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Immune infiltration; Spatial omics; Tertiary lymphoid structures Infiltración inmune; Ómica espacial; Estructuras linfoides terciarias Infiltració immune; Òmica espacial; Estructures limfoides terciàries The immune microenvironment of non-small cell lung cancer (NSCLC) is heterogeneous, which impedes the prediction of response to immune checkpoint inhibitors. We have mapped the expression of 49 proteins to spatial immune niches in 33 NSCLC tumors and report key differences in phenotype and function associated with the spatial context of immune infiltration. Tumor-infiltrating leukocytes (TIL), identified in 42% of tumors, had a similar proportion of lymphocyte antigens compared to stromal leukocytes (SL) but displayed significantly higher levels of functional, mainly immune suppressive, markers including PD-L1, PD-L2, CTLA-4, B7-H3, OX40L, and IDO1. In contrast, SL expressed higher levels of the targetable T-cell activation marker CD27, which increased with a longer distance to the tumor. Correlation analysis confirmed that metabolic-driven immune regulatory mechanisms, including ARG1 and IDO1, are present in the TIL. Tertiary lymphoid structures (TLS) were identified in 30% of patients. They displayed less variation in the expression profile and with significantly higher levels of pan lymphocyte and activation markers, dendritic cells, and antigen presentation compared to other immune niches. TLS also had higher CTLA-4 expression than non-structured SL, which may indicate immune dysfunction. Neither the presence of TIL nor TLS was associated with improved clinical outcomes. The apparent discrimination in functional profiles of distinct immune niches, independent of the overall level of leukocytes, illustrates the importance of spatial profiling to deconvolute how the immune microenvironment can dictate a therapeutic response and to identify biomarkers in the context of immunomodulatory treatment.

The tumor microenvironment (TME) and tertiary lymphoid structures (TLS) affect the occurrence and development of cancers. How the immune contexture interacts with the phenotype of clear cell renal cell carcinoma (ccRCC) remains unclear.We identified and evaluated TLS clusters in ccRCC using machine learning algorithms and the 12-chemokine gene signature for TLS. Analyses for functional enrichment, DNA variation, immune cell distribution, association with independent clinicopathological features and predictive value of CXCL13 in ccRCC were performed.We found a prominently enrichment of the 12-chemokine gene signature for TLS in patients with ccRCC compared with other types of renal cell carcinoma. We identified a prognostic value of CCL4, CCL5, CCL8, CCL19 and CXCL13 expression in ccRCC. DNA deletion of the TLS gene signature significantly predicted poor outcome in ccRCC compared with amplification and wild-type gene signature. We established TLS clusters (C1-4) and observed distinct differences in survival, stem cell-like characteristics, immune cell distribution, response to immunotherapies and VEGF-targeted therapies among the clusters. We found that elevated CXCL13 expression significantly predicted aggressive progression and poor prognosis in 232 patients with ccRCC in a real-world validation cohort.This study described a 12-chemokine gene signature for TLS in ccRCC and established TLS clusters that reflected different TME immune status and corresponded to prognosis of ccRCC. We confirmed the dense presence of TILs aggregation and TLS in ccRCC and demonstrated an oncogenic role of CXCL13 expression of ccRCC, which help develop immunotherapies and provide novel insights on the long-term management of ccRCC.

The presence of tertiary lymphoid structure (TLS) in tumor tissues has been reported to be a factor associated with a good prognosis in several types of cancers. However, the relationship between TLS formation and peripheral blood findings remains unclear. The purposes of the study were to evaluate the effect of the presence of TLS on survival and determine the peripheral blood characteristics associated with TLS formation in non-small cell lung cancer (NSCLC) patients. A total of 147 consecutive NSCLC patients who underwent lung resection at Fukushima Medical University Hospital between 2013 and 2017 were enrolled. TLS expression was evaluated, and the relationships between clinical parameters and outcomes were analyzed. Peripheral blood mononuclear cells (PBMCs) were further analyzed by mass cytometry to characterize the TLS-positive microenvironment. Forty-six patients had high TLS expression, and the remaining 101 patients had low TLS expression. In stage II to IV patients (n = 35), disease-free survival was longer in the high TLS expression group (p = 0.027). A low neutrophil to lymphocyte ratio (NLR)

BackgroundEpstein-Barr virus-associated gastric cancer (EBVaGC) exhibits unique histological characteristics within the immune-cell-rich microenvironment, but the role of tertiary lymphoid structure (TLS) in EBVaGC is not yet fully understood.MethodsWe retrospectively identified EBVaGC from 8517 consecutive GC cases from the two top cancer centers in China. Furthermore, we evaluated the prognostic value of TLS in 148 EBVaGC patients from our institute and then validated it in an external cohort (76 patients). TLS was quantified and its relationships with overall survival (OS) and therapeutic response were further analyzed. Multiplex immunofluorescence staining and targeted sequencing were used to characterize the composition of TLS and the genomic landscape, respectively.ResultsIn our study, EBVaGC was observed in 4.3% (190/4436) and 2.6% (109/4081) of GCs in the training and validation cohorts, respectively. TLS was identified in the intratumor (94.6%) and peritumor (77.0%) tissues with lymphoid aggregates, primary and secondary (i.e., mature TLSs) follicles in EBVaGC. Kaplan-Meier analysis showed that mature TLS in intratumoral tissues was associated with a favorable OS in the training and validation cohorts (p < 0.0001; p = 0.0108). Multivariate analyses demonstrated that intratumoral TLS maturation, pTNM, and PD-L1 expression were independent prognostic factors for OS (p < 0.05). Furthermore, the mature TLS was significantly associated with a good response to treatment in EBVaGC patients. Interestingly, the mutation frequency of SMARCA4 was significantly lower in the mature TLS groups.ConclusionsIntratumoral mature TLS was associated with a favorable prognosis and good therapeutic response, suggesting that it is a potential prognostic biomarker and predicts a good therapeutic response in EBVaGC patients.

BackgroundTertiary lymphoid structures (TLSs) are emerging as a potential predictor of prognosis and response to immunotherapy in some solid tumors. However, the comprehensive role of TLSs in bladder cancer remains unclear.MethodsEighteen bladder cancer (BCa) datasets were downloaded from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), ArratyExpress and IMvigor210. Based on 39 validated TLS signature genes (TSGs), we evaluated the TLS patterns in all patients, and correlated the TLS patterns with prognosis and tumor microenvironment (TME) cell-infiltrating characteristics. The cox regression model and principal component analysis (PCA) algorithms were used to construct the TLS score, which helps to quantify the TLS pattern in individuals.ResultsThe landscape of 39 validated TSGs in BCa was assessed first. Five distinct TLS patterns and four gene clusters were determined. TLS cluster C2 and gene cluster A were thought to be characterized by mature TLSs and showed better prognosis and higher immune cells infiltration than other clusters. The TLS score was discovered to be tightly correlated with the infiltration level of immune cells, and could predict the maturation status of TLSs to some extent. We found TLS score was an excellent predictor for prognosis in patients with BCa independent of tumor mutation burden (TMB), and low TLS score was related to better prognosis than high TLS score. Besides, low TLS score was correlated with a better response to immune checkpoint blockade (ICB) immunotherapy and commonly used chemotherapy drugs.ConclusionsOur work demonstrated the characteristics of TLSs in BCa. By using the TLS score, we could evaluate the TLS pattern in individuals. Better understanding of TLS pattern and the usage of TLS score could help instruct clinical strategy and precision medicine for BCa.

