Protein kinase Cι dictates tumor trajectory, cell plasticity, and immune surveillance in lung adenocarcinoma.

RORc-expressing immune cells negatively regulate tertiary lymphoid structure formation and support their pro-tumorigenic functions.

BackgroundTertiary lymphoid structures (TLS) are lymphoid aggregates that often form locally in tissues with chronic infection, autoimmune disease, and cancer. As such, TLS correlate with favorable prognosis in patients with...

Inducing tertiary lymphoid structure (TLS) formation can fuel antitumor immunity. It is necessary to create mouse models containing TLS to explore strategies of TLS formation. Oncolytic herpes simplex virus‐1 (oHSV) exhibited intense effects in preclinical and clinical trials. However, the role of oHSV in TLS formation remains to be elucidated. Here, we observed the presence of TLS in 4MOSC1 and MC38 subcutaneous tumour models. Interestingly, oHSV evoked TLS formation, and increased infiltration of B cells and stem‐like TCF1+CD8+ T cells proliferation. Mechanistically, oHSV increased the expression of TLS‐related chemokines, along with upregulated CXCL10/CXCR3 to facilitate TLS formation. Notably, CXCL10 and CXCR3 were favourable prognostic factors for cancer patients, and closely related with immune cells infiltration. Inhibiting CXCL10/CXCR3 reduced TCF1+CD8+ T cells and granzyme B expression, and impaired oHSV‐mediated TLS formation. Furthermore, oHSV‐mediated TLS formation revealed superior response and survival rate when combined with αPD‐1 treatment. Collectively, these findings indicate that oHSV recruits stem‐like TCF1+CD8+ T cells through CXCL10/CXCR3 pathway to propagate TLS formation, and warrants future antitumor immunity development.

Checkpoint blockade therapies that reactivate tumour-associated T cells can induce durable tumour control and result in the long-term survival of patients with advanced cancers1. Current predictive biomarkers for therapy response include high levels of intratumour immunological activity, a high tumour mutational burden and specific characteristics of the gut microbiota2,3. Although the role of T cells in antitumour responses has thoroughly been studied, other immune cells remain insufficiently explored. Here we use clinical samples of metastatic melanomas to investigate the role of Bcells in antitumour responses, and find that the co-occurrence of tumour-associated CD8+ T cells and CD20+ Bcells is associated with improved survival, independently of other clinical variables. Immunofluorescence staining of CXCR5 and CXCL13 in combination with CD20 reveals the formation of tertiary lymphoid structures in these CD8+CD20+ tumours. We derived a gene signature associated with tertiary lymphoid structures, which predicted clinical outcomes in cohorts of patients treated with immunecheckpoint blockade. Furthermore, B-cell-rich tumours were accompanied by increased levels of TCF7+ naive and/or memory T cells. This was corroborated by digital spatial-profiling data, in which T cells in tumours without tertiary lymphoid structures had a dysfunctional molecular phenotype. Our results indicate that tertiary lymphoid structures have a key role in the immune microenvironment in melanoma, by conferring distinct T cell phenotypes. Therapeutic strategies to induce the formation of tertiary lymphoid structures should be explored to improve responses to cancer immunotherapy.

Tertiary lymphoid structure, an ectopic lymphoid tissue induced under chronic inflammation, develops in various kidney diseases and is associated with poor prognosis. The immune system requires metabolic resources to support immune function and lymphocyte proliferation. Hence, dramatic metabolic alterations presumably occur during the formation of tertiary lymphoid structure. However, it remains unclear whether metabolic remodeling occurs during this formation and its underlying mechanism. In a murine model of renal tertiary lymphoid structures, we used imaging mass spectrometry and metabolome analysis to investigate the metabolic pathway that characterizes tertiary lymphoid structures. We also performed in situ hybridization with immunofluorescence and pharmacological inhibition to explore the expression and function of the key molecules governing the pivotal metabolic pathway. We analyzed urine samples from mice and humans to explore the metabolites estimating the presence of renal tertiary lymphoid structures. Significant glutathione accumulation and depletion of cysteine, which is essential for glutathione synthesis, was observed specifically within tertiary lymphoid structures. The kidneys with tertiary lymphoid structures exhibited higher glutathione concentrations than healthy kidneys. Tertiary lymphoid structures also showed significant accumulation of 4-HNE and 8-OHdG, markers of oxidative stress. Dendritic cells and fibroblasts within tertiary lymphoid structures expressed the cystine/glutamate transporter, that regulates glutathione synthesis, and supplied synthesized glutathione to lymphocytes, which lacked its expression. Pharmacological inhibition of the cystine/glutamate transporter prevented tertiary lymphoid structure formation in the kidney. Furthermore, enhanced glutathione synthesis within tertiary lymphoid structures was reflected in elevated urinary glutathione concentrations both in mice and humans, which effectively detected the presence of tertiary lymphoid structures in the kidney in IgA nephropathy patients. Glutathione significantly accumulated within tertiary lymphoid structures in the kidney. Inhibition of the cystine/glutamate transporter prevented the formation of tertiary lymphoid structures. Urinary glutathione served as a biomarker to detect tertiary lymphoid structures in the kidney.

