Abstract Influenza viruses remain a leading cause of global respiratory illness in humans. The suboptimal effectiveness of seasonal influenza vaccination underscores the need for a more comprehensive understanding of immune mediators of protection, especially in populations with diverse baseline immune profiles and exposure histories. While anti-influenza antibody titers are typically used to define correlates of protection, mounting evidence suggests a substantive role for innate and cellular immunity in determining tolerance and resistance during influenza virus infection. However, distinct cell subsets that correlate with protection against symptomatic influenza remain to be identified in humans. Here, we measured baseline cellular and serologic profiles in peripheral blood from 206 vaccinated or unvaccinated adult subjects enrolled in the 2018 SHIVERS-II cohort to determine how baseline variations in the cellular and humoral immune compartments contribute independently or synergistically to the risk of developing symptomatic influenza infection. Protection from symptomatic influenza correlated with increased individual frequencies of diverse and polyfunctional CD4 and CD8 T cells, cells associated with engagement of humoral responses including cTfh and mDCs, Th17 cells, and innate effector CD16-expressing cytotoxic and cytokine-producing NK cells. In contrast, increased susceptibility was predominantly attributed to nonspecific inflammatory populations including γδ T cells and activated CD16 neg NK cells, as well as TNFα + single-producing CD8 T cells. A trained random forest model categorizing symptomatic influenza cases identified that cellular covariates substantially improved model accuracy up to 86% over demographic and serologic factors alone (61%). A corresponding variable importance analysis showed cellular populations comprise 28 of the top 30 covariates (from 48 total), with the single most important factor being ICOS + cTfh cells. Lastly, using a multivariate logistic regression model considering participant demographics, anti-influenza antibody titers, vaccination status, and cell population covariates, we quantified how these factors contribute to risk of symptomatic influenza infection. Protection was associated with a combination of lymphocyte populations including naïve, CD107a + , and Th17 CD4 T cells, and serologic factors including antibodies targeting neuraminidase. Increased risk of symptomatic influenza (95% subtype A) was associated with elevated anti-hemagglutinin antibodies against influenza B (Yamagata), along with γδ T cells and TNFα + CD8 T cell single-cytokine producers. Together, these results demonstrate that the composition of pre-infection peripheral cell profiles is a stronger predictor of symptomatic influenza susceptibility than vaccination, demographics, or serology.
<div>Abstract<p>Current chimeric antigen receptor-modified (CAR) T-cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T-cell receptor (TCR) sequencing data set using pre- and postinfusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B-cell acute lymphoblastic leukemia. We identified cytotoxic postinfusion cells with identical TCRs to a subset of preinfusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared with other preinfusion cells, corresponding to an unexpected surface phenotype (TIGIT<sup>+</sup>, CD62L<sup>lo</sup>, CD27<sup>−</sup>). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor TOX. Collectively, these results demonstrate diverse effector potentials within preinfusion CAR T-cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytic framework for elucidating the mechanisms underlying effector development in CAR T-cell products.</p>Significance:<p>Utilizing clonal trajectories to define transcriptional potential, we find a unique signature of CAR T-cell effector precursors present in preinfusion cell products. Functional assessment of cells with this signature indicated early effector potential and resistance to exhaustion, consistent with postinfusion cellular patterns observed in patients.</p><p><i><a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-12-9-ITI" target="_blank">This article is highlighted in the In This Issue feature, p. 2007</a></i></p></div>
Abstract Regulatory T cells (Tregs) are key negative regulators of the immune system. Conventional T cells use their T cell receptor (TCR) to direct their effector function against specific targets. While much is known about the role of Tregs in their microenvironment, whether Tregs require this TCR specificity to elicit their effector functions is not clear. If so, Treg-specific antigens are also undefined. Investigation of the TCR repertoire can help to answer these questions as it provides insight into the degree of antigen recognition T cells experienced during a response. Since Tregs have been implicated in infection, autoimmunity, allergy, and cancer, we have chosen to characterize the Treg TCR repertoire in murine models for influenza, type I diabetes, asthma, neuroblastoma, and non-small cell lung carcinoma. Using an algorithm generated in our lab, TCRdist, we analyzed features of the TCR repertoire such as clonal expansion, TCR diversity, and V and J region biases. Our preliminary data show lung Tregs from asthmatic mice display the greatest antigen specificity while Tregs in influenza and neuroblastoma have minimal specificity based on clonal expansion and enrichment of TCR motifs. Additionally, we have identified Treg receptors that occur in more than one disease type. These data suggest that Treg TCR-dependency is condition-specific and may be determined by the local tissue as a pathological antigen. Future studies include associating these differences in the repertoire with TCR signaling and Treg functional capacities. Understanding the antigen-specificity of Tregs and identifying highly effective Tregs across diverse diseases can provide opportunities to develop broadly applicable, targeted therapeutics.