The immune checkpoint receptor PD-1 on T follicular helper (Tfh) cells promotes Tfh:B cell interactions and appropriate positioning within tissues. Here we examined the impact of regulation of PD-1 expression by the genomic organizer SATB1 on Tfh cell differentiation. Vaccination of CD4(Cre)Satb1(f/f) mice enriched for antigen-specific Tfh cells, and TGF-β-mediated repression of SATB1 enhanced Tfh differentiation of human T cells. Mechanistically, high Icos expression in Satb1(−/−) CD4(+) T cells promoted Tfh cell differentiation by preventing T follicular regulatory cell skewing, and resulted in increased isotype-switched B cell responses in vivo. Ovarian tumors in CD4(Cre)Satb1(f/f) mice accumulated tumor antigen-specific, LIGHT(+)CXCL13(+)IL-21(+) Tfh cells and tertiary lymphoid structures (TLS). TLS formation decreased tumor growth in a CD4(+) T cell and CXCL13-dependent manner. Transfer of Tfh cells, but not naïve CD4(+) T cells, induced TLS at tumor beds and decreased tumor growth. Thus, TGF-β-mediated silencing of Satb1 licenses Tfh cell differentiation, providing insight into the genesis of TLS within tumors.

Ectopic lymphoid follicles (ELFs), resembling germinal centre‐like structures, emerge in a variety of infectious and autoimmune and neoplastic diseases. ELFs can be found in the meninges of around 40% of the investigated progressive multiple sclerosis (MS) post‐mortem brain tissues and are associated with the severity of cortical degeneration and clinical disease progression. Of predominant importance for progressive neuronal damage during the progressive MS phase appears to be meningeal inflammation, comprising diffuse meningeal infiltrates, B‐cell aggregates and compartmentalized ELFs. However, the absence of a uniform definition of ELFs impedes reproducible and comparable neuropathological research in this field. In this review article, we will first highlight historical aspects and milestones around the discovery of ELFs in the meninges of progressive MS patients. In the next step, we discuss how animal models may contribute to an understanding of the mechanisms underlying ELF formation. Finally, we summarize challenges in investigating ELFs and propose potential directions for future research., ELFs are frequently found in the meninges of the deep sulci in about 40% of investigated progressive MS tissues. The typical structure of organized ELFs resembles the architecture of germinal centres in secondary lymphoid organs. In addition to compartmentalized B‐ and T‐cell zones, ELFs also feature specialized TFH cells, which are in close contact to B cells (predominantly CD27+ memory B cells), as well as follicular dendritic cells (FDC), which are essential for B‐cell differentiation and activation.

BackgroundTertiary lymphoid structures (TLS) have an effect on hepatocellular carcinoma (HCC), but the underlying mechanism remains to be elucidated.MethodsIntratumoral TLS (iTLS) was classified in the Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort using pathological sections from the Cancer Digital Slide Archive. Univariate and multivariate Cox regression analyses were performed to validate the effect of iTLS on overall survival (OS), relapse-free survival (RFS), and disease-free survival (DFS). The genes differentially expressed between the iTLS-negative and iTLS-positive groups were analyzed in combination with sequencing data. Gene set enrichment analysis (GSEA) was used to explore the signaling pathways affected by these differentially expressed genes. The random forest algorithm was used to identify genes with the highest correlation with the iTLS in the training set. Multivariate logistic regression was used to build a model to predict iTLS in tissue samples. Spearman’s correlation was used to analyze the relationship between TLS-associated chemokines and signature genes, and CIBERSORT was used to calculate immune infiltration scores. Copy number variation and its relationship with immune cell infiltration and signature genes were assessed using the gene set cancer analysis (GSCA). The Correlation R package was used for gene ontology (GO), disease ontology (DO), and gene mutation analyses. The GSCA was used for drug sensitivity analysis. LASSO regression was used to build prognostic models, and external data were used to validate the models.ResultsThere were 218 positive and 146 negative samples for iTLS. iTLS was significantly associated with better RFS and DFS according to Cox regression analysis. Twenty signature genes that were highly associated with iTLS positivity were identified. GO and mutation analyses revealed that the signature genes were associated with immunity. Most signature genes were sensitive to immune checkpoint inhibitors. Risk scores calculated using a characteristic gene-based prognostic model were found to be an independent prognostic factor for OS.ConclusionsThe improvement of RFS in HCC by iTLS was not limited to the early period as previously reported. iTLS improved DFS in patients. Characteristic genes are closely related to the formation of iTLS and TLS chemokines in HCC. These genes are closely related to immunity in terms of cellular infiltration, biological functions, and signaling pathways. Most are sensitive to immune checkpoint inhibitors, and their expression levels can affect prognosis.

Peripheral neurons comprise a critical component of the tumor microenvironment (TME). The role of the autonomic innervation in cancer has been firmly established. However, the effect of the afferent (sensory) neurons on tumor progression remains unclear. Utilizing surgical and chemical skin sensory denervation methods, we showed that afferent neurons supported the growth of melanoma tumors in vivo and demonstrated that sensory innervation limited the activation of effective antitumor immune responses. Specifically, sensory ablation led to improved leukocyte recruitment into tumors, with decreased presence of lymphoid and myeloid immunosuppressive cells and increased activation of T-effector cells within the TME. Cutaneous sensory nerves hindered the maturation of intratumoral high endothelial venules and limited the formation of mature tertiary lymphoid-like structures containing organized clusters of CD4+ T cells and B cells. Denervation further increased T-cell clonality and expanded the B-cell repertoire in the TME. Importantly, CD8a depletion prevented denervation-dependent antitumor effects. Finally, we observed that gene signatures of inflammation and the content of neuron-associated transcripts inversely correlated in human primary cutaneous melanomas, with the latter representing a negative prognostic marker of patient overall survival. Our results suggest that tumor-associated sensory neurons negatively regulate the development of protective antitumor immune responses within the TME, thereby defining a novel target for therapeutic intervention in the melanoma setting.

