Background T cell receptor (TCR) binding properties have been related to a wide range of pathological conditions, including infections, autoimmunity and cancer. Characterising the TCR repertoire is of great biomedical interest but it has been challenging due to its high structural diversity. Methods In situ sequencing (ISS) is a suitable technique for spatial cell typing and linking gene patterns directly to specific histopathological features of large biopsy areas. We applied ISS through the commercial Xenium platform, with the addition of a custom panel specifically designed for TCR gene detection. Based on the IMGT database, we selected unique target sequences for TCR genes encoding the constant, variable and joining TCR chains. Additionally, we developed an analysis pipeline for the assignment of putative clonotypes based on simultaneous expression of alpha and beta variable TCR chains (TCRVβ/Vα pairs) at the single-cell level. Findings Our approach captured specific immune cell distributions in relation to the individual sample clonality, as well as regional dominance of certain TCRVβ/Vα pairs in surgical non-small cell lung cancer (NSCLC) specimens and matching lymph node samples. Furthermore, we were able to study the spatiotemporal evolution of TCR repertoire on longitudinal FFPE biopsies from patients with breast cancer, during neoadjuvant treatment. Interpretation This study highlights the implementation of target-based spatially resolved transcriptomics for the spatial characterisation of TCRVβ/Vα pairs at the single-cell level, without the need for prior sequencing. Our approach allows for spatial immune characterisation of diagnostic tissue samples with emphasis on T cell biology and accompanying T cell diversity. Funding This study was supported from Cancerfonden (CAN 2021/1726), Swedish Research Council (Dnr: 2019-01238), U-CAN and the Trond Mohn Foundation.

Supplementary figure 4. Staining control for human samples and additional information about patients (A) Histology and (B) clinical parameters of glioma patients. MGMT-methylation status; TMZ-temozolomide; Adj-adjusted. WT – wild type.

Abstract Immune infiltration shapes anti-tumor responses and is associated with patient outcomes. Yet, comprehensive studies comparing infiltration patterns and their clinical impact across cancer types are scarce.We profiled the tumor immune microenvironment across 16 solid tumor types from more than 2,700 patients using multiplex immunofluorescence with pathologist-curated image analysis to quantify major lymphoid and myeloid subsets and their spatial organization in situ.Across all cancer types, lung, endometrial, and high-grade serous ovarian cancers were highly infiltrated, whereas prostate and ER-positive breast cancers were comparatively “immune cold”. Cancer-agnostic consensus clustering resolved four reproducible immune archetypes with distinct outcomes, including an immune-hot group enriched for CD4, CD8, and B cells that associated with the best survival (p<0.001). Notably, the tumor type accounted for only a part of the immune contexture, as most cancers spanned multiple immune signature groups. Nevertheless, a machine-learning classifier trained on immune compositions distinguished tumor types with high accuracy (AUC = 0.96), confirming that each cancer maintains a recognizable immune imprint even among pan-cancer archetypes.Analysis of survival associations of individual cell classes revealed a rare CD8+FOXP3+ T-cell phenotype that emerged as a strongest positive prognostic factor across cancers (HR=0.79, 95%CI:[0.70-0.89], p=0.002). Single-cell RNA sequencing data indicated that CD8+FoxP3+ cells exhibit two distinct gene programs, with regulatory and cytotoxic capacities. Spatial mapping suggested context-dependency in the function of CD8+FoxP3+ cells, as their proximity to CD8 T cells was linked to better survival (enrichment at 10um: HR=0.76, 95%CI:[0.67-0.90], p=0.0006), whereas proximity to tumor cells was associated with worse prognosis (enrichment at 10um: HR=1.29, 95%CI:[1.12-1.49], p=0.0005).Together, our findings define pan-cancer immune archetypes that are both shared and disease-specific, explaining why “immune hot” and “cold” states are not synonymous with pure infiltration quantities. We identified CD8+FoxP3+ immune cells as a cell type with strong biomarker potential independent of cancer type. Citation Format: Artur Mezheyeuski, Emma Sandberg, Max Backman, Ali Teymur Kahraman, Amanda Lindberg, Carina Strell, Hans Brunnström, Jutta Huvila, Malin Sund, Fredrik Wärnberg, Bengt Glimelius, Ina Hrynchyk, Siarhei Mauchanski, Salome Khelashvili, Klara Hammarström, Margret Agnarsdottir, Gemma Garcia-Vicién, DAVID G. MOLLEVI, Aine O´Reilly, Sara Corvigno, Hanna Dahlstrand, Johan Botling, Ulrika Segersten, Agnieszka Krzyzanowska, Anders Bjartell, Jacob Elebro, Margareta Heby, Sebastian Lundgren, Charlotta Hedner, David Borg, Jenny Brändstedt, Hanna Sartor, Per-Uno Malmström, Martin Johansson, Anna Portyanko, Björn Nodin, Cecilia Lindskog, Karin Leandersson, Karin Jirström, Tobias Sjöblom, Patrick Micke. Tumor-agnostic analysis identified pan-cancer immune archetypes and CD8+FoxP3+ cells as novel in situ predictors of survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 193.

