic to those dependent on IL-15. Gene expression and functional genetic approaches reveal that Hhex regulates NK cell survival by directly binding Bcl2l11 (Bim) and repressing expression of this key apoptotic mediator. These data implicate Hhex as a transcriptional regulator of NK cell homeostasis and immunity.

The cancer-natural killer cell immunity cycle.

Immunotherapy with checkpoint blockade induces rapid and durable immune control of cancer in some patients and has driven a monumental shift in cancer treatment. Neoantigen-specific CD8+ T cells are at the forefront of current immunotherapy strategies, and the majority of drug discovery and clinical trials revolve around further harnessing these immune effectors. Yet the immune system contains a diverse range of antitumour effector cells, and these must function in a coordinated and synergistic manner to overcome the immune-evasion mechanisms used by tumours and achieve complete control with tumour eradication. A key antitumour effector is the natural killer (NK) cells, cytotoxic innate lymphocytes present at high frequency in the circulatory system and identified by their exquisite ability to spontaneously detect and lyse transformed or stressed cells. Emerging data show a role for intratumoural NK cells in driving immunotherapy response and, accordingly, there have been renewed efforts to further elucidate and target the pathways controlling NK cell antitumour function. In this Review, we discuss recent clinical evidence that NK cells are a key immune constituent in the protective antitumour immune response and highlight the major stages of the cancer-NK cell immunity cycle. We also perform a new analysis of publicly available transcriptomic data to provide an overview of the prognostic value of NK cell gene expression in 25 tumour types. Furthermore, we discuss how the role of NK cells evolves with tumour progression, presenting new opportunities to target NK cell function to enhance cancer immunotherapy response rates across a more diverse range of cancers.

Harnessing Natural Killer Immunity in Metastatic SCLC.

INTRODUCTION: SCLC is the most aggressive subtype of lung cancer, and though most patients initially respond to platinum-based chemotherapy, resistance develops rapidly. Immunotherapy holds promise in the treatment of lung cancer; however, patients with SCLC exhibit poor overall responses highlighting the necessity for alternative approaches. Natural killer (NK) cells are an alternative to T cell-based immunotherapies that do not require sensitization to antigens presented on the surface of tumor cells. METHODS: We investigated the immunophenotype of human SCLC tumors by both flow cytometry on fresh samples and bioinformatic analysis. Cell lines generated from murine SCLC were transplanted into mice lacking key cytotoxic immune cells. Subcutaneous tumor growth, metastatic dissemination, and activation of CD8+ T and NK cells were evaluated by histology and flow cytometry. RESULTS: Transcriptomic analysis of human SCLC tumors revealed heterogeneous immune checkpoint and cytotoxic signature profiles. Using sophisticated, genetically engineered mouse models, we reported that the absence of NK cells, but not CD8+ T cells, substantially enhanced metastatic dissemination of SCLC tumor cells in vivo. Moreover, hyperactivation of NK cell activity through augmentation of interleukin-15 or transforming growth factor-ß signaling pathways ameliorated SCLC metastases, an effect that was enhanced when combined with antiprogrammed cell death-1 therapy. CONCLUSIONS: These proof-of-principle findings provide a rationale for exploiting the antitumor functions of NK cells in the treatment of patients with SCLC. Moreover, the distinct immune profiles of SCLC subtypes reveal an unappreciated level of heterogeneity that warrants further investigation in the stratification of patients for immunotherapy.

NK Cell Priming From Endogenous Homeostatic Signals Is Modulated by CIS.

Natural killer (NK) cell activation is controlled by a balance of activating and inhibitory signals and cytokines such as IL-15. We previously identified cytokine-inducible SH2-containing protein (CIS) as a negative regulator of IL-15 signaling in NK cells under inflammatory conditions. While the functional effect of Cish-deficiency in NK cells was obvious by their increased anti-tumor immunity and hyper-proliferative response to IL-15, it remained unclear how CIS regulates NK cell biology in steady-state. Here, we investigated the role of CIS in the homeostatic maintenance of NK cells and found CIS-ablation promoted terminal differentiation of NK cells and increased turnover, suggesting that under steady-state conditions, CIS plays a role in maintaining IL-15 driven regulation of NK cells in vivo. However, hyper-responsiveness to IL-15 did not manifest in NK cell accumulation, even when the essential NK cell apoptosis mediator, Bcl2l11 (BIM) was deleted in addition to Cish. Instead, loss of CIS conferred a lower activation threshold, evidenced by augmented functionality on a per cell basis both in vitro and in vivo without prior priming. We conclude that Cish regulates IL-15 signaling in NK cells in vivo, and through the rewiring of several activation pathways leads to a reduction in activation threshold, decreasing the requirement for priming and improving NK cell anti-tumor function. Furthermore, this study highlights the tight regulation of NK cell homeostasis by several pathways which prevent NK cell accumulation when IL-15 signaling and intrinsic apoptosis are dysregulated.

