Sup. Fig. S5. Description of most immature T cells precursors, Early Thymic Precursors (ETP) CD3-CD34+ cells from Alive DUMP-CD45+: CD3-CD34+ cells.

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

Sup. Fig. S8. Characteristics of CD8+ cells and clonal populations

Sup. Fig. S10. Characterization of Thymic Epithelial Cells in different datasets

Sup. Fig. S12. Alteration of the antigen processing and presentation machinery

Sup. Fig. S11. Tumoral Thymic Epithelial Cells display oncogenic characteristics

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

Thymomas are rare thymic epithelial tumors harboring a high but variable proportion of lymphocytes without obvious function. Autoimmunity is present in one third of patients at diagnosis. In this study, we performed a phenotypic, single-cell RNA sequencing and spatial analysis of both the T cells and tumoral cells. T cells at all stages of T-cell development-from immature to mature-were present in the tumor, suggesting active thymopoiesis in thymoma. However, data generated through multiple approaches suggested a maturation blockade at the double-negative to double-positive stage of T-cell development. In the mature T-cell compartment, the frequency of regulatory T cells was strongly decreased. The single-cell RNA sequencing analysis showed that the transcriptome of tumoral thymic epithelial cells (TEC) was most similar to that of nontumoral medullary TEC, but the expression of key molecules involved in positive and negative selection was defective. Multiplexed IHC consecutive staining revealed a loss of the cortex-medulla zoning in thymoma, which may be related to a decrease in the expression of T cell-targeted chemokines by tumoral TEC. Altogether, these results suggest that the thymopoiesis present in thymoma is abnormal and may be the cause of the prevalent autoimmunity observed in this disease.

Sup. Fig. S14. Decrease of Treg (CD4+CD8-FOXP3+) frequency in thymoma.

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. To provide additional insights into the biology of CD4 + T cells in cancer, here the authors report a multi-omics analysis of CD4 + T cells from matched blood, tumor invaded lymph nodes (LNs), and tumor tissues of patients with non-small cell lung cancer, showing that tumor-invaded LNs and tumors harbor clonally linked and plastic follicular CD4+T cell subsets.

Sup. Fig. S13. MICSSS (Multiplexed Immunohistochemical Consecutive Staining on Single Slide)

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

Supplementary Table S3: Quality and chain metrics for scTCR-seq (paired with scRNA-seq)

Sup. Fig. S6. Current trajectory predictions from early immature DN populations towards DP and SP mature T cells are not driven by spliceosome dynamic modification

Supplementary Table S1: Cohort description

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

486 Background: Management of locally advanced gastric and gastroesophageal junction (GOJ) cancer relies on peri-operative chemotherapy and radical surgery. Response to neoadjuvant chemotherapy can be evaluated by Mandard Tumor Regression Grade (TRG) performed on the removed specimens. The aim of this study was to investigate the prognostic value of TRG, in terms of post-surgical disease-free survival (DFS) and overall survival (OS). Methods: Retrospective analysis of all consecutive patients (Jan 2010 to Dec 2021) who underwent oncological gastrectomy for gastric or GOJ adenocarcinoma after neoadjuvant chemotherapy was performed. TRG was evaluated according to Mandard classification, ranging from 1 (complete pathological response) to 5 (absence of response), TRG 1-2 being considered as “good responders” and TRG 3-5 as “bad responders”. Univariate and multivariate analysis were performed for DFS and OS. Results: One hundred and ninety-nine patients were included, 157 treated with FOLFOX and 42 with FOLFOX + Taxanes (either FLOT or FOLFOX + Abraxane). TRG 1-2 was observed in 30% in patients (pts) treated with FOLFOX and 24% in pts treated with FOLFOX + Taxanes. With a median follow-up over 5 years (yrs), median DFS and OS were not reached (DFS rates at 3, 5 and 10yrs of 66.5%, 64.1% and 55.7%, respectively; OS rates at 3, 5 and 10yrs of 75.6%, 66.8% and 60.4%, respectively). In univariate analysis, TRG 1-2 was significantly associated with favorable outcome compared to TRG 3-5 in terms of DFS (HR 2.4, p-value 0.0056) and OS (HR 2.6, p-value 0.0061), as well as ECOG at diagnosis (0 vs. 1), ypT stage (yp T0-2 vs. yp T3-4), ypN stage (ypN- vs. ypN+), tumor localization (antrum + body vs. cardia + esophagus), and lymphatic, vascular and peri nervous embolisms (negative vs. positive) for both DFS and OS. However, in multivariate analysis (selection backward Cox-model), only ypT stage and vascular embolisms for DFS, and ypT and ypN stages for OS were found to be independent prognostic factors. Conclusions: Although TRG is a prognostic factor in patients with gastric or GOJ cancer, it does not appear as an independent factor for DFS and OS in this series of 199 pts. These results raise the issue of “good responder” definition, especially for TRG 3. Prognostic impact of TRG needs to be investigated for patients treated with combination of immune checkpoint inhibitors and chemotherapy in neoadjuvant regimens.