Supplementary figure 5. FH, ICOSL and ICOS dependence on glioma patient survival. The survival data of n = 509 patients from TCGA provisional dataset brain lower-grade glioma, analyzed with cBioPortal. Statistical tests: Logrank Test.

Supplementary figure 3. Direct effect of FH on glioma cells Proliferation of PIGPC cells treated with medium only, 25- or 100-μg/mL FH (A) and H4 mock or FH-transfected (B) was analyzed after 24, 48, 72 and 96 hours using CyQUANT assay. Data obtained at 24 hours was used for the normalization. (C) Correlation between FH and Ki67 gene expression in tumor cells from glioma patients, obtained from GEO database. Survival of PIGPC cells pretreated with 100 μg/mL FH (D-F) and H4 mock or FH-transfected (G-I) rendered apoptotic by treatment with 0.75 uM staurosporine for 24 h. Viability was assessed by Annexin V and Via-Probe staining. Data are means ± SD of (D, E, F) n = 4, (A, B, G, H, I) n = 3 independent experiments. Statistical tests: Two-way ANOVA with Bonferroni's multiple comparisons test (A, B), one-way ANOVA with Tukey´s multiple comparison test (D-I) Spearman’s rho correlation (C). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, nonsignificant; CTRL, control.

Supplementary figure 6. FH expression in glioma correlates with pro-tumorigenic markers

Supplementary figure 2. FH in Ntv-a mouse model (A) Mouse FH binds to mouse derived primary Tregs. Tregs were incubated for 2 h at 4oC with fluorescently labeled 25 or 100 μg/mL FH. The binding was detected using flow cytometry. (B) Western blot detecting FH in supernatants of tumor cells isolated from Ntv-a mice. FH was partially depleted with antibody against mouse FH bound to Dynabeads. (C) FH-rendered increase in survival of mouse Tregs. The cells were incubated with tumor cell derived supernatant and FH-depleted supernatant. After 7 days viability was assessed by Annexin V and Via-Probe staining. (D) FH was successfully knockdown with shFH. DF1 cells were transfected with three different shFH constructs and Gl2 shRNA. The knockdown of FH in NIH3T3 cells, transfected with supernatants from the DF1-RCAS-shFH/DF-1-RCAS-shGl2 cells was detected with goat anti-FH antiserum by western blot. (E) Control staining of mouse tumor sections. Samples were incubated with goat anti-FH antibody, goat IgG isotype control, and anti-goat IgG secondary antibody or only secondary antibody. Nucleus was stained with DAPI. Data are means ± SD of (A) n = 4, (C) n = 3 independent experiments. Representative blot (D) of n = 3 and picture (E) of n = 3 independent experiments. Statistical tests: two- Kruskal-Wallis with Dunn´s multiple comparison test (A, C). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns -, nonsignificant; CTRL, control.