Approval of B-cell-depleting therapies signifies an important advance in the treatment of multiple sclerosis (MS). However, it is unclear whether the administration route of anti-CD20 monoclonal antibodies (mAbs) alters tissue distribution patterns and subsequent downstream effects. This study aimed to investigate the distribution and efficacy of radiolabeled ofatumumab and ocrelizumab in humanized-CD20 (huCD20) transgenic mice following subcutaneous (SC) and intravenous (IV) administration. For distribution analysis, huCD20 and wildtype mice (n = 5 per group) were imaged by single-photon emission computed tomography (SPECT)/CT 72 h after SC/IV administration of ofatumumab or SC/IV administration of ocrelizumab, radiolabeled with Indium-111 (111In-ofatumumab or 111In-ocrelizumab; 5 µg, 5 MBq). For efficacy analysis, huCD20 mice with focal delayed-type hypersensitivity lesions and associated tertiary lymphoid structures (DTH-TLS) were administered SC/IV ofatumumab or SC/IV ocrelizumab (7.5 mg/kg, n = 10 per group) on Days 63, 70 and 75 post lesion induction. Treatment impact on the number of CD19+ cells in select tissues and the evolution of DTH-TLS lesions in the brain were assessed. Uptake of an 111In-labelled anti-CD19 antibody in cervical and axillary lymph nodes was also assessed before and 18 days after treatment initiation as a measure of B-cell depletion. SPECT/CT image quantification revealed similar tissue distribution, albeit with large differences in blood signal, of 111In-ofatumumab and 111In-ocrelizumab following SC and IV administration; however, an increase in both mAbs was observed in the axillary and inguinal lymph nodes following SC versus IV administration. In the DTH-TLS model of MS, both treatments significantly reduced the 111In-anti-CD19 signal and number of CD19+ cells in select tissues, where no differences between the route of administration or mAb were observed. Both treatments significantly decreased the extent of glial activation, as well as the number of B- and T-cells in the lesion following SC and IV administration, although this was mostly achieved to a greater extent with ofatumumab versus ocrelizumab. These findings suggest that there may be more direct access to the lymph nodes through the lymphatic system with SC versus IV administration. Furthermore, preliminary findings suggest that ofatumumab may be more effective than ocrelizumab at controlling MS-like pathology in the brain.

Inducing a full antitumor immune response in the tumor microenvironment (TME) is essential for successful cancer immunotherapy. Here, we report that an oncolytic adenovirus carrying mIL-15 (Ad-IL15) can effectively induce antitumor immune response and inhibit tumor growth in a mouse model of cancer. We found that Ad-IL15 facilitated the activation and infiltration of immune cells, including dendritic cells (DCs), T cells and natural killer (NK) cells, in the TME. Unexpectedly, we observed that Ad-IL15 also induced vascular normalization and tertiary lymphoid structure formation in the TME. Moreover, we demonstrated these Ad-IL15-induced changes in the TME were depended on the Ad-IL15-induced activation of the STING-TBK1-IRF3 pathway in DCs. Taken together, our findings suggest that Ad-IL15 is a candidate for cancer immunotherapy that promotes immune cell activation and infiltration, tumor vascular normalization and tertiary lymphoid structure formation in the TME.

OBJECTIVE Our aim was to compare transcriptome and phenotype profiles of CD4+ T cells and CD19+ B cells in patients with Takayasu arteritis (TAK), patients with giant cell arteritis (GCA), and healthy donors. METHODS Gene expression analyses, flow cytometry immunophenotyping, T cell receptor (TCR) gene sequencing, and functional assessments of cells from peripheral blood and arterial lesions from TAK patients, GCA patients, and healthy donors were performed. RESULTS Among the most significantly dysregulated genes in CD4+ T cells of TAK patients compared to GCA patients (n = 720 genes) and in CD4+ T cells of TAK patients compared to healthy donors (n = 1,447 genes), we identified a follicular helper T (Tfh) cell signature, which included CXCR5, CCR6, and CCL20 genes, that was transcriptionally up-regulated in TAK patients. Phenotypically, there was an increase in CD4+CXCR5+CCR6+CXCR3- Tfh17 cells in TAK patients that was associated with a significant enrichment of CD19+ B cell activation. Functionally, Tfh cells helped B cells to proliferate, differentiate into memory cells, and secrete IgG antibodies. Maturation of B cells was inhibited by JAK inhibitors. Locally, in areas of arterial inflammation, we found a higher proportion of tertiary lymphoid structures comprised CD4+, CXCR5+, programmed death 1+, and CD20+ cells in TAK patients compared to GCA patients. CD4+CXCR5+ T cells in the aortas of TAK patients had an oligoclonal α/β TCR repertoire. CONCLUSION We established the presence of a specific Tfh cell signature in both circulating and aorta-infiltrating CD4+ T cells from TAK patients. The cooperation of Tfh cells and B cells might be critical in the occurrence of vascular inflammation in patients with TAK.

High endothelial venules (HEVs) are specialized blood vessels mediating lymphocyte trafficking to lymph nodes (LNs) and other secondary lymphoid organs. By supporting high levels of lymphocyte extravasation from the blood, HEVs play an essential role in lymphocyte recirculation and immune surveillance for foreign invaders (bacterial and viral infections) and alterations in the body’s own cells (neoantigens in cancer). The HEV network expands during inflammation in immune-stimulated LNs and is profoundly remodeled in metastatic and tumor-draining LNs. HEV-like blood vessels expressing high levels of the HEV-specific sulfated MECA-79 antigens are induced in non-lymphoid tissues at sites of chronic inflammation in many human inflammatory and allergic diseases, including rheumatoid arthritis, Crohn’s disease, allergic rhinitis and asthma. Such vessels are believed to contribute to the amplification and maintenance of chronic inflammation. MECA-79+ tumor-associated HEVs (TA-HEVs) are frequently found in human tumors in CD3+ T cell-rich areas or CD20+ B-cell rich tertiary lymphoid structures (TLSs). TA-HEVs have been proposed to play important roles in lymphocyte entry into tumors, a process essential for successful antitumor immunity and lymphocyte-mediated cancer immunotherapy with immune checkpoint inhibitors, vaccines or adoptive T cell therapy. In this review, we highlight the phenotype and function of HEVs in homeostatic, inflamed and tumor-draining lymph nodes, and those of HEV-like blood vessels in chronic inflammatory diseases. Furthermore, we discuss the role and regulation of TA-HEVs in human cancer and mouse tumor models.

Although activation of adaptive immunity is a common pathological feature of chronic obstructive pulmonary disease (COPD), particularly during later stages of the disease, the underlying mechanisms are poorly understood. In small airways of COPD patients, we found that localized disruption of the secretory immunoglobulin A (SIgA)-containing mucosal immunobarrier correlated with lymphocyte accumulation in airway walls and development of tertiary lymphoid structures (TLS) around small airways. In SIgA-deficient mice, we observed bacterial invasion into the airway epithelial barrier with lymphocytic infiltration and TLS formation, which correlated with the progression of COPD-like pathology with advanced age. Depletion of either CD4+ or CD8+ T lymphocytes reduced the severity of emphysema in SIgA-deficient mice, indicating that adaptive immune activation contributes to progressive lung destruction. Further studies revealed that lymphocyte infiltration into the lungs of SIgA-deficient mice was dependent on monocyte-derived dendritic cells (moDCs), which were recruited through a CCR2-dependent mechanism in response to airway bacteria. Consistent with these results, we found that moDCs were increased in lungs of COPD patients, along with CD4+ and CD8+ effector memory T cells. Together, these data indicate that endogenous bacteria in SIgA-deficient airways orchestrate a persistent and pathologic T lymphocyte response through monocyte recruitment and moDC differentiation.

