Eliane Piaggio

Tumor-invaded lymph nodes (LNs) are critical but poorly studied hubs of anti-tumor immunity. Using single-cell transcriptome, clonotype, and chromatin profiling, we analyze CD4 + T cells from blood, LNs, and tumors of NSCLC patients. LNs and tumors are enriched in follicular regulatory T cells (Treg-Tfr), follicular-like conventional T cells (Tconv-Tfh and Tconv-CXCL13), and tissue-resident Tregs (Treg-Trm). These populations are predicted to share a transcriptional program of activation and tissue adaptation, driven by BATF. Additionally, these subsets show extensive LN-tumor clonal sharing, indicating recirculation, and are enriched in transcriptional signatures of tumor reactivity. Treg-Tfr cells function as progenitors bifurcating into Treg-Trm or ex-Tregs with a Tfh-like CXCL13+ effector phenotype. Follicular subsets in LNs and tumors are transcriptionally and epigenetically similar and localize in germinal-center-like niches. Overall, tumor-invaded LNs and tumors coordinate the generation and maintenance of tumor-reactive CD4+ lineages, identifying highly plastic follicular T cells as therapeutic checkpoints to reinforce anti-tumor responses.

In approximately one third of patients, resected head and neck squamous cell carcinoma will recur. We postulated that the induction of tumor neoantigen-specific T cell responses could prevent relapse. To this end, we developed TG4050, an individualized neoantigen therapeutic vaccine encoding up to 30 patient-specific predicted tumor neoantigens delivered by a Modified Vaccinia Ankara viral vector. We tested adjuvant TG4050 as single agent in a randomized phase I trial comparing treatment with TG4050 immediately after standard of care adjuvant therapy versus watchful waiting and treatment with TG4050 after recurrence (NCT04183166). The primary endpoint was safety, secondary endpoints included feasibility and efficacy, and immunogenicity was exploratory. TG4050 was well tolerated. Of 16 evaluable patients randomized the immediate treatment arm, none relapsed after a median follow-up of 30 months, while 3 of 16 relapsed in the control arm. T cell responses to vaccine neoantigens were detected in 73.3% of patients treated with TG4050 immediately, with a median of 3 neoantigens per responder. These responses were maintained throughout treatment and persisted for over one year after the last dose. Vaccine neoantigen-specific CD8 ⁺ T cells had an effector phenotype, displayed high expression of cytotoxic and tissue-resident markers, were polyclonal and comprised both de novo responses and amplification of pre-existing tumor-infiltrating T cell clones. Together, these translational data are consistent with the hypothesis in which single-agent delivery of TG4050 induces long-lasting tumor neoantigen-specific cytotoxic T cell responses that can prevent tumor recurrence.

One of the first-line treatments for advanced non-small cell lung cancer (NSCLC) are immune checkpoint inhibitors (ICI), which activate the antitumor immune response. Despite their success, ICI remain ineffective in many patients, highlighting the need for strategies to overcome resistance. Most efforts have focused on promoting immune cell infiltration into refractory tumors to improve ICI efficacy. In this work, we mobilize this approach by focusing on Argonaute 2 (Ago2), a pivotal member of the RNA interference pathway. Using two murine models of immunorefractory NSCLC, we demonstrate that tumoral Ago2 suppresses interferon signaling, leading to poor immunogenicity and failure of ICI therapy. Genetic deletion of Ago2 in cancer cells restores interferon signaling and supports immune infiltration of the tumor. Consequently, whereas wild-type tumors are resistant to ICI, tumors devoid of Ago2 become sensitive to treatment. In NSCLC patients treated with ICI, high Ago2 expression and a low interferon signature in tumors correlate with reduced survival. Ago2 is thus a driver of the immunorefractory phenotype observed in NSCLC and may represent a therapeutic target when aiming to sensitize patients to ICI.

Introduction: Humanized immune system (HIS) mouse models, generated by engrafting tumors and hematopoietic cells of human (Hu) origin into immunodeficient host mice, effectively recapitulate key aspects of the crosstalk between human immune cells and tumors. These models represent a valuable tool for the preclinical evaluation of immunotherapies. Methods: In this study, we provide a comprehensive comparison of two widely used HIS models: the Hu-CD34+ model, which engrafts Hu-hematopoietic cells derived from Hu-CD34+ hematopoietic stem cells (HSCs), and the Hu-PBMC model, which utilizes Hu-peripheral blood mononuclear cells (PBMCs). Results: We assess the kinetics, quality and extent of immune cell engraftment, as well as the development of graft-versus-host disease (GVHD). Additionally, we investigate the impact of different immunodeficient host mouse strains on immune cell reconstitution in the Hu-CD34+ model. Both HIS models were engrafted with human tumors derived from either cell lines or patient-derived xenografts (PDX), revealing distinct immune-tumor interactions that influenced antitumor responses. Notably, tumor responses to T-cell-directed therapies, including anti-PD1 antibodies, IL-2-anti-IL-2 antibody complexes, and T-cell engagers, varied across these models. Discussion: Our findings provide novel insights into the properties and limitations of HIS models, offering a critical resource for optimizing next-generation immuno-oncology strategies and guiding the design of future therapeutic interventions.

Sup. Fig. S9. Gating strategy for sorting EPCAM+CD45- cells after TEC digestion

Sup. Fig. S2. Quality control for T cells scRNA-seq

Supp. Fig. S3. Filtering of Thymocytes dataset

Sup. Fig. S1. Gating strategy for the characterization of hematopoietic CD45+ cells (pediatric sample Ped4 shown as example).

Supplementary table S2D : Raw DEG of scRNA-seq from TEC

Supplementary Table S5. Conditions for antibodies for the MICSSS (Multiplexed Immunohistochemical Consecutive Staining on Single Slide).