INTRODUCTION:The immune microenvironment plays a crucial role in the progression of colorectal cancer (CRC), but its variation across primary tumors and metastatic sites remains incompletely understood. In this study, we compared the composition and prognostic significance of the immune microenvironment in lung metastases, paired primary tumors, and liver metastases from a retrospective cohort of 216 patients with metastatic CRC who underwent pulmonary metastasectomy in a single Swedish institution. METHODS:Immunohistochemistry was applied on tissue microarrays to evaluate CD3+, CD8+, CD20+, FoxP3+ immune cells, PD-L1+ immune cells and tumor cells, and tertiary lymphoid-like structures (TLLS). RESULTS:Lung metastases exhibited significantly higher infiltration of all immune cell subsets compared to both primary tumors and liver metastases. FoxP3+ cell density was lower in liver metastases than in primary tumors. Immune cell infiltration in lung metastases was largely unaffected by neoadjuvant chemotherapy, except for lower FoxP3+ cell infiltration in treated cases. None of the immune markers bore prognostic significance in the overall analysis. However, high infiltration of FoxP3+, CD20+, and PD-L1+ immune cells, and presence of TLLSs in lung metastases were associated with improved overall survival in patients receiving adjuvant chemotherapy, with a significant prognostic treatment interaction. None of the immune markers were prognostic in primary tumors or liver metastases. CONCLUSION:These findings underscore a distinct, more immunologically active immune microenvironment in CRC lung metastases compared to primary tumors and liver metastases, that suggestedly also predicts response to adjuvant chemotherapy. Immune profiling of lung metastases may thus pave the way for improved personalized treatment of these patients.

Supplementary figure 1. FH binds to T-cells via ICOS

FH in glioma correlates with Treg occurrence and increased Treg viability. FH expression positively correlates with infiltration of Tregs in lower-grade glioma (A) and GBM (B). C, Western blot detecting FH in supernatants of FH-transfected glioma cell lines and depletion of FH with MRC-OX24-bound Dynabeads. D, Tregs incubated with supernatants of FH-transfected H4 glioma cell line exhibited increased survival, and the effect is weaker when FH is partially depleted. Correlation analysis of the immune infiltration was performed using TIMER2.0, based on n = 516 samples from lower-grade glioma and n = 599 samples from patients with GBM. Data are means ± SD of n = 4 (D) independent experiments. Representative blot of n = 4 (C) independent experiments. Statistical tests: Spearman’s rho (A and B), Kruskal–Wallis with Dunn’s multiple comparison test (D). ***, P < 0.001; ****, P < 0.0001.

ICOS is associated with poor prognosis of patients with glioma. A, ICOS expression correlates with decreased survival of patients with glioma. Expression of ICOS correlates with neoplasm histological grade (B), cancer type (C), and EGFR mutations (D). ICOS positively correlates with infiltration of Tregs (E) and negatively with infiltration of CD8+ T cells (F) and CD4+ T cells (G) in lower-grade glioma. H, In a patient cohort stratified based on FH expression, ICOS correlates with worse survival only in the FH-high group. The survival estimates and correlation with other clinical parameters were analyzed in cBioPortal and based on the TCGA provisional dataset brain lower-grade glioma. The cases were set to include samples with mRNA data from n = 508 patients (A–D and H). Correlation analysis of the immune infiltration was performed using TIMER2.0, based on n = 516 samples from patients with lower-grade glioma. Statistical tests: logrank test (A–H), χ2 test (B and C), one-sided Fisher exact test (D), and Spearman’s rho correlation (E–G).

