Single-Cell Profiling Reveals a Naive-Memory Relationship between CD56 bright and Adaptive Human Natural Killer Cells.

Development of antigen-specific memory upon pathogen exposure is a hallmark of the adaptive immune system. While natural killer (NK) cells are considered part of the innate immune system, humans exposed to the chronic viral pathogen cytomegalovirus (CMV) often possess a distinct NK cell population lacking in individuals who have not been exposed, termed "adaptive" NK cells. To identify the "naïve" population from which this "memory" population derives, we performed phenotypic, transcriptional, and functional profiling of NK cell subsets. We identified immature precursors to the Adaptive NK cells that are equally present in both CMV+ and CMV-individuals, resolved an Adaptive transcriptional state distinct from most mature NK cells and sharing a common gene program with the immature CD56 bright population, and demonstrated retention of proliferative capacity and acquisition of superior IFNγ production in the Adaptive population. Furthermore, we distinguish the CD56 bright and Adaptive NK populations by expression of the transcription factor CXXC5, positioning these memory NK cells at the inflection point between innate and adaptive lymphocytes.

Immune profiling after allogeneic hematopoietic cell transplantation in pediatric acute myeloid leukemia.

Although allogeneic hematopoietic cell transplant (allo-HCT) is curative for high-risk pediatric acute myeloid leukemia (AML), disease relapse remains the primary cause of posttransplant mortality. To identify pressures imposed by allo-HCT on AML cells that escape the graft-versus-leukemia effect, we evaluated immune signatures at diagnosis and posttransplant relapse in bone marrow samples from 4 pediatric patients using a multimodal single-cell proteogenomic approach. Downregulation of major histocompatibility complex class II expression was most profound in progenitor-like blasts and accompanied by correlative changes in transcriptional regulation. Dysfunction of activated natural killer cells and CD8+ T-cell subsets at relapse was evidenced by the loss of response to interferon gamma, tumor necrosis factor α signaling via NF-κB, and interleukin-2/STAT5 signaling. Clonotype analysis of posttransplant relapse samples revealed an expansion of dysfunctional T cells and enrichment of T-regulatory and T-helper cells. Using novel computational methods, our results illustrate a diverse immune-related transcriptional signature in posttransplant relapses not previously reported in pediatric AML.

Human Cytomegalovirus Infection Promotes Expansion of a Functionally Superior Cytoplasmic CD3+ NK Cell Subset with a Bcl11b-Regulated T Cell Signature.

Human CMV (HCMV) is a ubiquitous pathogen that indelibly shapes the NK cell repertoire. Using transcriptomic, epigenomic, and proteomic approaches to evaluate peripheral blood NK cells from healthy human volunteers, we find that prior HCMV infection promotes NK cells with a T cell-like gene profile, including the canonical markers CD3ε, CD5, and CD8ß, as well as the T cell lineage-commitment transcription factor Bcl11b. Although Bcl11b expression is upregulated during NK maturation from CD56bright to CD56dim, we find a Bcl11b-mediated signature at the protein level for FcεRIγ, PLZF, IL-2Rß, CD3γ, CD3δ, and CD3ε in later-stage, HCMV-induced NK cells. BCL11B is targeted by Notch signaling in T cell development, and culture of NK cells with Notch ligand increases cytoplasmic CD3ε expression. The Bcl11b-mediated gain of CD3ε, physically associated with CD16 signaling molecules Lck and CD247 in NK cells is correlated with increased Ab-dependent effector function, including against HCMV-infected cells, identifying a potential mechanism for their prevalence in HCMV-infected individuals and their prospective clinical use in Ab-based therapies.

Human cytomegalovirus expands a CD8+ T cell population with loss of BCL11B expression and gain of NK cell identity.

