At the turn of the last century, the emerging field of medical oncology chose a cytotoxic approach to cancer therapy over an immune-centered approach at a time when evidence in support of either paradigm did not yet exist. Today, nearly 120 years of data have established that (a) even the best cytotoxic regimens only infrequently cure late-stage malignancy and (b) strategies that supplement and augment existing antitumor immune responses offer the greatest opportunities to potentiate durable remission in cancer. Despite widespread acceptance of these paradigms today, the ability of the immune system to recognize and fight cancer was a highly controversial topic for much of the twentieth century. Why this modern paradigmatic mainstay should have been both dubious and controversial for such an extended period is a topic of considerable interest that merits candid discussion. Herein, we review the literature to identify and describe the watershed events that ultimately led to the acceptance of immunotherapy as a viable regimen for the treatment of neoplastic malignancy. In addition to noting important clinical discoveries, we also focus on research milestones and the development of critical model systems in rodents and dogs including the advanced modeling techniques that allowed development of patient-derived xenografts. Together, their use will further our understanding of cancer biology and tumor immunology, allow for a speedier assessment of the efficacy and safety of novel approaches, and ultimately provide a faster bench to beside transition.

Key Points Cytomegalovirus reactivation and interleukin 15 are major contributors to NK cell repertoire diversity and maturation after CBT. An immature NK cell subset characterized by low diversity index and poor effector function was highly predictive of relapse after CBT.

Chronic lymphocytic leukemia (CLL) cells possess regulatory functions comparable to those of normal B10 cells, a regulatory B cell subset that suppresses effector T-cell function through STAT3-mediated IL-10 production. However, the mechanisms governing IL-10 production by CLL cells are not fully understood. Here, we show that the CXC chemokine ligand 12 (CXCL12)–CXCR4–STAT3 axis regulates IL-10 production by CLL cells and their ability to suppress T-cell effector function through an IL-10 mediated mechanism. Knockdown of STAT3 significantly impaired the ability of CLL cells to produce IL-10. Furthermore, experiments to assess the role of lenalidomide, an immunomodulatory agent with direct antitumor effect as well as pleiotropic activity on the immune system, showed that this agent prevents a CXCL12-induced increase in p-S727-STAT3 and the IL-10 response by CLL cells. Lenalidomide also suppressed IL-10-induced Y705-STAT3 phosphorylation in healthy T cells, thus reversing CLL-induced T-cell dysfunction. We conclude that the capacity of CLL cells to produce IL-10 is mediated by the CXCL12–CXCR4–STAT3 pathway and likely contributes to immunodeficiency in patients. Lenalidomide appears to be able to reverse CLL-induced immunosuppression through including abrogation of the CXCL12–CXCR4–S727–STAT3-mediated IL-10 response by CLL cells and prevention of IL-10-induced phosphorylation of Y705-STAT3 in T cells.

Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)–grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.

Glioblastoma, the most aggressive brain cancer, often recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. Here, we show that patient-derived GSCs, but not normal astrocytes, are highly sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. In contrast, single cell analysis of autologous, tissue infiltrating NK cells isolated from surgical samples of high-grade glioblastoma patient tumors using mass cytometry and single cell RNA sequencing revealed an abnormal phenotype associated with impaired lytic function compared with peripheral blood NK cells from GBM patients or healthy donors. This immunosuppression was attributed to an integrin-TGF-β mechanism, activated by direct cell-cell contact between GSCs and NK cells. Treatment of GSCengrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGFβ signaling, or with TGF-β receptor 2 gene-edited NK cells prevented GSC-induced NK cell dysfunction and tumor growth. Collectively, our findings reveal a novel mechanism of NK cell immune evasion by GSCs and implicate the integrin-TGF-β axis as a useful therapeutic target to eliminate GSCs in this devastating tumor. RESULTS GSCs are susceptible to NK cell-mediated killingThe GSCs can be distinguished from their mature tumor progeny at the transcriptional, epigenetic and metabolic levels 16,17 , raising the question of whether these cells can be recognized and killed by NK cells. We therefore asked whether patient-derived GSCs, defined as being capable of self-renewal, pluripotent differentiation, and tumorigenicity when implanted into an animal host, are susceptible to NK cell cytotoxic activity as compared with healthy human astrocytes. We derived GSCs from patients with various glioblastoma subtypes including mesenchymal (GSC20, GSC267), classical (GSC231, GSC6-27), and proneural (GSC17, GSC8-11, GSC262) while also showing heterogeneity in the O(6)-Methylguanine-DNA methyltransferase (MGMT) methylation status (methylated: GSC231, GSC8-11, GSC267; indeterminate: GSC6-26, GSC17, GSC262).K562 targets were used as positive control because of their marked sensitivity to NK cell mediated killing due to lack of expression of HLA class I 18 . Across all effector:target (E:T) ratios, healthy donor NK cells killed GSCs (n=6) and K562 cells with equal efficiency and much more readily than healthy human astrocytes (n=6), which displayed a relative resistance to NK cell-mediated killing (Fig. 1A). Multi-parametric flow cytometry was then used to analyze the expression of NK cell activating or inhibitory receptor ligands on GSCs. GSCs (n=6) expressed normal levels of HLA-class I and HLA-E (both ligands for inhibitory NK receptors), at levels similar to those observed on healthy human astrocytes (n=3) (Fig. 1B). In contrast, the ligands for activating NK receptors, such as CD155 (ligand for DNAM1 and TIGIT), MICA/B and ULBP1/2/3 (ligands for NKG2D) and B7 -H6 (ligand for NKp30) were upregulated on GSCs but not on healthy human astrocytes (Fig. 1B). To as...