FH enhances the immunosuppressive function of Tregs. A, FH increases the immunosuppressive effect of Tregs. Tregs were incubated in medium only or supplemented with 150 μg/mL FH. CD4+ T cells were isolated from the same donor and cocultured with Tregs in the presence of CD2/CD3/CD28 MACSiBeads and 3H-thymidine. Secretion of IL10 (B and D) and TGFβ (C) by Tregs incubated in medium only, 150 μg/mL α1-AT, FH, or dFH. Cells were stimulated with anti-CD3 and anti-CD28 microbeads in the presence of IL2, and secreted cytokines were measured by ELISA after 48 hours (B) or 72 hours (C). D, Frequency of IL10-producing Tregs, either unstimulated, or stimulated for 16 hours with TransAct, TransAct + FH, TransAct + dFH, or TransAct + SEA, relative to ICOS expression, was assessed with IL10 catch. Data are means ± SD of n = 7 (A), n = 6 (B), n = 4 (C) independent experiments; representative plots of n = 5 (D) independent experiments. Statistical tests: two-way ANOVA (A) and one-way ANOVA (B and C) with Tukey’s multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. AF, Alexa Fluor; CPM, count per minute; CTRL, control; ns, nonsignificant; SEA, staphylococcal enterotoxin A; Tresp, responder CD4+ T cells.

FH knockdown is associated with a lower number of ICOS+ Tregs in a murine glioma model. FH expression was investigated in murine gliomas generated using the RCAS/tv-a vectors to induce expression of PDGFB (A and B) and shp53 (B). C, Schematic illustration of the mouse model. Increased survival (D), decrease in number of Olig2+ tumor cells (E), changes in infiltration of T cells (F–J), and macrophages (K) as well as number of astrocytes (L) and changes in proliferation of tumor cells (M) were investigated in GBM model with shRNA-induced FH knockdown. Vector targeting Gl2 was used as a control. Statistical analysis: logrank test (D), Student t test with Welch correction (E–G and J–M), Student t test paired (H). Data are means ± SD of n = 15 (D), n = 13 (E–H and J–M) mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. CTRL, control; Gl2, firefly luciferase; RCAS/tv-a, replication-competent ASLV long terminal repeat with a tv-a splice acceptor. (C, Created with BioRender.com.)

FH binds to T cells via ICOS. A, Biotinylated FH binds to CD4+ T cells upon incubation for 2 hours with TexMACS medium alone and supplemented with 50 and 100 μg/mL FH. B, Fractionation detecting FH binding but not internalization into CD4+ T cells, incubated with 150 μg/mL FH for 2 hours at 4°C or 37°C. C, HuProt microarray showing top-specific proteins binding FH CCP19-20. D and E, PLA showing FH binding to ICOS. CD4+ T cells and activated CD4+ T cells were incubated with FH for 2 hours at 37°C, and the interaction between ICOS and FH was detected with specific antibodies and visualized as white dots. F, ELISA detection of dose-dependent binding of FH to ICOS or α1-AT. CD4+ T cells were incubated for 2 hours at 37°C with biotinylated FH or α1-AT and lysed. The microtiter plate was coated with an anti-ICOS antibody, and bound complexes from the lysates were detected with streptavidin. G, Preincubation with anti-ICOS antibody reduced FH binding to CD4+ T cells in a dose-dependent manner. Isotype control was used at the same concentration, without affecting the binding. Representative blot (H) and quantification (I and J) of increased phosphorylation of Akt and GSK in Tregs upon incubation with 150 μg/mL FH. ICOS blocking antibody but not isotype control inhibited this effect. Data are means ± SD of n = 8 (A), n = 5 (E–G Iso-CTRL), n = 7 (G α-ICOS), n = 4 (I and J) independent experiments; representative blot of (B and H) n = 4 independent experiments; representative pictures of (D) n = 5 independent experiments. Statistical test: Kruskal–Wallis with Dunn’s multiple comparison test (A and E), two-way ANOVA (F and G), and one-way ANOVA (I and J) with Tukey’s multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. act, activated; AF, Alexa Fluor; CTRL, control; GSK, glycogen synthase kinase-3; iso, isotype; ns, nonsignificant; PLA, proximity ligation assay.