CD8+ T cells not only are critical mediators of adaptive immunity but also may exhibit innate-like properties such as surface expression of NKG2C, an activating receptor typically associated with natural killer (NK) cells. We demonstrate that, similar to NK cells, NKG2C+TCRαß+CD8+ T cells are associated with prior human cytomegalovirus (HCMV) exposure. In addition to expressing several NK cell markers such as CD56 and KIR, NKG2C+CD8+ T cells are oligoclonal and do not up-regulate PD-1 even in response to persistent activation. Furthermore, we found that NKG2C+CD8+ T cells from some individuals exhibited strong effector function against leukemia cells and HCMV-infected fibroblasts, which was dictated by both NKG2C and TCR specificity. Transcriptomic analysis revealed that the transcription factor BCL11B, a regulator of T cell developmental fate, is down-regulated in NKG2C+CD8+ T cells when compared with conventional NKG2C−CD8+ T cells. BCL11B deletion in conventional CD8+ T cells resulted in the emergence of a similar innate-like CD56+CD94+DAP12+NKG2C+CD45RA+CCR7−PD-1−/low T cell population with activity against HLA-E+ targets. On the basis of their intrinsic capacity to recognize diseased cells coupled with lack of PD-1 induction, NKG2C+CD8+ T cells represent a lymphocyte population that resides at the boundary between innate and adaptive immunity, presenting an attractive alternative for cellular therapy, including CAR T cell­based therapies.

Establishing a model system for evaluating CAR T cell therapy using dogs with spontaneous diffuse large B cell lymphoma.

Multiple rodent and primate preclinical studies have advanced CAR T cells into the clinic. However, no single model accurately reflects the challenges of effective CAR T therapy in human cancer patients. To evaluate the effectiveness of next-generation CAR T cells that aim to overcome barriers to durable tumor elimination, we developed a system to evaluate CAR T cells in pet dogs with spontaneous cancer. Here we report on this system and the results of a pilot trial using CAR T cells to treat canine diffuse large B cell lymphoma (DLBCL). We designed and manufactured CD20-targeting, second-generation canine CAR T cells for functional evaluation in vitro and in vivo using lentivectors to parallel human CAR T cell manufacturing. A first-in-species trial of five dogs with DLBCL treated with CAR T was undertaken. Canine CAR T cells functioned in an antigen-specific manner and killed CD20+ targets. Circulating CAR T cells were detectable post-infusion, however, induction of canine anti-mouse antibodies (CAMA) was associated with CAR T cell loss. Specific selection pressure on CD20+ tumors was observed following CAR T cell therapy, culminating in antigen escape and emergence of CD20-disease. Patient survival times correlated with ex vivo product expansion. Altering product manufacturing improved transduction efficiency and skewed toward a memory-like phenotype of canine CAR T cells. Manufacturing of functional canine CAR T cells using a lentivector is feasible. Comparable challenges to effective CAR T cell therapy exist, indicating their relevance in informing future human clinical trial design.

Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas.

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells' efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.

Feasibility and Safety of RNA-transfected CD20-specific Chimeric Antigen Receptor T Cells in Dogs with Spontaneous B Cell Lymphoma.

Preclinical murine models of chimeric antigen receptor (CAR) T cell therapy are widely applied, but are greatly limited by their inability to model the complex human tumor microenvironment and adequately predict safety and efficacy in patients. We therefore sought to develop a system that would enable us to evaluate CAR T cell therapies in dogs with spontaneous cancers. We developed an expansion methodology that yields large numbers of canine T cells from normal or lymphoma-diseased dogs. mRNA electroporation was utilized to express a first-generation canine CD20-specific CAR in expanded T cells. The canine CD20 (cCD20) CAR expression was efficient and transient, and electroporated T cells exhibited antigen-specific interferon-gamma (IFN-γ) secretion and lysed cCD20+ targets. In a first-in-canine study, autologous cCD20-ζ CAR T cells were administered to a dog with relapsed B cell lymphoma. Treatment was well tolerated and led to a modest, but transient, antitumor activity, suggesting that stable CAR expression will be necessary for durable clinical remissions. Our study establishes the methodologies necessary to evaluate CAR T cell therapy in dogs with spontaneous malignancies and lays the foundation for use of outbred canine cancer patients to evaluate the safety and efficacy of next-generation CAR therapies and their optimization prior to translation into humans.