Lymph node stromal cells coordinate the adaptive immune response in secondary lymphoid organs, providing both a structural matrix and soluble factors that regulate survival and migration of immune cells, ultimately promoting Ag encounter. In several inflamed tissues, resident fibroblasts can acquire lymphoid-stroma properties and drive the formation of ectopic aggregates of immune cells, named tertiary lymphoid structures (TLSs). Mature TLSs are functional sites for the development of adaptive responses and, consequently, when present, can have an impact in both autoimmunity and cancer conditions. In this review, we go over recent findings concerning both lymph node stromal cells and TLSs function and formation and further describe what is currently known about their role in disease, particularly their potential in tolerance.

Tertiary lymphoid structures (TLSs) are transient ectopic lymphoid aggregates whose formation might be caused by chronic inflammation states, such as cancer. However, how TLSs are induced in the tumor microenvironment (TME) and how they affect patient survival are not well understood. We investigated TLS distribution in relation to tumor infiltrating lymphocytes (TILs) and related gene expression in high grade serous ovarian cancer (HGSC) specimens. CXCL13 gene expression correlated with TLS presence and the infiltration of T cells and B cells, and was a favorable prognostic factor for HGSC patients. Coexistence of CD8⁺ T cells and B-cell lineages in the TME significantly improved the prognosis of HGSC and was correlated with the presence of TLSs. CXCL13 expression was predominantly coincident with CD4⁺ T cells in TLSs and CD8⁺ T cells in TILs, and shifted from CD4⁺ T cells to CD21⁺ follicular dendritic cells as TLS matured. In a mouse ovarian cancer model, recombinant CXCL13 induced TLSs and enhanced survival by the infiltration of CD8⁺ T cells. These results suggest that TLS formation was associated with CXCL13-producing CD4⁺ T cells and that TLSs facilitated the coordinated antitumor response of cellular and humoral immunity in ovarian cancer., 卵巣がんにおける新たな免疫の仕組みを発見 --三次リンパ様構造の形成メカニズムと予後への影響を解明--. 京都大学プレスリリース. 2022-08-05.

BackgroundTertiary lymphoid structures (TLSs) are crucial in promoting and maintaining positive anti-tumor immune responses. The tumor stroma has a powerful immunosuppressive function that could exclude tumor-infiltrating lymphocytes from the tumor beds and lead to a “cold” phenotype. TLSs and tumor stroma percentage (TSP) are significantly associated with the prognosis of patients with certain cancers. However, the exact roles of TLSs and TSP and their intrinsic relationship are still largely unknown in colorectal cancer (CRC).MethodsTLSs and TSP were assessed using hematoxylin-eosin (H&E) and/or immunohistochemistry (IHC) staining from 114 CRC patients in the training set and 60 CRC patients in the external validation set. The correlation between TILs, TLS and clinicopathological characteristics and their prognostic values were assessed. Finally, we plotted a Nomogram including the TLS, TSP and tumor-node-metastasis (TNM) stage to predict the probability of recurrence-free survival (RFS) at 2- and 5-years in non-metastatic colorectal cancer (nmCRC) patients.ResultsPeritumoral TLS (P-TLS), intratumoral TLS (In-TLS) and high TSP (H-TSP, >50%) were present in 99.1%, 26.3% and 41.2% patients, respectively. H-TSP tumor tends to be associated with lower P-TLS density (P =0.0205). The low P-TLS density (< 0.098/mm2) was significantly associated with reduced RFS (HR=6.597 95% CI: 2.882-15.103, P <0.001) and reduced overall survival (OS) (HR=6.628 95% CI: 2.893-15.183, P < 0.001) of nmCRC patients. In-TLS was not of significance in evaluating the clinical outcomes of nmCRC patients. H-TSP was significantly associated with reduced RFS (HR=0.126 95% CI: 0.048-0.333, P <0.001) and reduced OS (HR=0.125 95% CI: 0.047-0.332, P <0.001) of nmCRC patients. The 5-year RFS of the high P-TLS, low-TLS, H-TSP, and L-TSP groups were 89.7%, 47.2%, 53.2%, and 92.5%, respectively. The P-TLS density, TSP and TNM stage were independent prognosis factors of nmCRC patients. The Nomogram, including the P-TLS density, TSP and TNM stage, outperformed the TNM stage.ConclusionsHigh P-TLS density and low TSP (L-TSP) were independent and favorable prognostic factors of nmCRC patients, which might provide new directions for targeted therapy in the CRC tumor microenvironment, especially the tumor immune microenvironment.

BackgroundTertiary lymphoid structures (TLSs) have been proven to be predictive biomarkers of favorable clinical outcomes and response to immunotherapies in several solid malignancies. Nevertheless, the effect of TLSs in patients with breast cancer (BC) remains controversial. The objective of the current study is to investigate the clinicopathological and prognostic significance of TLSs in BC. Given the unique difficulties for detecting and quantifying TLSs, a TLS-associated gene signature based on The Cancer Genome Atlas (TCGA) BC cohort was used to validate and supplement our results.MethodsElectronic platforms (PubMed, Web of Science, EMBASE, the Cochrane Library, CNKI, and Wanfang) were searched systematically to identify relevant studies as of January 11, 2022. We calculated combined odds ratios (ORs) with 95% confidence intervals (CIs) to determine the relationship between clinicopathological parameters and TLSs. The pooled hazard ratios (HRs) and 95% CIs were also calculated to evaluate the prognostic significance of TLSs. The TLS signature based on the TCGA BC cohort was applied to validate and supplement our results.ResultsFifteen studies with 3,898 patients were eligible for enrollment in our study. The combined analysis indicated that the presence of TLSs was related to improved disease-free survival (DFS) (HR = 0.61, 95% CI: 0.41–0.90, p < 0.05) and overall survival (OS) (HR = 1.66, 95% CI: 1.26–2.20, p < 0.001). Additionally, the presence of TLSs was positively correlated with early tumor TNM stage and high tumor-infiltrating lymphocytes. TLS presence was positively related to human epidermal growth factor receptor 2 (HER-2) and Ki-67 but inversely correlated with the status of estrogen and progesterone receptor. Simultaneously, our study found that tumor immune microenvironment was more favorable in the high-TLS signature group than in the low-TLS signature group. Consistently, BC patients in the high-TLS signature group exhibited better survival outcomes compared to those in the low-TLS signature group, suggesting that TLSs might be favorable prognostic biomarkers.ConclusionsTLS presence provides new insight into the clinicopathological features and prognosis of patients with BC, whereas the factors discussed limited the evidence quality of this study. We look forward to consistent methods to define and characterize TLSs, and more high-quality prospective clinical trials designed to validate the value of TLSs alone or in combination with other markers.