Glioma, characterized by limited lymphocytic infiltration, constitutes an "immune-desert" tumor displaying insensitivity to various immunotherapies. This study aims to explore therapeutic strategies for inducing tertiary lymphoid structure (TLS) formation within the glioma microenvironment (GME) to transition it from an immune resistant to an activated state. TLS formation in GME was successfully induced by intracranial administration of Toll-like receptor (TLR) agonists (OK-432, TLR2/4/9 agonist) and glioma antigens (i.c. αTLR-mix). We employed staining analysis, antibody neutralization, single-cell RNA sequencing (scRNA-Seq), and BCR/TCR sequencing to investigate the underlying mechanisms of TLS formation and its role in anti-glioma immunity. Additionally, a preliminary translational clinical study was conducted. TLS formation correlated with increased lymphocyte infiltration in GME and led to improved prognosis in glioma-bearing mice. In the study of TLS induction mechanisms, certain macrophages/microglia and Th17 displayed markers of "LTo" and "LTi" cells, respectively, interaction through LTα/β-LTβR promoted TLS induction. Post-TLS formation, CD4 + and CD8 + T cells but not CD19 + B cells contributed to anti-glioma immunity. Comparative analysis of B/T cells between brain and lymph node showed that brain B/T cells unveiled the switch from naïve to mature, some B cells highlighted an enrichment of class switch recombination (CSR)-associated genes, V gene usage, and clonotype bias were observed. In related clinical studies, i.c. αTLR-mix treatment exhibited tolerability, and chemokines/cytokines assay provided preliminary evidence supporting TLS formation in GME. TLS induction in GME enhanced anti-glioma immunity, improved the immune microenvironment, and controlled glioma growth, suggesting potential therapeutic avenues for treating glioma in the future.

The infiltration and activation of immune cells in the tumor microenvironment (TIME) affect the prognosis of patients with cancer. Tertiary lymphoid structure (TLS) formation favors tumour- infiltrating-lymphocyte (TIL) recruitment and is regarded as an important indicator of good prognosis associated with immunotherapy in patients with tumors. Chemotherapy is currently one of the most commonly used clinical treatment methods. However, there have been no clear report to explore the effects of different types of chemotherapy on TLS formation in the TIME. This study examined the effects of immunogenic cell death (ICD)-inducing chemotherapeutics on immune cells, high-endothelial venules (HEV), and TLSs in mouse melanomas. Doxorubicin (an ICD inducer), gemcitabine (non-ICD inducer), and a combination of the two drugs was delivered intra-peritoneally to B16F1-loaded C57BL/6 mice. The infiltration of immune cells into tumor tissues was evaluated using flow cytometry. HEV and TLS formation was assessed using immunohistochemistry and multiple fluorescent immunohistochemical staining. Doxorubicin alone, gemcitabine alone, and the two-drug combination all slowed tumor growth, with the combined treatment demonstrating a more pronounced effect. Compared with the control group, the doxorubicin group showed a higher infiltration of CD8+ T cells and tissue-resident memory T cells (TRM) and an increase in the secretion of interferon-γ, granzyme B, and perforin in CD8+ T subsets and activation of B cells and dendritic cells. Doxorubicin alone and in combination with gemcitabine decreased regulatory T cells in the TIME. Moreover, doxorubicin treatment promoted the formation of HEV and TLS. Doxorubicin treatment also upregulated the expression of programmed cell death protein (PD)-1 in CD8+ T cells and programmed cell death protein ligand (PD-L)1 in tumor cells. These results indicate that doxorubicin with an ICD reaction promotes TLS formation and increases PD-1/PD-L1 expression in tumor tissues. The results demonstrate the development of a therapeutic avenue using combined immune checkpoint therapy.