Tumor-invaded lymph nodes (LNs) serve as critical hubs for anti-tumor immunity, yet their role in orchestrating immune responses remains poorly understood. Using integrated single-cell RNA sequencing, T cell receptor sequencing, and chromatin accessibility profiling, we analyzed CD4+ T cells from matched blood, tumor-invaded LNs, and tumors of treatment-naïve non-small cell lung cancer patients. We identified distinct immunological landscapes across these compartments. Compared to blood, tumor-invaded LNs and tumors were enriched for follicular regulatory T cells (Treg-Tfr), conventional T cell subsets with Tfh-like characteristics (Tconv-Tfh and Tconv-CXCL13), and tissue-resident memory Tregs (Treg-Trm). These populations share a BATF-dependent transcriptional program that governs T-cell activation and tissue adaptation, while simultaneously engaging distinct, subset-specific regulatory networks. Integrative TCR-RNA analysis revealed that tumor-reactive, neoantigen-specific T cell clones were enriched within these subsets and demonstrated extensive LN-tumor clonal sharing, indicating active recirculation between compartments. Through clonal coupling analysis and trajectory inference, we uncovered that Treg-Tfr cells function as multipotent progenitors that bifurcate into tissue-resident Treg-Trm or into ex-Tregs adopting a Tfh-like CXCL13+ ewector phenotype. Remarkably, follicular CD4+ T subsets from LNs and tumor were transcriptionally and epigenetically similar and localized to analogous germinal center niches. These findings establish tumor-invaded LNs as functional extensions of the tumor microenvironment that generate and maintain tumor-reactive CD4+ lineages. The identification of tissue-resident Treg-Tfr plasticity reveals a critical developmental checkpoint that could be therapeutically targeted to redirect immunosuppressive programs toward anti-tumor ewector responses. ### Competing Interest Statement E.P. is co-founder of Egle-Tx. S.L., M.P. are employees from Egle-Tx. J.T. and J.W. were consultants for Egle-Tx. ANR, ANR-10-INBS-09-08, ANR-10-EQPX-03 ITMO-Cancer Aviesan (Plan Cancer III) INCA, SiRIC Grant INCa-DGOS-465, INCa-DGOSInserm_12554 ICGex NGS platform

BACKGROUND AND PURPOSE:Radiation toxicities, such as pneumonitis and fibrosis, are major limitations affecting patients' quality of life. Developed a decade ago, FLASH radiotherapy is an innovative method that, by delivering radiation at ultrafast dose rate, reduces radiation toxicities on healthy tissue while preserving the anti-tumoral effect of radiotherapy. This so-called FLASH effect has been described in different preclinical models but has not been observed in human tissue. This study aims to determine if FLASH irradiation can induce a sparing effect on human healthy lung tissue. MATERIALS AND METHODS:To address this question, precision-cut lung slices (Hu-PCLS) were prepared from healthy lung samples collected from 19 lung cancer patients undergoing lobectomy. These Hu-PCLS were irradiated ex vivo at a dose of 9 Gy using the ElectronFLASH (SIT) device operated either in conventional or FLASH mode. We monitored cell division for each patient and performed RNAseq analysis to uncover some mechanistic insights. RESULTS:Analysis of cell division 24 h after treatment with conventional or ultra-high dose rate showed a higher proportion of dividing cells in Hu-PCLS after FLASH irradiation. Consistently, RNAseq analysis from irradiated lung samples confirmed an attenuated cell cycle checkpoint inhibition, p53 pro-apoptotic genes, DNA damage, and antioxidant pathways after ultra-high dose rate compared to conventional treatment. CONCLUSION:Altogether, this study shows that, using freshly isolated patient-derived lung samples, cell proliferation can serve as an early marker of the normal lung response to FLASH irradiation. These findings hold great promises for future applications of FLASH radiotherapy in the clinic.

Erdheim-Chester disease (ECD) is a rare histiocytic disorder with localized presentations or multisystem disease. Clinical presentations of ECD are usually non-specific and depends on the site of involvement. ECD can involve one or several organs. Clinical manifestations range from asymptomatic lesions to severe and life-threatening organ dysfunction. Hence, accurate and timely diagnosis is challenging given the rarity and varied presentation of ECD. The most common clinical manifestations are bone pain related to osteosclerosis, usually in the lower limbs. We report here a case with no obvious clinical manifestation of ECD preceding initial recurrent pleural effusions. The diagnosis of ECD was suggested based on pleural thickening revealed by relapsing pleural effusions combined with radiological finding of a coated aorta and slight perirenal infiltrate. Pleural biopsy revealed collagen fibrosis, and immunohistochemistry with the anti-CD163 antibody showed an important infiltration by histiocytes, strong cytoplasmic phosphorylated ERK in the lesional cells, and positive factor XIIIa staining. A cell-free DNA from peripheral blood revealed negative BRAF mutation and the presence of MAP2K1 mutation, a key driver mutation in ECD. The diagnosis is often suggested based on clinic-radiological presentation but requiring histopathology to establish a final diagnosis of ECD. Plasma cell-free DNA is a promising and non-invasive tool to detect key driver mutations.