Showing papers by "Lewis L. Lanier published in 2022"

Diminished cell proliferation promotes natural killer cell adaptive-like phenotype by limiting FcεRIγ expression

Abstract: Diminished human NK cell proliferation limits FcεRIγ expression, leading to an adaptive-like NK cell phenotype, and may have an early prognostic value for COVID-19 disease severity, associated with increased inflammation and higher TGFβ and IFNα levels.

Mass cytometry reveals single-cell kinetics of cytotoxic lymphocyte evolution in CMV-infected renal transplant patients

Abstract: Significance Memory-like NK cells (NKG2C+CD57+FcεRIγ–) are established during CMV infection. Here, mass cytometry tracked the in vivo kinetics of CMV-induced memory NK cells generation and identified a unique subset of NKG2C+CD57+FcεRIγlow–dim as potentially prememory-like NK cells in CMV-infected kidney transplant patients. The study demonstrated that prememory-like NK cells with a high cytotoxic profile proliferate along with accumulation of new memory-like NK cells, whereas preexisting memory-like NK cells decreased in the peripheral blood after transplantation. Moreover, NKG2C+CD8+ T cells and cytotoxic γδ T cells also expand during CMV infection. This interplay of three different cytotoxic lymphocytes demonstrates a combinatorial immune response against CMV infection, which may contribute to preventing CMV-associated complication in organ transplantation. 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+FcεRIγlow–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.

The CD3ζ adaptor structure determines functional differences between human and mouse CD16 Fc receptor signaling

Abstract: CD16 is a potent activating receptor in human NK cells but is weakly responsive in mouse NK cells. Aguilar et al. demonstrate that this is due to differences in amino acids in the transmembrane of CD3ζ that facilitate receptor assembly and function.

Combined protein and transcript single-cell RNA sequencing in human peripheral blood mononuclear cells

Abstract: Cryopreserved peripheral blood mononuclear cells (PBMCs) are frequently collected and provide disease- and treatment-relevant data in clinical studies. Here, we developed combined protein (40 antibodies) and transcript single-cell (sc)RNA sequencing (scRNA-seq) in PBMCs.Among 31 participants in the Women's Interagency HIV Study (WIHS), we sequenced 41,611 cells. Using Boolean gating followed by Seurat UMAPs (tool for visualizing high-dimensional data) and Louvain clustering, we identified 50 subsets among CD4+ T, CD8+ T, B, NK cells, and monocytes. This resolution was superior to flow cytometry, mass cytometry, or scRNA-seq without antibodies. Combined protein and transcript scRNA-seq allowed for the assessment of disease-related changes in transcriptomes and cell type proportions. As a proof-of-concept, we showed such differences between healthy and matched individuals living with HIV with and without cardiovascular disease.In conclusion, combined protein and transcript scRNA sequencing is a suitable and powerful method for clinical investigations using PBMCs.

Differential IL-12 signaling induces human natural killer cell activating receptor-mediated ligand-specific expansion

Abstract: Low IL-12 concentrations are sufficient to induce expansion of human NK cells by innate-activating NK receptor-orchestrated differential IL-12 signaling.

Influence of Self–MHC Class I Recognition on the Dynamics of NK Cell Responses to Cytomegalovirus Infection

Abstract: Although interactions between inhibitory Ly49 receptors and their self–MHC class I ligands in C57BL/6 mice are known to limit NK cell proliferation during mouse CMV (MCMV) infection, we created a 36-marker mass cytometry (CyTOF) panel to investigate how these inhibitory receptors impact the NK cell response to MCMV in other phenotypically measurable ways. More than two thirds of licensed NK cells (i.e., those expressing Ly49C, Ly49I, or both) in uninfected mice had already differentiated into NK cells with phenotypes indicative of Ag encounter (KLRG1+Ly6C−) or memory-like status (KLRG1+Ly6C+). These pre-existing KLRG1+Ly6C+ NK cells resembled known Ag-specific memory NK cell populations in being less responsive to IL-18 and IFN-α stimulation in vitro and by selecting for NK cell clones with elevated expression of a Ly49 receptor. During MCMV infection, the significant differences between licensed and unlicensed (Ly49C−Ly49I−) NK cells disappeared within both CMV-specific (Ly49H+) and nonspecific (Ly49H−) responses. This lack of heterogeneity carried into the memory phase, with only a difference in CD16 expression manifesting between licensed and unlicensed MCMV-specific memory NK cell populations. Our results suggest that restricting proliferation is the predominant effect licensing has on the NK cell population during MCMV infection, but the inhibitory Ly49–MHC interactions that take place ahead of infection contribute to their limited expansion by shrinking the pool of licensed NK cells capable of robustly responding to new challenges. Key Points Naive C57BL/6 mice have a pre-existing memory-like NK cell population. The pre-existing memory-like population comprises predominantly licensed NK cells. Ly49I has limited direct effect on NK cell responses to MCMV beyond proliferation.