Cancer immunotherapy of glioma, the most common primary brain tumor in adults, is constrained by an immunosuppressive microenvironment. Tertiary lymphoid structures (TLSs) are ectopic lymphoid structures that mediate immune responses and provide a local site for tumor antigen presentation. Here, we show for the first time that TLSs are present in human low-grade glioma and that they can be induced in murine glioma models. Agonistic CD40 antibodies induced lymphotoxin α expression in B cells and enhanced the formation of TLSs in mice bearing orthotopic GL261 or CT-2A gliomas. TLSs consisting of B cells, T-cells and antigen presenting cells formed around CD31+ vessels proximal to the tumor tissue and adjacent to the meninges. Agonistic CD40 antibody treatment improved the CD8+ T-cell / Treg ratio in tumor-bearing mice, but there was no survival benefit. A decreased CD69 activation status on CD8+ T-cells, an increase of B cells with a CD1d+CD5+ regulatory phenotype and a high infiltration of Tregs in TLSs indicate that agonistic CD40 antibodies induce an immunosuppressive microenvironment in glioma. Consistent with this, the efficacy of both PD-1 and CTLA-4 checkpoint blockade was significantly decreased by co-administration of agonistic CD40 antibodies. These results demonstrate that TLSs are present and can be induced in glioma, but that they at least under certain conditions are associated with immunosuppression, shedding light on new mechanisms of immune regulation in the brain microenvironment. Citation Format: Luuk van Hooren, Alessandra Vaccaro, Maria Georganaki, Mohanraj Ramachandran, Hua Huang, Joey Lau, Anja Smits, Magnus Essand, Anna Dimberg. Agonistic CD40 antibody therapy induces formation of tertiary lymphoid structures in glioma and inhibits the response to immune-checkpoint blockade [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A159.

Tertiary lymphoid structures (TLS) develop in the kidneys of lupus-prone mice and systemic lupus erythematosus (SLE) patients with lupus nephritis (LN). Here we investigated the presence of mesenchymal stem cells (MSCs) in the development of TLS in murine LN, as well as the role of human MSCs as lymphoid tissue organizer (LTo) cells on the activation of CD4+ T cells from three groups of donors including Healthy, SLE and LN patients. Mesenchymal stem like cells were detected within the pelvic wall and TLS in kidneys of lupus-prone mice. An increase in LTβ, CXCL13, CCL19, VCAM1 and ICAM1 gene expressions were detected during the development of murine LN. Human MSCs stimulated with the pro-inflammatory cytokines TNF-α and IL-1β significantly increased the expression of CCL19, VCAM1, ICAM1, TNF-α, and IL-1β. Stimulated MSCs induced proliferation of CD4+ T cells, but an inhibitory effect was observed when in co-culture with non-stimulated MSCs. A contact dependent increase in Th2 and Th17 subsets were observed for T cells from the Healthy group after co-culture with stimulated MSCs. Our data suggest that tissue-specific or/and migratory MSCs could have pivotal roles as LTo cells in accelerating early inflammatory processes and initiating the formation of kidney specific TLS in chronic inflammatory conditions.