41Immune checkpoint therapy has produced remarkable improvements in the outcome for 42 certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a 43 strategy that couples targeting of the cytokine-inducible SH2-containing (CIS) protein, a 44 key negative regulator of interleukin (IL)-15 signaling, with chimeric antigen receptor 45 (CAR) engineering of natural killer (NK) cells. This combined strategy boosted NK cell 46 effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, 47 resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this 48 combined approach improved NK cell anti-tumor activity more than either alteration 49 alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We 50 conclude that combining CIS checkpoint deletion with CAR engineering promotes the 51 metabolic fitness of NK cells in an otherwise suppressive tumor microenvironment. This 52 approach, together with the prolonged survival afforded by CAR modification, represents 53 a promising milestone in the development of the next generation of NK cells for cancer 54 immunotherapy. 55 56 57 Introduction 58 59Adoptive cellular therapy using autologous T cells transduced with a chimeric antigen 60 receptor (CAR) has proved to be a powerful approach in the treatment of human cancers, 61 especially B cell leukemias and lymphomas. 1,2 However, ongoing efforts to consolidate 62 and extend these gains face a number of obstacles: (i) uncoupling cytotoxicity against 63 tumor cells from systemic toxicity, (ii) finding ways to reduce target antigen negative 64 relapses, (iii) overcoming the inhibitory effects of checkpoint molecules in the infused 65 immune effector cells, and (iv) developing universal off-the-shelf cell therapy products 66 that avoid the logistic hurdles of generating autologous products, as well as several of the 67 pitfalls of allogeneic T-cell therapy, such as graft-versus-host disease (GvHD). 3 68 69 Natural killer (NK) cells are attractive candidates for the next wave of effective cancer 70 immunotherapies. They mediate potent cytotoxicity against a range of tumor cells 4 and, 71 unlike T cells, lack the capacity to induce GvHD in the allogeneic setting. 5 Moreover, 72 their ready availability in high numbers from various sources, such as umbilical cord 73 blood (CB), boosts their potential as an off-the-shelf product for widespread clinical 74 scalability. 6,7 One of the most intriguing recent advances in the development of NK cell-75 based immunotherapy was the demonstration that genetic modification of these cells to 76 express a CAR can enhance their effector function. 8 This led to the realization that one 77 might overcome some of the limitations of NK cell immunotherapy in cancer, such as the 78 lack of antigen specificity and poor persistence, by exploiting current genetic engineering 79 tools. In our experience, transducing NK cells with a retroviral vector encoding a CD19-80 4 specific CAR, the interleukin (IL)-15 cytokine and ...

BackgroundNatural killer (NK) cells are a nascent cellular immunotherapy for hematologic malignancies. Target recognition of NK cell-resistant cancers remains a substantial barrier to broad application of NK cell therapy. One solution are bispecific engagers that trigger NK cells via an NK activating receptor when simultaneously engaging a tumor-specific antigen.MethodsHere, we investigated single NK cell responses stimulated by the tetravalent bispecific innate cell engager (ICE®) AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on several types of NK cells.ResultsMultidimensional mass cytometry revealed heterogeneity within AFM13-directed conventional (c)NK cell responses, as well as consistent polyfunctional activation of mature terminally differentiated NK cells across donors. The source of NK cells also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from Hodgkin lymphoma patients. IL-12, IL-15, and IL-18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared to cNK cells. Cord-blood expanded NK cells that were pre-activated with IL-12, IL-15 and IL-18 also exhibited enhanced killing with AFM13 stimulation, via upregulation of signaling pathways related to NK cell effector function. These cells were stably pre-loaded with AFM13 enhancing responses to CD30+ lymphomas in vitro and in vivo in immunodeficient NSG mouse models.ConclusionsCollectively, these data identify promising combinations of AFM13 with cytokine-activated adult blood or cord blood NK cells against CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

Mast cells (MC) are key effectors cells of the immune system. MC infiltration has been reported in a variety of renal diseases including lupus nephritis. Recently it has been argued that MCs are key contributors to a common pathway of progressive renal injury, however, the chemotaxic mechanism of renal MC recruitment are largely unknown. We observed a progressive age-depended MC infiltrate in kidneys of MRL/lpr mice. MRL/lpr bone marrow-derived MC (BMMC) had a high chemotatic capacity in response to MRL/lpr serum. In order to characterize the factors involved in MCs recruitment in MRL/lpr mice we performed expression array analysis in stimulated and un-stimulated MRL/lpr MCs. Serum stimulation increased BMMC expression of the integrin alpha E and chemokine receptors CCR5 and CXCR4. Kidney immunofluorescence staining confirmed these finding in situ. BMMC chemotaxis in vitro was enhanced in response to CCR5 ligands (MIP-1α and RANTES) and CXCR4 ligand (SDF-1) and was blocked by using a non-peptide chemokine receptor antagonist TAK-779. In vivo, TAK-779 significantly blocked the accumulation of renal MC in MRL/lpr mice. Our study indicate that the chemokine receptors CCR5 and CXCR4 and the integrin alpha E are important mediators of renal MC recruitment.