Abstract: Natural killer (NK) cells are innate lymphocytes capable of distinguishing between healthy and pathogenic cells using a myriad of receptors expressed on their cell surface. The CD16 receptor detects the Fc portion of IgG on antibody-coated cells resulting in NK cell activation. The importance of this receptor on human NK cell biology has long been appreciated; however, how CD16 functions in mouse NK cells remains poorly understood. Here, we report drastic differences between human and mouse CD16 functions in NK cells. We demonstrate that one of the adaptor molecules that CD16 associates with and signals through, CD3ζ, plays a critical role in these functional differences. Using a systematic approach, we demonstrate that key residues in the transmembrane domain of the mouse CD3ζ molecule prevent efficient complex formation with mouse CD16, thereby dampening receptor function. Mutating these residues in mouse CD3ζ to those encoded by human CD3ζ resulted in rescue of CD16 receptor function. Supported by grants from the Cancer Research Institute (CRI), Burroughs Wellcome Fund (BWF), the Parker Institute for Cancer Immunotherapy (PICI) and NIH grants AI068129 and AI146581.

Natural killer (NK) cells can detect antibody-coated cells through recognition by the CD16 Fc receptor. The importance of CD16 in human NK cell biology has long been appreciated, but how CD16 functions in mouse NK cells remains poorly understood. Here, we report drastic differences between human and mouse CD16 functions in NK cells. We demonstrate that one of the adaptor molecules that CD16 associates with and signals through, CD3ζ, plays a critical role in these functional differences. Using a systematic approach, we demonstrate that residues in the transmembrane domain of the mouse CD3ζ molecule prevent efficient complex formation with mouse CD16, thereby dampening receptor function. Mutating these residues in mouse CD3ζ to those encoded by human CD3ζ resulted in rescue of CD16 receptor function. We reveal that the mouse CD3ζ transmembrane domain adopts a tightly packed confirmation, preventing association with CD16, whereas human CD3ζ adopts a versatile configuration that accommodates receptor assembly.

CMV causes mostly asymptomatic but lifelong infection. Primary infection or reactivation in immunocompromised individuals can be life-threatening. CMV viremia often occurs in solid organ transplant recipients and associates with decreased graft survival and higher mortality. Furthering understanding of impaired immunity that allows CMV reactivation is critical to guiding antiviral therapy and examining the effect of CMV on solid organ transplant outcomes. This study characterized longitudinal immune responses to CMV in 31 kidney transplant recipients with CMV viremia and matched, nonviremic recipients. Recipients were sampled 3 and 12 months after transplant, with additional samples 1 week and 1 month after viremia. PBMCs were stained for NK and T cell markers. PBMC transcriptomes were characterized by RNA-Seq. Plasma proteins were quantified by Luminex. CD8+ T cell transcriptomes were characterized by single-cell RNA-Seq. Before viremia, patients had high levels of IL-15 with concurrent expansion of immature CD56bright NK cells. After viremia, mature CD56dim NK cells and CD28-CD8+ T cells upregulating inhibitory and NK-associated receptors were expanded. Memory NK cells and NK-like CD28-CD8+ T cells were associated with control of viremia. These findings suggest that signatures of innate activation may be prognostic for CMV reactivation after transplant, while CD8+ T cell functionality is critical for effective control of CMV.

Cytomegalovirus (CMV) infection is associated with graft rejection in renal transplantation. Memory-like natural killer (NK) cells expressing NKG2C and lacking FcεRIγ are established during CMV infection. Additionally, CD8⁺ T cells expressing NKG2C have been observed in some CMV-seropositive patients. However, in vivo kinetics detailing the development and differentiation of these lymphocyte subsets during CMV infection remain limited. Here, we interrogated the in vivo kinetics of lymphocytes in CMV-infected renal transplant patients using longitudinal samples compared with those of nonviremic (NV) patients. Recipient CMV-seropositive (R+) patients had preexisting memory-like NK cells (NKG2C⁺CD57⁺FcεRIγ^-) at baseline, which decreased in the periphery immediately after transplantation in both viremic and NV patients. We identified a subset of prememory-like NK cells (NKG2C⁺CD57⁺FcεRIγ^low-dim) that increased during viremia in R+ viremic patients. These cells showed a higher cytotoxic profile than preexisting memory-like NK cells with transient up-regulation of FcεRIγ and Ki67 expression at the acute phase, with the subsequent accumulation of new memory-like NK cells at later phases of viremia. Furthermore, cytotoxic NKG2C⁺CD8⁺ T cells and γδ T cells significantly increased in viremic patients but not in NV patients. These three different cytotoxic cells combinatorially responded to viremia, showing a relatively early response in R+ viremic patients compared with recipient CMV-seronegative viremic patients. All viremic patients, except one, overcame viremia and did not experience graft rejection. These data provide insights into the in vivo dynamics and interplay of cytotoxic lymphocytes responding to CMV viremia, which are potentially linked with control of CMV viremia to prevent graft rejection.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.