e21509 Background: Tertiary lymphoid structures (TLS) are transient ectopic lymphoid aggregates composed of immune cells such as B cells, T cells, and dendritic cells (DCs), and have been observed in various solid tumors. TLS are associated with favorable clinical outcomes. While TLS are believed to contribute to adaptive antitumor cellular and humoral responses, the mechanisms driving TLS formation and their immune regulatory functions in tumor immune microenvironment (TIME) remain poorly understood. Methods: We collected formalin-fixed paraffin-embedded (FFPE) tissue sections from 86 MM patients and performed hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and multiplex IHC (mIHC) to assess TLS density, maturation, and distribution. Clinical data were integrated to investigate the relationship between TLS and patient prognosis in MM. Additionally, single-cell data from 20 patients were analyzed to explore the abundance of immune cell types and subtypes in the TIME at single-cell resolution, along with potential intercellular interactions. Results: Based on histopathological analysis of MM samples, patients were stratified into two groups: those poccessing TLS (TLS+, n = 44) and those lacking TLS (TLS-, n = 42). Across the entire cohort, the TLS+ group showed a trend toward improved overall survival (OS), though not statistically significant (p = 0.27). A higher proportion of advanced-stage (T3 and T4) patients in the TLS+ group (T1-T3a: TLS- = 12, TLS+ = 5; T3a-T4b: TLS- = 17, TLS+ = 21) may have influenced the prognostic value of TLS in the overall patient cohort. Further supporting this notion, subgroup analysis restricted to patients with AJCC stage IIB and IIC revealed significantly improved OS in the TLS+ group (p = 0.046). Single-cell data revealed significant differences in the abundance of immune-related cells between the TLS+ and TLS- groups. All B cell subtypes (naïve, memory, germinal center (GC), and plasma cells) were enriched in the TLS+ group compared to the TLS- group. CD4+ and CD8+ naïve T cells also exhibited similar trends, whereas the distribution of CD4+ and CD8+ memory T cells was comparable between the two groups. Furthermore, plasma cells in the TLS+ group were predominantly IgG-producing. Intercellular communication analysis with B cells identified a CAF subpopulation highly expressing TLS-associated genes. A CCL19-CCR7 and CCL21-CCR7 chemokine axis from this CAF subpopulation was observed exclusively in TLS+ tumors but was deficient in TLS- tumors. Multiple T cell subsets exhibited high expression of the CXCL13-CXCR5 axis when communicating with B cells. Conclusions: Our findings highlight the prognostic value of TLS in patients within MM. TLS formation may be associated with specific CAF and T cell subtypes and their interactions with B cells.

Background The prognosis of patients with non-small cell lung cancer (NSCLC) is closely related to the tumor microenvironment. Studies have shown that the status of tertiary lymphoid structures (TLS) is an important factor affecting the tumor microenvironment. B cell cluster is the main component of TLS, and the C-X-C motif chemokine ligand 13 (CXCL13) is a selective attractant for B cells. However, the relationship between CXCL13 and the formation of TLS remains to be explored. Methods A hundred NSCLC patients who underwent surgical resection in Tianjin Chest Hospital in 2018 were retrospectively recruited. TLS number and maturity were assessed by HE staining and immunohistochemical staining, respectively. The relationship between TLS in tumor tissue and clinicopathological characteristics and prognosis of NSCLC patients was analyzed. The CXCL13 mRNA and protein expression levels were detected by RT-PCR and immunohistochemical staining, respectively. The correlation between CXCL13 in tumor tissue and TLS in tumor microenvironment was analyzed by Spearman correlation.COX regression analysis and Kaplan-Meier survival curve were used to analyze the prognostic effects of TLS and CXCL13 on patients. Results There was no correlation between TLS number or maturity and gender, age, smoking history, histological type, differentiation, pleural invasion, or TNM stage. TLS number and maturity were positively correlated with CD8+ T cell infiltration in tumor tissue (P < 0.05). Kaplan-Meier survival analysis showed that OS was significantly better in patients with greater TLS count, TLS maturity, and CXCL13 expression than those of relatively low values (P < 0.05). The results of multivariate COX regression analysis showed that TLS maturity (P = 0.002) and TNM stage (P < 0.001) were independent predictors of OS in NSCLC patients. Spearman correlation analysis showed that the number and maturity of TLS were positively correlated with CXCL13 mRNA and protein levels. Conclusion The large number of TLS, high TLS maturity, and high CXCL13 expression are associated with good prognosis of NSCLC patients. TLS maturity can be used as a predictor of patient prognosis. Moreover, CXCL13 mRNA and protein expression levels were significantly correlated with TLS number and maturity. CXCL13 may induce B cells to infiltrate the tumor immune microenvironment to promote the formation of TLS in tumor tissues.