High endothelial venules (HEVs) are specialized post-capillary venules that recruit naïve lymphocytes to lymph nodes. HEVs are essential for the development of adaptive immunity. HEVs can also develop in tumors where they are thought to be important for recruiting naïve T cells and B cells into the tumors and locally enhancing antitumor immunity by supporting the formation of tertiary lymphoid structures. Herein, we used comparative transcriptome analysis of human breast cancer to investigate genes differentially expressed between tumor-associated HEVs and the rest of the tumor vasculature. Tumor vessels highly expressing HEV-upregulated genes, such as the homeobox gene MEOX2 and the tetraspanin gene TSPAN7, were associated with extensive infiltration of T and B cells and the occurrence of tertiary lymphoid structures, which is known to predict therapeutic responses to immune checkpoint inhibitors. Moreover, high transcript counts of these genes in clinical tumor specimens were associated with a significant survival benefit in advanced breast cancer. The molecular signature of HEVs identified herein may be useful for guiding immunotherapies and provides a new direction for investigating tumor-associated HEVs and their clinical significance.

Chronic inflammation is the result a persistent increase in the expression of several proinflammatory pathways with impaired inflammatory resolution. Ectopic lymphoid organs (ELOs), untypical lymphoid annexes, emerge during chronic inflammation and contribute to the physiopathology of chronic inflammatory disorders. This review discusses the pathophysiological role of ELOs in the progression of immune-mediated inflammatory diseases (IMIDs), including multiple sclerosis (MS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atherosclerosis, and Sjögren syndrome (SSj). The molecular pathways underlying the emergence of ELOs are of interest for the development of novel pharmacological approaches for the management of chronic inflammatory diseases.

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that phenotypically resemble conventional secondary lymphoid organs and are commonly found at sites of chronic inflammation. They are also found in a wide variety of primary and metastatic human tumors. The presence of tumor-associated TLS (TA-TLS) is associated with prolonged patient survival, higher rates of disease-free survival, and a favorable response to current cancer therapies. However, the immune responses that occur in these structures, and how they contribute to improved clinical outcomes, remain incompletely understood. In addition, it is unknown how heterogeneity in TA-TLS cellular composition, structural organization, and anatomic location influences their functionality and prognostic significance. Understanding more about TA-TLS development, formation, and function may offer new therapeutic options to modulate antitumor immunity.

Background: Although the importance of ectopic lymphoid tissues (eLTs) in the pathophysiology of nasal polyps (NPs) is increasingly appreciated, the mechanisms underlying their formation remain unclear. Objective: To study the role of IL-17A, CXCL13 and lymphotoxin (LT) in eLT formation in NPs. Methods: The expression of CXCL13 and LT as well as their receptors, and the phenotypes of stromal cells in NPs were studied by flow cytometry, immunostaining, and RT-PCR. Purified nasal stromal cells and polyp B cells were cultured and a murine model with nasal type 17 inflammation was established for the mechanistic study. Results: Excessive CXCL13 production was found in NPs and correlated with enhanced IL-17A expression. Stromal cells, with an expansion of CD31-Pdpn+ fibroblastic reticular cell (FRC) type, were the major source of CXCL13 in NPs without eLTs. IL-17A induced FRC expansion and CXCL13 production in nasal stromal cells. In contrast, B cells were the main source of CXCL13 and LTα1β2 in NPs with eLTs. CXCL13 upregulated LTα1β2 expression on polyp B cells, which in turn promoted CXCL13 production from polyp B cells and nasal stromal cells. LTα1β2 induced expansion of FRCs and CD31+Pdpn+ lymphoid endothelial cells, corresponding to the phenotypic characteristic of stromal cells in NPs with eLTs. IL-17A gene knockout, and CXCL13 and LTβR blockage diminished nasal eLT formation in the murine model. Conclusion: We identified an important role of IL-17A-induced stromal cell remodeling in the initiation, and crosstalk between B and stromal cells via CXCL13 and LTα1β2 in the enlargement of eLTs in NPs.

CXCL13-producing PD-1hiCXCR5-CD4+ T cells were discovered in synovial tissues of rheumatoid arthritis (RA). Further intensive analysis of RA samples led to the proposal of PD-1hiCXCR5-CD4+ T cells as a pathogenic CD4 subset, peripheral helper T (Tph) cells. Tph cells are upregulated also in the peripheral blood of seropositive RA patient and exert B-cell helper activities. Furthermore, Tph cells are associated with other inflammatory conditions including systemic lupus erythematosus, IgG4-related diseases, type 1 diabetes, inflammatory bowel diseases, and malignant tumors. In this review, molecular feature and pathogenic and beneficial functions of Tph cells in inflammatory conditions are discussed.

Esophageal squamous cell carcinoma (ESCA) exhibits high intratumoral molecular heterogeneity posing a challenge to cancer therapy. Immune checkpoint blockade therapy has been approved for this disease, but with modest results. RNA-Seq data from paired tumor and surrounding nonmalignant tissue from 14 patients diagnosed with ESCA without previous treatment and from The Cancer Genome Atlas-ESCA cohort were analyzed. Herein, we investigated ESCA immune landscape including mutation-derived neoantigens and immune cell subpopulations. Tumor-associated antigen expression was determined by in silico analyses and confirmed by immunohistochemistry showing that PRAME, CEACAM4, and MAGEA11 proteins are expressed on tumors. Immune checkpoint molecules gene expression was higher in the tumor compared with surrounding nonmalignant tissue, but its expression varies greatly among patients. TCR repertoire and BCR transcripts analysis evidenced low clonal diversity with one TCR clone predicted to be specific for a MAGEA11-derived peptide. A high number of B-cell clones infiltrating the tumors and the abundance of these cells in tertiary lymphoid structures observed in ESCA tumors support B cells as a potential immune modulator in this tumor.

Ectopic lymphoid structures (ELS) or tertiary lymphoid organs are structures with the organization similar to the one of secondary lymphoid organs, formed in non-lymphoid tissues. They are considered to be an important site for the lymphocytic physiological and pathological role in conditions such are chronic infections, autoimmune diseases, cancer, and allograft rejection. Although similar to the secondary lymphoid tissues, the initiation of ELS formation is not preprogramed and requires chronic inflammation, expression of homeostatic chemokines, and lymphotoxin beta receptor activation. Importantly, while ELS formation may be considered beneficiary in antimicrobial and antitumor immunity, the persistence of these active lymphoid structures within the tissue increase the chance for development of autoimmunity and lymphoma. This paper is providing an overview of biological mechanisms involved in ELS formation, as well as the overview of the pathophysiological role of these structures. In addition, the paper discusses the possibility to therapeutically target ELS formation, bearing in mind their bivalent nature and role in different pathophysiological conditions.