Sex Differences in Coronary Artery Disease and Diabetes Revealed by scRNA-Seq and CITE-Seq of Human CD4+ T Cells

Abstract: Background Despite the decades-old knowledge that diabetes mellitus (DM) is a major risk factor for cardiovascular disease (CVD), the reasons for this association are only partially understood. Among the immune cells involved in CVD development, accumulating evidence supports the critical role of T cells as drivers and modifiers of this condition. CD4+ T cells are commonly found in atherosclerotic plaques. The activity and distribution of CD4+ T cell subsets differs between the sexes. Methods Peripheral blood mononuclear cells (PBMCs) of 61 men and women who underwent cardiac catheterization were interrogated by single cell RNA sequencing (scRNA-Seq, ∼200,000 cells) combined with 49 protein markers (CITE-Seq). Coronary artery disease (CAD) was quantified using Gensini scores, with scores above 30 considered CAD+ and below 6 considered CAD-. Four pairs of groups were matched for clinical and demographic parameters. To test how DM changed cell proportions and gene expression, we compared matched groups of diabetic and non-diabetic subjects. We analyzed 41,782 single CD4+ T cell transcriptomes for sex differences in 61 mostly statin-treated coronary artery disease patients with and without DM. Results We identified 16 clusters in CD4 T cells. The proportion of cells in CD4 cluster 8 (CD4T8, CCR2+ Em) was significantly decreased in CAD+, especially among DM+ participants. The proportions of cells in CD4T2, CD4T11, CD4T16 were increased and CD4T13 was decreased in CAD+ among DM+Statin+ participants. CD4T12 was increased in DM+ participants. In female participants, CD4T8, 12, and 13 were decreased compared to in male participants. In CD4 T cells, 31 genes showed significant and coordinated upregulation in both CAD and DM. The DM gene signature was partially additive to the CAD gene signature. Conclusions We conclude that CAD and DM are clearly reflected in PBMC transcriptomes and that significant differences exist between women and men and between subjects treated with statins or not.

NKG2D receptor activation drives primary graft dysfunction severity and poor lung transplantation outcomes

Abstract: Clinical outcomes after lung transplantation, a life-saving therapy for patients with end-stage lung diseases, are limited by primary graft dysfunction (PGD). PGD is an early form of acute lung injury with no specific pharmacologic therapies. Here, we present a large multicenter study of plasma and bronchoalveolar lavage (BAL) samples collected on the first posttransplant day, a critical time for investigations of immune pathways related to PGD. We demonstrated that ligands for NKG2D receptors were increased in the BAL from participants who developed severe PGD and were associated with increased time to extubation, prolonged intensive care unit length of stay, and poor peak lung function. Neutrophil extracellular traps (NETs) were increased in PGD and correlated with BAL TNF-α and IFN-γ cytokines. Mechanistically, we found that airway epithelial cell NKG2D ligands were increased following hypoxic challenge. NK cell killing of hypoxic airway epithelial cells was abrogated with NKG2D receptor blockade, and TNF-α and IFN-γ provoked neutrophils to release NETs in culture. These data support an aberrant NK cell/neutrophil axis in human PGD pathogenesis. Early measurement of stress ligands and blockade of the NKG2D receptor hold promise for risk stratification and management of PGD.

Abstract 3602: Mechanisms of CAR T cell dysfunction and identification of transcription factors that drive the exhaustion phenotype