NK cell-derived GM-CSF potentiates inflammatory arthritis and is negatively regulated by CIS.

Despite increasing recognition of the importance of GM-CSF in autoimmune disease, it remains unclear how GM-CSF is regulated at sites of tissue inflammation. Using GM-CSF fate reporter mice, we show that synovial NK cells produce GM-CSF in autoantibody-mediated inflammatory arthritis. Synovial NK cells promote a neutrophilic inflammatory cell infiltrate, and persistent arthritis, via GM-CSF production, as deletion of NK cells, or specific ablation of GM-CSF production in NK cells, abrogated disease. Synovial NK cell production of GM-CSF is IL-18-dependent. Furthermore, we show that cytokine-inducible SH2-containing protein (CIS) is crucial in limiting GM-CSF signaling not only during inflammatory arthritis but also in experimental allergic encephalomyelitis (EAE), a murine model of multiple sclerosis. Thus, a cellular cascade of synovial macrophages, NK cells, and neutrophils mediates persistent joint inflammation via production of IL-18 and GM-CSF. Endogenous CIS provides a key brake on signaling through the GM-CSF receptor. These findings shed new light on GM-CSF biology in sterile tissue inflammation and identify several potential therapeutic targets.

Drug target validation in primary human natural killer cells using CRISPR RNP.

The ability to genetically modify CD8 T cells using viral gene delivery has facilitated the development of next generation of cancer immunotherapies such as chimeric Ag receptor (CAR) T cells engineered to specifically kill tumor cells. Development of immunotherapies targeting NK cells have stalled in part by their resistance to traditional viral gene delivery systems. Here, an efficient approach is described to genetically edit human NK cells by electroporation and CRISPR-Cas9 ribonucleoprotein (RNP) complexes. Electroporation pulse codes and buffer optimization for protein uptake by human NK cells and viability, and the efficiency of this approach over other methods are detailed. To highlight the transformative step this technique will have for NK cell immunotherapy drug discovery, NCR1 and CISH are deleted in primary human NK cells and murine findings are validated on their key roles in regulating NK cell antitumor function.

miR17~92 restrains pro-apoptotic BIM to ensure survival of haematopoietic stem and progenitor cells.

The miR17~92 cluster plays important roles in haematopoiesis. However, it is not clear at what stage of differentiation and through which targets miR17~92 exerts this function. Therefore, we generated miR17~92fl/fl; RosaCreERT2 mice for inducible deletion of miR17~92 in haematopoietic cells. Bone marrow reconstitution experiments revealed that miR17~92-deleted cells were not capable to contribute to mature haematopoietic lineages, which was due to defects in haematopoietic stem/progenitor cells (HSPCs). To identify the critical factor targeted by miR17~92 we performed gene expression analysis in HSPCs, demonstrating that mRNA levels of pro-apoptotic Bim inversely correlated with the expression of the miR17~92 cluster. Strikingly, loss of pro-apoptotic BIM completely prevented the loss of HSPCs caused by deletion of miR17~92. The BIM/miR17~92 interaction is conserved in human CD34+ HSPCs, as miR17~92 inhibition or blockade of its binding to the BIM 3'UTR reduced the survival and growth of these cells. Despite the prediction that miR17~92 functions by impacting a plethora of different targets, the absence of BIM alone is sufficient to prevent all defects caused by deletion of miR17~92 in haematopoietic cells.

Tumor inherent interferon regulators as biomarkers of long-term chemotherapeutic response in TNBC.

Patients diagnosed with triple negative breast cancer (TNBC) have an increased risk of rapid metastasis compared to other subtypes. Predicting long-term survival post-chemotherapy in patients with TNBC is difficult, yet enhanced infiltration of tumor infiltrating lymphocytes (TILs) has been associated with therapeutic response and reduced risk of metastatic relapse. Immune biomarkers that predict the immune state of a tumor and risk of metastatic relapse pre- or mid-neoadjuvant chemotherapy are urgently needed to allow earlier implementation of alternate therapies that may reduce TNBC patient mortality. Utilizing a neoadjuvant chemotherapy trial where TNBC patients had sequential biopsies taken, we demonstrate that measurement of T-cell subsets and effector function, specifically CD45RO expression, throughout chemotherapy predicts risk of metastatic relapse. Furthermore, we identified the tumor inherent interferon regulatory factor IRF9 as a marker of active intratumoral type I and II interferon (IFN) signaling and reduced risk of distant relapse. Functional implications of tumor intrinsic IFN signaling were demonstrated using an immunocompetent mouse model of TNBC, where enhanced type I IFN signaling increased anti-tumor immunity and metastasis-free survival post-chemotherapy. Using two independent adjuvant cohorts we were able to validate loss of IRF9 as a poor prognostic biomarker pre-chemotherapy. Thus, IRF9 expression may offer early insight into TNBC patient prognosis and tumor heat, allowing for identification of patients that are unlikely to respond to chemotherapy alone and could benefit from further immune-based therapeutic intervention.