Aging has multifaceted effects on the immune system, but how aging affects tissue-specific immunity is not well-defined. Bladder diseases characterized by chronic inflammation are highly prevalent in older women, but mechanisms by which aging promotes these pathologies remain unknown. Tissue transcriptomics of unperturbed, young and aged bladders identified a highly altered immune landscape as a fundamental feature of the aging female bladder. Detailed mapping of immune cells using single cell RNA-sequencing revealed novel subsets of macrophages and dendritic cells and unique changes to the immune repertoire in the aged bladder. B and T cells are highly enriched in aged bladders and spontaneously form organized bladder tertiary lymphoid tissues (bTLTs). Naïve, activated, and germinal center B cells and IgA+ plasma cells are found within bTLT and associated with increased urinary IgA concentrations. bTLTs form with increasing age and their formation is dependent on TNFα. Microbiota are not required to form bTLT form, as aged germfree mice harbor them. Thus, bTLTs require age-dependent TNFα but are independent of the microbiota. Our results indicate that chronic, age-associated inflammation underlies a fundamental alteration to the bladder and establishes a resource for further investigation of the bladder immune system in homeostasis, aging, and disease., One Sentence Summary: Mice develop bladder tertiary lymphoid tissue (bTLT) during aging that is dependent on TNFα

Tuberculosis prevalence is significantly higher among men than women. We have previously revealed an increased susceptibility of male C57BL/6 mice towards Mycobacterium tuberculosis (Mtb) H37Rv. In the current study, we confirm the male bias for infection with the Beijing strain HN878. Males succumbed to HN878 infection significantly earlier than females. In both models, premature death of males was associated with smaller B cell follicles in the lungs. Analysis of homeostatic chemokines and their receptors revealed differences between H37Rv and HN878 infected animals, indicating different immune requirements for follicle formation in both models. However, expression of IL-23, which is involved in long-term containment of Mtb and lymphoid follicle formation, was reduced in male compared to female lungs in both models. Our study reveals sex differences in the formation of B cell follicles in the Mtb infected lung and we propose that impaired follicle formation is responsible for accelerated disease progression in males.

IL-33, an epithelial-derived cytokine, functions as an alarmin for the immune system in the tumor microenvironment (TME). However, the expression and role of IL-33 on cervical cancer remain unclear. The aim of this study was to investigate the expression of IL-33 and its relationship with clinicopathologic features, tertiary lymphoid structures (TLS), and programmed cell death 1 (PD-1)/programmed cell death 1 ligand (PD-L1) immune checkpoints by immunohistochemistry in 93 cervical cancer patient specimens. Down-regulation of IL-33 expression was observed in tumor tissues compared with adjacent tissues. More importantly, IL-33 was detected in the cytoplasm of tumor fraction. IL-33 expression in tumor cytoplasm was associated with tumor size and the invasive depth of tumors (p

In this issue of Immunity, Meylan et al. (2022) uses spatial transcriptomics to examine B cell immunity within intratumoral tertiary lymphoid structures (TLSs). They find that B cells expand and mature into plasma cells (PCs) within the TLS, migrate along fibroblastic tracks to tumor beds, and produce IgG antibodies that target cancer cells.

Immune checkpoint inhibitors (ICI) have provided significant improvement in clinical outcomes for some patients with solid tumors. However, for patients with head and neck cancer, the response rate to ICI monotherapy remains low, leading to the exploration of combinatorial treatment strategies. In this preclinical study, we use an oncolytic adenovirus (Ad5/3) encoding hTNFα and hIL-2 and non-replicate adenoviruses (Ad5) encoding mTNFα and mIL-2 with ICI to achieve superior tumor growth control and improved survival outcomes. The in vitro effect of Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 was characterized through analyses of virus replication, transgene expression and lytic activity using head and neck cancer patient derived cell lines. Mouse models of ICI naïve and refractory oral cavity squamous cell carcinoma were established to evaluate the local and systemic anti-tumor immune response upon ICI treatment with or without the non-replicative adenovirus encoding mTNFα and mIL-2. We delineated the mechanism of action by measuring the metabolic activity and effector function of CD3+ tumor infiltrating lymphocytes (TIL) and transcriptomic profile of the CD45+ tumor immune compartment. Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 demonstrated robust replicative capability in vitro across all head and neck cell lines screened through potent lytic activity, E1a and transgene expression. In vivo, in both ICI naïve and refractory models, we observed improvement to tumor growth control and long-term survival when combining anti-PD-1 or anti-PD-L1 with the non-replicative adenovirus encoding mTNFα and mIL-2 compared to monotherapies. This observation was verified by striking CD3+ TIL derived mGranzyme b and interferon gamma production complemented by increased T cell bioenergetics. Notably, interrogation of the tumor immune transcriptome revealed the upregulation of a gene signature distinctive of tertiary lymphoid structure formation upon treatment of murine anti-PD-L1 refractory tumors with non-replicative adenovirus encoding mTNFα and mIL-2. In addition, we detected an increase in anti-tumor antibody production and expansion of the memory T cell compartment in the secondary lymphoid organs. In summary, a non-replicative adenovirus encoding mTNFα and mIL-2 potentiates ICI therapy, demonstrated by improved tumor growth control and survival in head and neck tumor-bearing mice. Moreover, the data reveals a potential approach for inducing tertiary lymphoid structure formation. Altogether our results support the clinical potential of combining this adenovirotherapy with anti-PD-1 or anti-PD-L1.

International audience; The presence of intratumoral tertiary lymphoid structures (TLS) is associated with positive clinical outcomes and responses to immunotherapy in cancer. Here, we used spatial transcriptomics to examine the nature of B cell responses within TLS in renal cell carcinoma (RCC). B cells were enriched in TLS, and therein, we could identify all B cell maturation stages toward plasma cell (PC) formation. B cell repertoire analysis revealed clonal diversification, selection, expansion in TLS, and the presence of fully mature clonotypes at distance. In TLS+ tumors, IgG- and IgA-producing PCs disseminated into the tumor beds along fibroblastic tracks. TLS+ tumors exhibited high frequencies of IgG-producing PCs and IgG-stained and apoptotic malignant cells, suggestive of anti-tumor effector activity. Therapeutic responses and progression-free survival correlated with IgG-stained tumor cells in RCC patients treated with immune checkpoint inhibitors. Thus, intratumoral TLS sustains B cell maturation and antibody production that is associated with response to immunotherapy, potentially via direct anti-tumor effects.

Tertiary lymphoid structure (TLS) and tumor-resident memory T cells (TRM) play crucial roles in the anti-tumor immune response, facilitating a good prognosis in patients with cancer. However, there have been no reports on the relationship between TRM and TLS maturity. In this study, we detected TRM and the maturity of TLS by immunofluorescence staining and analyzed the relationship between their distribution and proportion in patients with lung adenocarcinoma (LUAD). The proportion of TRM within TLSs was significantly higher than that outside and was positively correlated with the survival of patients. In addition, the proportions of CD4+CD103+ TRM and CD8+CD103+ TRM were significantly increased with the gradually maturation of TLSs. We divided the patients into three levels (grade 1, grade 2, and grade 3) according to the presence of increasing maturation of TLSs. The proportion of CD103+ TRM in grade 3 patients was significantly higher than that in grade 1 and grade 2 patients, suggesting a close relationship between CD103+ TRM and TLS maturity. Furthermore, positive prognosis was associated with grade 3 patients that exhibited CD103+TRMHigh phenotype.