How the genomic landscape of a tumor shapes the formation of tertiary lymphoid structure (TLS) and how might TLS alter the clinical outcome or response to immunotherapy had not been systematically explored. Utilizing the genomic and transcriptome data of solid tumors on TCGA, we quantified TLS based on a previous identified 12-chemokine signature and evaluated its correlation with mutation/neoantigen burden, functional mutation of oncogenes and the presence of viral infection. Clinical data was integrated to decide the prognostic significance of TLS for different cancers after surgical treatment. Publicly available data (clinical and transcriptome data) of immunotherapy clinical trials involving melanoma and lung cancer were also collected to evaluate TLS’s association with therapeutic outcome. Mutation burden and predicted neoantigen counts were positively correlated with TLS scoring in multiple cancer types. Mutation in tumor suppressor genes (KEAP1, PBRM1) and genes involved in extrinsic apoptosis (CASP8), antigen-presentation (HLA-A, HLA-B), immune regulation (SMAD4) or DNA repair (BRCA1, BRCA2, TP53BP1) correlated with TLS alteration in multiple tumor types, indicating the interaction between mutation landscape and TLS formation. Epstein-Barr virus (EBV) infection in gastric cancer and human papillomavirus (HPV) infection in Head and Neck squamous cell carcinoma were associated with increased TLS scoring. High TLS scoring predicted favorable prognosis in certain cancer after surgical treatment and improved response to immunotherapy in lung cancer and melanoma. Our findings unraveled the genomic properties associated with TLS formation in different solid tumors and highlighted the prognostic and predictive significance of TLS in surgical treatment and immunotherapy.

<h3>Background</h3> Tertiary lymphoid structures (TLS) may develop in non-lymphoid tissues in response to a variety of different stimuli and can serve as foci for generating anti-tumor immunity.¹ TLS formation is...

Tertiary lymphoid structures (TLS) are organized immune cell aggregates that arise in chronic inflammatory conditions. In cancer, TLS are associated with better prognosis and enhanced response to immunotherapy, making these structures attractive therapeutic targets. However, the mechanisms regulating TLS formation and maintenance in cancer are incompletely understood. Using spatial transcriptomics and multiplex imaging across various human tumors, we found an enrichment of mature dendritic cells (DC) expressing high levels of CCR7 in TLS, prompting us to investigate the role of DC in the formation and maintenance of TLS in solid tumors. To address this, we developed a novel murine model of non-small cell lung cancer (NSCLC) that forms mature TLS, containing B cell follicles with germinal centers and T cell zones with T follicular helper cells (TFH) and TCF1+PD-1+ progenitor exhausted CD8+ T cells (Tpex). Here we show that, during the early stages of tumor development, TLS formation relies on IFNγ-driven maturation of the conventional DC type 1 (cDC1) subset, their migration to tumor-draining lymph nodes (tdLN), and recruitment of activated T cells to the tumor site. As tumors progress, TLS maintenance becomes independent of T cell egress from tdLN, coinciding with a significant reduction of cDC1 migration to tdLN. Instead, mature cDC1 accumulate within intratumoral CCR7 ligand-enriched stromal hubs. Notably, timed depletion of cDC1 or disruption of their migration to these stromal hubs after TLS are formed alters TLS maintenance. Importantly, we found that cDC1-mediated antigen presentation to both CD4+ and CD8+ T cells and intact CD40 signaling, is critical for the maintenance of TLS, the preservation of the TFH cell pool, the formation of germinal center and the production of tumor-specific IgG antibodies. These findings underscore the key role of mature cDC1 in establishing and maintaining functional TLS within tumor lesions and highlight the potential for cDC1-targeting therapies as a promising strategy to enhance TLS function and improve anti-tumor immunity in patients with cancer.

Inflammatory immune cells recruited at the site of chronic inflammation form structures that resemble secondary lymphoid organs (SLO). These are characterized by segregated areas of prevalent T- or B-cell aggregation, differentiation of high endothelial venules, and local activation of resident stromal cells, including lymphatic endothelial cells. B-cell proliferation and affinity maturation toward locally displayed autoantigens have been demonstrated at these sites, known as tertiary lymphoid structures (TLS). TLS formation during chronic inflammation has been associated with local disease persistence and progression, as well as increased systemic manifestations. While bearing a similar histological structure to SLO, the signals that regulate TLS and SLO formation can diverge and a series of pro-inflammatory cytokines have been ascribed as responsible for TLS formation at different anatomical sites. Moreover, for a long time the structural compartment that regulates TLS homeostasis, including survival and recirculation of leucocytes has been neglected. In this chapter, we summarize the novel data available on TLS formation, structural organization, and the functional and anatomical links connecting TLS and SLOs.