Abstract: Chimeric antigen receptor (CAR) T cell immunotherapy is FDA approved for the treatment of a subset of B cell malignancies but has shown limited clinical success in solid tumor therapy. T cell exhaustion is an important factor involved in treatment failure, and can, in part, result from continuous stimulation of the CAR by tumor cells expressing the cognate antigen. To gain deeper understanding of CAR T cell exhaustion induced by chronic antigen exposure (CAE), we developed and validated a robust in vitro model, in which mesothelin-redirected CAR T cells (M5CAR) were continuously stimulated with mesothelin-expressing AsPC-1 pancreatic tumor cells such that tumor cells were never cleared, and we characterized these CAE CAR T cells by gene expression at population and single-cell levels, and by epigenetic analyses. CAE M5CAR T cells recapitulate the hallmarks of T cell exhaustion, including reduced proliferation, down-modulation of surface CAR, decreased cytokine production, and reduced cytotoxicity. In addition, CAR T cells undergoing CAE have a transcriptional signature and an epigenetic landscape consistent with exhaustion. Further, transcriptomic analysis revealed that CAE M5CAR T cells undergo a transition from T cells to a post-thymic NK-like T cell phenotype. This plasticity was confirmed by TCR lineage tracing and was also detected in CD19 CAR T cells analyzed from post-infusion blood from DLBCL patients and in M5CAR T cells infiltrating relapsed tumors derived from a xenograft NSG/AsPC-1 mouse model. The dysfunctional signature and the NK phenotype were further detected in vivo by transcriptomic analysis in Ly95 TCR-specific TILs infiltrating NY-ESO-1 tumors. Among the genes included in the dysfunctional signature, the transcription factors SOX4 and ID3 were identified as potential regulators of dysfunction by differential gene expression and pathway analyses. To determine their role in the establishment of a dysfunctional phenotype, we generated ID3KO.M5 and SOX4KO.M5 CAR T cells using CRISPR technology in primary human lymphocytes. The cytotoxic potential of the M5 CAR T cell product generated was not modified by the genetic disruption of the transcription factors. However, when CAR T cells were challenged with chronic antigen stimulation, KO CAR T cells showed a significant reduction of the dysfunctional signature and the NK-like T cell gene expression. Importantly, CAE KO CAR T cells exhibit improved cytotoxicity as compared to Mock.M5CAR T cells. In summary, we have developed a robust in vitro model that recapitulates the hallmarks of T cell exhaustion and that facilitated the identification of a gene signature defining CAR dysregulation, a T-to-NK-like-T cell transition as a novel feature of CAR T cell dysfunction and the transcription factors SOX4 and ID3 as key regulators of CAR T cell exhaustion. Citation Format: M. Angela Aznar, Charly R. Good, Shunichiro Kuramitsu, Parisa Samareh, Sangya Agarwal, Greg Donahue, Kenichi Ishiyama, Nils Wellhausen, Austin K. Rennels, Yujie Ma, Lifeng Tian, Sonia Guedan, Katherine A. Alexander, Zhen Zhang, Philipp C. Rommel, Nathan Singh, Karl M. Glastad, Max W. Richardson, Keisuke Watanabe, Janos L. Tanyi, Mark H. O’Hara, Marco Ruella, Simon F. Lacey, Edmund K. Moon, Stephen J. Schuster, Steven M. Albelda, Lewis L. Lanier, Regina M. Young, Shelley L. Berger, Carl H. June. Mechanisms of CAR T cell dysfunction and identification of transcription factors that drive the exhaustion phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3602.

CD3ζ adaptor structure determines functional differences between human and mouse CD16 Fc-gamma receptor signaling in natural killer cells

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.

Cutting Edge: Heterogeneity in cell age contributes to functional diversity of natural killer cells

Abstract: Heterogeneity among naïve adaptive lymphocytes determines their individual functions and fate decisions during an immune response. Natural killer (NK) cells are innate lymphocytes capable of generating ‘adaptive’ responses during infectious challenges. However, the factors that govern various NK cell functions are not fully understood. Here, we use a reporter mouse model to permanently ‘time-stamp’ NK cells and type 1 innate lymphoid cells (ILC1s) in order to characterize the dynamics of their homeostatic turnover. We found that the homeostatic turnover of tissue-resident ILC1s is much slower than that of circulating NK cells. NK cell homeostatic turnover is further accelerated without the transcription factor Eomes. Finally, heterogeneity in NK cell age diversifies NK cell function, with ‘older’ NK cells exhibiting more potent IFN-γ production to activating stimuli, and more robust adaptive responses during cytomegalovirus infection. These results provide insight into how the functional response of an NK cell varies over its lifespan.

Modeling CD16 signal kinetics reveals the role of adaptor CD3ζ in NK cell signaling in humans and mice

Abstract: —Natural killer (NK) cells provide important protection against viral infections and tumors. Stimulation of CD16, an activating NK receptor that recognizes antibodies, leads to lysis of antibody-coated target cells by NK cells. However, engagement of CD16 in mouse NK cell produces weaker cytotoxic responses than in human NK cell [1]. We developed a CD16 signaling model to investigate the above differences in NK cell responses between humans and mice. Our study suggests higher number of ITAMs in CD3ζ, along with increased association between CD16 and CD3ζ are crucial for greater recruitment of downstream signaling molecules, resulting in higher cytotoxic responses. signaling pathways for different mutants of CD3ζ and FcεR1γ and computed the kinetics of the abundance of phosphorylated ZAP70 (pZAP70) and Ca 2+ flux. Comparison of the Ca 2+ flux kinetics between our model and experiments indicates that the increase in the number of ITAMs plays a crucial role in increasing Ca 2+ flux. CD3ζ, which has 6 ITAMs are more efficient in producing phosphorylated ZAP70 compared to FcεR1γ, which has 2 ITAMs. We further explain Ca 2+ signals from experiments using our model for different mutants of CD3ζ. Our model suggests, mutants that are better at producing Ca 2+ signal have higher affinities to associate with the CD16 receptor. Our model can be extended to describe signaling kinetics for various receptors that can associate with multiple types of adaptors.