Abstract The survival rate of patients with glioma has not significantly increased in recent years despite aggressive treatment and advances in immunotherapy. The limited response to treatments is partially attributed to the immunosuppressive tumor microenvironment, in which regulatory T cells (Treg) play a pivotal role in immunologic tolerance. In this study, we investigated the impact of complement factor H (FH) on Tregs within the glioma microenvironment and found that FH is an ICOS ligand. The binding of FH to this immune checkpoint molecule promoted the survival and function of Tregs and induced the secretion of TGFβ and IL10 while suppressing T-cell proliferation. We further demonstrated that cancer cells in human and mouse gliomas directly produce FH. Database investigations revealed that upregulation of FH expression was associated with the presence of Tregs and correlated with worse prognosis for patients with glioma. We confirmed the effect of FH on glioma development in a mouse model, in which FH knockdown was associated with a decrease in the number of ICOS+ Tregs and demonstrated a tendency of prolonged survival (P = 0.064). Because the accumulation of Tregs represents a promising prognostic and therapeutic target, evaluating FH expression should be considered when assessing the effectiveness of and resistance to immunotherapies against glioma.

T-cell activation and clonal expansion are essential for the efficacy of immunotherapy in non-small cell lung cancer (NSCLC) patients. Since the distribution of T-cell clones might provide insights into immunogenic mechanisms, we determined the α/β TCR clonality using RNA-sequencing from frozen tumor tissue of 182 NSCLC patients and paired the results with extensive in situ image and sequence analyses of the immune microenvironment of NSCLC. TCR clonality (Gini index) patterns ranged from high T-cell clone diversity with high evenness (Gini index low) to clonal dominance with low evenness (Gini index high). TCR clonality in cancer tissue was lower than in matched normal lung (p=0.021). High Gini index correlated strongly with distinct mutations (EGFR, P53), tumor mutation burden (p<0.001), and inflamed tumor phenotypes (PRF1, GZMA, GZMB, INFG) with exhaustion signatures (LAG3, TIGIT, IDO1, PD-1, PD-L1). Correspondingly, PD-1+, CD3+, CD8A+, CD163+, and CD138+ immune cells infiltrated cancer tissue with high TCR clonality. In situ sequencing revealed that dominant T-cell clones were more often of CD8-subtype and tended to approximate the tumor cell compartment (p<0.03). In a checkpoint inhibitor-treated NSCLC patient cohort, high TCR clonality was associated with therapy response (p=0.016) and prolonged survival (p=0.003, median survival 13.8 vs 2.9 months). Our robust analysis pipeline revealed diverse TCR repertoires related to genotypes and immune phenotypes. The in situ positioning of expanded T-cell clones indicated functional impact, which was clinically confirmed in NSCLC patients receiving immunotherapy. ### Competing Interest Statement The authors have declared no competing interest.

The vast majority of patients with pancreatic cancer present with unresectable disease and precision medicine is lagging behind. Circulating tumor DNA (ctDNA) has emerged as a promising tool, both as a proxy for tumor burden and for capturing tumor heterogeneity, but optimal gene panels and prognostic cutoffs remain to be determined. Herein, we applied ultra-deep ctDNA sequencing using a customized panel targeting 23 genes and six frequently altered chromosomes on plasma samples obtained before, during and after chemotherapy from 60 patients enrolled in a prospective clinical study. At baseline, positive versus negative ctDNA was not prognostic, neither in the adjuvant nor in the palliative setting, but in palliative patients, an independent prognostic cutoff could be calculated from the absolute number of mutated DNA molecules. Median overall survival was 3.7 months in the ctDNAhigh compared to 11.9 months in the ctDNAlow group (p < 0.0001), and the cutoff remained prognostic at one and three months. Moreover, relevant genetic alterations were highly concordant in ctDNA and paired tumor tissue. These findings demonstrate the potential clinical utility of a customized and focused gene panel for prognostication and target identification over time in patients with newly diagnosed pancreatic cancer, in particular in the palliative setting.ClinicalTrials.gov number: NCT03724994.