<div>Abstract<p>Immune cells regulate tumor growth by mirroring their function as tissue repair organizers in normal tissues. To understand the different facets of immune–tumor collaboration through genetics, spatial transcriptomics, and immunologic manipulation with noninvasive, longitudinal imaging, we generated a penetrant double oncogene–driven autochthonous model of neuroblastoma. Spatial transcriptomic analysis showed that CD4<sup>+</sup> and myeloid populations colocalized within the tumor parenchyma, while CD8<sup>+</sup> T cells and B cells were peripherally dispersed. Depletion of CD4<sup>+</sup> T cells or CCR2<sup>+</sup> macrophages, but not B cells, CD8<sup>+</sup> T cells, or natural killer (NK) cells, prevented tumor formation. Tumor CD4<sup>+</sup> T cells displayed unconventional phenotypes and were clonotypically diverse and antigen independent. Within the myeloid fraction, tumor growth required myeloid cells expressing arginase-1. Overall, these results demonstrate how arginine-metabolizing myeloid cells conspire with pathogenic CD4<sup>+</sup> T cells to create permissive conditions for tumor formation, suggesting that these protumorigenic pathways could be disabled by targeting myeloid arginine metabolism.</p>Significance:<p>A new model of human neuroblastoma provides ways to track tumor formation and expansion in living animals, allowing identification of CD4<sup>+</sup> T-cell and macrophage functions required for oncogenesis.</p></div>
<div>Abstract<p>Current chimeric antigen receptor-modified (CAR) T-cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T-cell receptor (TCR) sequencing data set using pre- and postinfusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B-cell acute lymphoblastic leukemia. We identified cytotoxic postinfusion cells with identical TCRs to a subset of preinfusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared with other preinfusion cells, corresponding to an unexpected surface phenotype (TIGIT<sup>+</sup>, CD62L<sup>lo</sup>, CD27<sup>−</sup>). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor TOX. Collectively, these results demonstrate diverse effector potentials within preinfusion CAR T-cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytic framework for elucidating the mechanisms underlying effector development in CAR T-cell products.</p>Significance:<p>Utilizing clonal trajectories to define transcriptional potential, we find a unique signature of CAR T-cell effector precursors present in preinfusion cell products. Functional assessment of cells with this signature indicated early effector potential and resistance to exhaustion, consistent with postinfusion cellular patterns observed in patients.</p><p><i><a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-12-9-ITI" target="_blank">This article is highlighted in the In This Issue feature, p. 2007</a></i></p></div>
Abstract Current chimeric antigen receptor-modified (CAR) T cell therapy products are evaluated in bulk, without assessment of the possible heterogeneity in effector potential between cells. Conceivably, only a subset of the pre-infusion product differentiates into optimal effectors. We generated a comprehensive single-cell gene expression and T cell receptor (TCR) sequencing dataset using both pre- and post-infusion CD19-CAR T cells from peripheral blood and bone marrow of pediatric patients with B cell acute lymphoblastic leukemia (B-ALL). We identified potent effector post-infusion cells with identical TCRs to a subset of pre-infusion CAR T cells. Effector precursor CAR T cells exhibited a unique transcriptional profile compared to other pre-infusion cells, and the number of effector precursor cells infused correlated with peak CAR T cell expansion. Additionally, we identified an unexpected cell surface phenotype (TIGIT+, CD62Llo, CD27−), conventionally associated with inhibiting effective T cell responses, that we used to successfully enrich for subsequent effector potential. Collectively, these results demonstrate that highly diverse effector potentials are present among cells in pre-infusion cell products, which can be exploited for diagnostic and therapeutic applications. Furthermore, we provide an integrative experimental and analytical framework for elucidating the biological mechanisms underlying effector development in other CAR T cell therapy products. This work was supported by the National Institutes of Health (NIH)/National Cancer Institute grant P30CA021765, NIH grants U01AI150747 and R01AI136514 (PGT), the American Society of Transplantation and Cellular Therapy (AT), the American Society of Hematology (AT), the Key for a Cure Foundation (PGT), the Mark Foundation ASPIRE Award (PGT), and the American Lebanese Syrian Associated Charities (SG, PGT). Part of the laboratory studies were performed by the Center for Translational Immunology and Immunotherapy (CeTI2), which is supported by SJCRH. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
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