BackgroundThe aims of this study were (1) to clarify the impact of tertiary lymphoid structure (TLS) status on the outcome and immunogenomic profile of human clear cell renal cell carcinoma (ccRCC) and (2) to determine phenotypic differences in TLSs between different types of genitourinary cancer, that is, urinary ccRCC and bladder cancer.MethodsWe performed a quantitative immunohistological analysis of ccRCC tissue microarrays and conducted integrated genome mutation analysis by next-generation sequencing and methylation array analysis. Since the tumor immune microenvironment of ccRCC often differs from that of other cancer types, we analyzed the phenotypic differences in TLSs between ccRCC and in-house bladder cancer specimens.ResultsVarying distribution patterns of TLSs were observed throughout ccRCC tumors, revealing that the presence of TLSs was related to poor prognosis. An analysis of genomic alterations based on TLS status in ccRCC revealed that alterations in the PI3K-mTOR pathway were highly prevalent in TLS-positive tumors. DNA methylation profiling also revealed distinct differences in methylation signatures among ccRCC samples with different TLS statuses. However, the TLS characteristics of ccRCC and bladder cancer markedly differed: TLSs had the exact opposite prognostic impact on bladder cancer as on ccRCC. The maturity and spatial distribution of TLSs were significantly different between the two cancer types; TLSs were more mature with follicle-like germinal center organization and likely to be observed inside the tumor in bladder cancer. Labeling for CD8, FOXP3, PD-1, and PD-L1 showed marked differences in the diversity of the immune microenvironment surrounding TLSs. The proportions of CD8-, FOXP3-, and PD-L1-positive cells were significantly higher in TLSs in bladder cancer than in TLSs in ccRCC; rather the proportion of PD-1-positive cells was significantly higher in TLSs in ccRCC than in TLSs in bladder cancer.ConclusionThe immunobiology of ccRCC is unique, and various cancerous phenomena conflict with that seen in other cancer types; therefore, comparing the TLS characteristics between ccRCC and bladder cancer may help reveal differences in the prognostic impact, maturity and spatial distribution of TLSs and in the immune environment surrounding TLSs between the two cancers.

Lymphoid neogenesis gives rise to tertiary lymphoid structures (TLS) in the periphery of multiple cancer types including muscle invasive bladder cancer (MIBC) where it has positive prognostic and predictive associations. Here, we explored molecular, clinical, and histological data of The Cancer Genome Atlas, as well as the IMvigor210 dataset to study factors associated with TLS development and function in the tumor microenvironment (TME) of MIBC. We also analyzed tumor immune composition including TLS in an independent, retrospective MIBC cohort. We found that the combination of TLS density and tumor mutational burden provides a novel independent prognostic biomarker in MIBC. Gene expression profiles obtained from intratumoral regions that rarely contain TLS in MIBC showed poor correlation with the prognostic TLS density measured in tumor periphery. Tumors with high TLS density showed increased gene signatures as well as infiltration of activated lymphocytes. Intratumoral B-cell and CD8+ T-cell co-infiltration was frequent in TLS-high samples, and such regions harbored the highest proportion of PD-1+TCF1+ progenitor-like T cells, naïve T cells, and activated B cells when compared to regions predominantly infiltrated by either B cells or CD8+ T cells alone. We found four TLS maturation subtypes; however, differences in TLS composition appeared to be dictated by the TME and not by the TLS maturation status. Finally, we identified one downregulated and three upregulated non-immune cell-related genes in TME with high TLS density, which may represent candidates for tumor-intrinsic regulation of lymphoid neogenesis. Our study provides novel insights into TLS-associated gene expression and immune contexture of MIBC and indicates towards the relevance of B-cell and CD8+ T-cell interactions in anti-tumor immunity within and outside TLS.

B cells constitute a major component of infiltrating immune cells in colorectal cancer (CRC). However, the characteristics of B cells and their clinical significance remain unclear. In this study, using single-cell RNA sequencing and multicolour immunofluorescence staining experiments, we identified five distinct subtypes of B cells with their marker genes, distribution patterns and functional properties in the CRC tumour microenvironment. Meanwhile, we found a higher proportion of IgG plasma cells in tumour sites than that in adjacent normal mucosal tissues. In addition, the CXCL13-producing CD8

Repeated short-term intranasal instillation of lupus-prone mice with crystalline silica (cSiO2) induces inflammatory gene expression and ectopic lymphoid neogenesis in the lung, leading to early onset of systemic autoimmunity and rapid progression to glomerulonephritis. These responses are suppressed by dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). Here, we tested the hypothesis that dietary DHA supplementation suppresses cSiO2-induced inflammatory proteins in bronchoalveolar alveolar lavage fluid (BALF) and plasma of lupus-prone mice. Archived tissue fluid samples were used from a prior investigation in which 6 wk-old lupus-prone female NZBWF1 mice were fed isocaloric diets containing 0 or 10 g/kg DHA for 2 wks and then intranasally instilled with 1 mg cSiO2or vehicle once weekly for 4 wks. Cohorts were terminated at 1, 5, 9 or 13 wk post-instillation (PI). BALF and plasma from each cohort were analyzed by high density multiplex array profiling of 200 inflammatory proteins. cSiO2time-dependently induced increases in the BALF protein signatures that were highly reflective of unresolved lung inflammation, although responses in the plasma were much less robust. Induced proteins in BALF included chemokines (e.g., MIP-2, MCP-5), enzymes (e.g., MMP-10, granzyme B), adhesion molecules (e.g., sE-selectin, sVCAM-1), co-stimulatory molecules (e.g., sCD40L, sCD48), TNF superfamily proteins (e.g., sTNFRI, sBAFF-R), growth factors (e.g., IGF-1, IGFBP-3), and signal transduction proteins (e.g., MFG-E8, FcgRIIB), many of which were blocked or delayed by DHA supplementation. The BALF inflammatory proteome correlated positively with prior measurements of gene expression, pulmonary ectopic lymphoid tissue neogenesis, and induction of autoantibodies in the lungs of the control and treatment groups. Ingenuity Pathway Analysis (IPA) revealed that IL-1β, TNF-α, and IL-6 were among the top upstream regulators of the cSiO2-induced protein response. Furthermore, DHA’s effects were associated with downregulation of cSiO2-induced pathways involving i) inhibition of ARE‐mediated mRNA decay, ii) bacterial and viral pattern recognition receptor activation, or iii) TREM1, STAT3, NF-κB, and VEGF signaling and with upregulation of PPAR, LXR/RXR and PPARα/RXRα signaling. Altogether, these preclinical findings further support the contention that dietary DHA supplementation could be applicable as an intervention against inflammation-driven autoimmune triggering by cSiO2or potentially other environmental agents.