Metastasis to lymph nodes is strongly associated with reduced survival in breast cancer patients. To increase the understanding on how lymph node metastasis impairs the local immune response in affected lymph nodes, we here studied spatial proteomic changes of critical lymph node immune populations in uninvolved lymph nodes (UnLN) and paired lymph nodes with metastases (LNM) from five breast cancer patients. The proteome was analyzed for cortical lymphocyte compartments, subcapsular sinus (SCS) and medullary sinus (MS) CD169+ macrophages, using the Digital Spatial Profiling (DSP) platform from NanoString. Our results identified a stable proteome of SCS CD169+ macrophages in LNM, with the exception for downregulation of the anti-apoptotic protein Bcl-xL and FAPα, but a clear reduction in numbers of SCS CD169+ macrophages in LNM. In contrast, the proteome of MS CD169+ macrophages, B-cell compartments and interfollicular T-cells showed altered immune signatures in LNM, indicating that the decline in SCS CD169+ macrophages coincide with a malfunction in the local, anti-tumor immune responses. The findings from our study support the notion that metastasis to lymph nodes in breast cancer patients modifies local immune responses. These changes may contribute to explain unsuccessful therapeutic responses, and thereby worsened prognosis, for breast cancer patients with LNM.

FH increases the viability of Tregs. FH-rendered increase in survival of CD4+ (A) but not CD8+ (B) or naïve CD4+ (C) T cells. FH increased survival of Tregs (D) and did not increase survival of Treg-depleted CD4+ T cells (E). Cells were incubated with medium, 150 μg/mL FH, or α1-AT for 7 days, assessed by Annexin V and Via-Probe staining. F, Gating of Tregs according to ICOS expression. FH rescues survival of ICOS+ but not ICOS− Tregs incubated with medium alone and supplemented with 150 μg/mL FH. G, Incubation with 2 and 10 μg/mL of ICOS blocking antibody decreased the FH-rendered increase in Treg viability in a dose-dependent manner. H, The p110δ-specific PI3Kδ inhibitor CAL-101 negates the increase in Treg survival induced by 150 μg/mL of FH. Data are means ± SD of n = 4 (A–D and H), n = 3 (F), n = 5 (G) independent experiments. Statistical tests: two-way ANOVA with Tukey’s multiple comparison test (A–C and F–H), Kruskal–Wallis with Dunn’s multiple comparison test (D and E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****P < 0.0001; AF, Alexa Fluor; CTRL, control; iso, isotype; ns, nonsignificant.

Tumor associated neutrophils (TANs) exert dual and opposing functions in tumors, acting pro-tumorigenic and anti-tumorigenic, depending on tumor progression, polarization state and subtype. Consequently, the prognostic impact of TANs in breast cancer is also contradictory. Since neutrophils are critically needed to fight infections in cancer patients, the mediators leading to tumor progression need more investigation as potential future targets. The neutrophil derived mediator myeloperoxidase (MPO) is a peroxidase with dual functions in tumors, acting both immune enhancing and suppressing. Patients with metastatic breast cancer (MBC) have aggressive tumors with a dismal prognosis and urgently need novel treatment strategies. Therefore, we here aimed to investigate the prognostic impact of TANs, MPO+ TANs and MPO+ non-neutrophils using a cohort with newly diagnosed MBC patients specifically. We show that high infiltration of MPO+ TANs and MPO+ non-neutrophils in the primary tumor (PT), was associated with clinicopathological features and worse prognosis in patients with MBC. However, only infiltration of MPO+ TANs showed independent prognostic impact in multivariable analysis adjusting for other prognostic factors in MBC. The results need to be validated in a larger cohort but suggests that MPO targeting strategies could be relevant in breast cancer patients with aggressive disease.