The clinical success of immunotherapy has revolutionized the treatment of cancer patients, bringing renewed attention to tumor-infiltrating lymphocytes (TILs) of various cancer types. Immune checkpoint blockade is effective in patients with mismatched repair defects and high microsatellite instability (dMMR-MSI-H) in metastatic colorectal cancer (CRC), leading the FDA to accelerate the approval of two programmed cell death 1 (PD-1) blocking antibodies, pembrolizumab and nivolumab, for treatment of dMMR-MSI-H cancers. In contrast, patients with proficient mismatch repair and low levels of microsatellite stability or microsatellite instability (pMMR-MSI-L/MSS) typically have low tumor-infiltrating lymphocytes and have shown unsatisfied responses to the immune checkpoint inhibitor. Different TILs environments reflect different responses to immunotherapy, highlighting the complexity of the underlying tumor-immune interaction. Profiling of TILs fundamental Indication would shed light on the mechanisms of cancer-immune evasion, thus providing opportunities for the development of novel therapeutic strategies. In this review, we summarize phenotypic diversities of TILs and their connections with prognosis in CRC and provide insights into the subsets-specific nature of TILs with different MSI status. We also discuss current clinical immunotherapy approaches based on TILs as well as promising directions for future expansion, and highlight existing clinical data supporting its use.

TIM4 has previously been associated with antitumor immunity, yet the pattern of expression and the function of this receptor across human cancer tissues remain poorly explored. Here we combined extensive immunolabeling of human tissues with in silico analysis of pan-cancer transcriptomic data sets to explore the clinical significance of TIM4 expression. Our results unveil that TIM4 is expressed on a fraction of cavity macrophages (CATIM4+MΦ) of carcinoma patients. Moreover, we uncover a high expression of TIM4 on macrophages of the T-cell zone of the carcinoma-associated tertiary lymphoid structures (TLSTIM4+MΦ). In silico analysis of a pan-cancer data set revealed a positive correlation between TIM4 expression and markers of B cells, effector CD8+ T cells, and a 12-chemokine signature defining tertiary lymphoid structure. In addition, TLSTIM4+MΦ were enriched in cancers displaying microsatellite instability and high CD8+ T-cell infiltration, confirming their association with immune-reactive tumors. Both CATIM4+MΦ and TLSTIM4+MΦ express FOLR2, a marker of tissue-resident MΦ. However, CATIM4+MΦ had a higher expression of the immunosuppressive molecules TREM2, IL10, and TGFβ as compared with TLSTIM4+MΦ. By analyzing a scRNA sequence data set of tumor-associated myeloid cells, we identified two TIM4+FOLR2+ clusters coherent with CATIM4+MΦ and TLSTIM4+MΦ. We defined specific gene signatures for each subset and found that the CATIM4+ MΦ signature was associated with worse patient survival. In contrast, TLSTIM4+MΦ gene signature positively correlates with a better prognosis. Together, these data illustrate that TIM4 marks two distinct macrophage populations with distinct phenotypes and tissue localization and that may have opposing roles in tumor immunity.

Simple Summary Adenoid cystic carcinoma (ACC) is a rare tumor with late occurring metastases and recurrences, which makes it necessary for patients to be monitored closely even beyond the usual five years. So far, there is hardly any effective treatment in the palliative situation and trials on immunotherapeutic drugs have not been successful. We sought to find possible prognostic markers by analyzing patterns of tumor infiltrating immune cells and additionally to learn more about possible reasons for this lack of response to immunotherapy. It appears from our data that it is not relevant for prognosis if the tumor is infiltrated by immune cells. This might also be the reason that immunotherapies do not work in this particular disease, which suggests that ACC is not recognized by infiltrating immune cells. Therefore, the tumor would have to be made visible to the immune system, for example, through vaccinations. Abstract Adenoid cystic carcinoma (ACC) is a rare malignancy in the head and neck. The prognosis remains poor and late recurrences often occur after 5 years and later. To date, there are no reliable prognostic markers for ACC. In several solid tumors, tertiary lymphoid structures (TLS) are associated with improved survival. This study aims to investigate the role of distribution patterns of tumor infiltrating immune cells (TIL) in ACC. A cohort of 50 patients from three different cancer centers was available for analysis. Sections were stained for CD3, CD4, CD8 and CD20 and evaluated with regard to their distribution of TIL. Patterns were determined as infiltrated-excluded, infiltrated-inflamed and presence of tertiary lymphoid structures. About half of the cases showed an infiltrated-excluded TIL pattern and only a minority of six cases had TLS present within the tumor. Within the inflamed phenotype CD3+ cells were by far the most abundant lymphocyte subtype, and within this compartment, CD8+ T cells were predominant. There was no influence on overall or disease-free survival by any of the TIL patterns. This indicates that ACC is a tumor with very low immunogenicity and even abundance of lymphocytes does not seem to improve prognosis for this disease. Therefore, the observed lack of response towards immunotherapy is not surprising and other methods to induce recognition of ACC by the immune system must be found., publishedVersion

Nivolumab, an immune checkpoint blocker, has been approved for advanced gastric cancer (GC), but predictive factors of nivolumab’s efficacy in patients with GC, especially immune cells such as tissue-resident memory T cells or those forming tertiary lymphoid structures (TLS), remain unclear. Tissue samples were obtained from surgically resected specimens of patients with GC who were treated with nivolumab as third-line or later treatment. Immunohistochemical staining was performed to detect the presence of TLS and CD103+T cells and assess the association between TLSs and response to nivolumab treatment. A total of 19 patients were analyzed. In patients with partial response (PR) to nivolumab, numerous TLS were observed, and CD103+T cells were found in and around TLS. Patients with many TLS experienced immune-related adverse events more often than those with few TLS (p= 0.018). The prognosis of patients with TLS high was better than those with TLS low. Patients with a combination of TLS high and CD103 high tended to have a better prognosis than other groups. Our results suggested that TLS status might be a predictor of nivolumab effectiveness.

Pancreatic ductal adenocarcinoma (PDA) is usually unresponsive to immunotherapeutic approaches. However, tertiary lymphoid structures (TLS) are associated with favorable patient outcomes in PDA. A better understanding of the B cell infiltrate and biological features of TLS formation is needed to further explore their potential and improve patient management. We analyzed tumor tissues (n = 55) and corresponding blood samples (n = 51) from PDA patients by systematical immunohistochemistry and multiplex cytokine measurements. The tissue was compartmentalized in “tumor” and “stroma” and separately examined. Clinical patient information was used to perform survival analyses. We found that the mere number of B cells is not associated with patient survival, but formation of TLS in the peritumoral stroma is a prognostic favorable marker in PDA patients. TLS-positive tissues show a higher density of CD8+ T cells and CD20+ B cells and a higher IL2 level in the peritumoral stroma than tissues without TLS. Compartmental assessment shows that gradients of IL2 expression differ with regard to TLS formation: TLS presence is associated with higher IL2 levels in the stromal than in the tumoral compartment, while no difference is seen in patients without TLS. Focusing on the stroma-to-serum gradient, only patients without TLS show significantly higher IL2 levels in the serum than in stroma. Finally, low circulatory IL2 levels are associated with local TLS formation. Our findings highlight that TLS are prognostic favorable and associated with antitumoral features in the microenvironment of PDA. Also, we propose easily accessible serum IL2 levels as a potential marker for TLS prediction.