Lack of sustained response to oncogenic Kras (Kras*) inhibition in preclinical models and patients with pancreatic ductal adenocarcinoma (PDAC) emphasizes the need to identify impactful synergistic combination therapies to achieve robust clinical benefit. Kras* targeting results in an influx of global T cell infiltrates including Tregs, effector CD8 + T cells and exhausted CD8 + T cells expressing several immune checkpoint molecules in PDAC. Here, we probe whether the T cell influx induced by diverse Kras* inhibitors open a therapeutic window to target the adaptive immune response in PDAC. We show a specific synergy of anti-CTLA4 immune checkpoint blockade with Kras* targeting primed by Kras G12D allele specific inhibitor, MRTX1133 and multi-selective pan-RAS inhibitor, RMC-6236, both currently in clinical testing phase. In contrast, attempted therapeutic combination following Kras* targeting with multiple checkpoint inhibitors, including anti-PD1, anti-Tim3, anti-Lag3, anti-Vista and anti-4-1BB agonist antibody failed due to compensatory mechanisms mediated by other checkpoints on exhausted CD8 + T cells. Anti-CTLA4 therapy in Kras* targeted PDAC transcriptionally reprograms effector T regs to a naïve phenotype, reverses CD8 + T cell exhaustion and is associated with recruitment of tertiary lymphoid structures (TLS) containing interferon (IFN)-stimulated/ activated B cells and germinal center B cells to enable immunotherapy efficacy and overcome resistance with long-term survival. Single cell ATAC sequencing analysis revealed that transcriptional reprogramming of Tregs is epigenetically regulated by downregulation of AP-1 family of transcription factors including Fos, Fos-b, Jun-b, Jun-d in the IL-35 promoter region. This study reveals an actionable vulnerability in the adaptive immune response in Kras* targeted PDAC with important clinical implications.

Breast cancer, a highly prevalent global cancer, poses significant challenges, especially in advanced stages. Prognostic models are crucial to enhance patient outcomes. Tertiary lymphoid structures (TLS) within the tumor microenvironment have been associated with better prognostic outcomes.

We analyzed data from 13 independent breast cancer cohorts, totaling over 9,551 patients. Using single-cell RNA sequencing and machine learning algorithms, we identified critical TLS-associated genes and developed a TLS-based predictive model. This model stratified patients into high and low-risk groups. Genomic alterations, immune infiltration, and cellular interactions within the tumor microenvironment were assessed.

The TLS-based model demonstrated superior accuracy compared to traditional models, predicting overall survival. High TLS patients had higher tumor mutation burden and more chromosomal alterations, correlating with poorer prognosis. High-risk patients exhibited a significant depletion of CD4+ T cells, CD8+ T cells, and B cells, as evidenced by single-cell and bulk transcriptomic analyses. In contrast, immune checkpoint inhibitors demonstrated greater efficacy in low-risk patients, whereas chemotherapy proved more effective for high-risk individuals.

The TLS-based prognostic model is a robust tool for predicting breast cancer outcomes, highlighting the tumor microenvironment's role in cancer progression. It enhances our understanding of breast cancer biology and supports personalized therapeutic strategies.

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.

Tertiary lymphoid structures (TLS), formerly recognized as Crohn's-like structures, serve as crucial biomarkers for evaluating the progression of colorectal cancer (CRC). Understanding their spatial distribution, cellular composition, and interactions within CRC is paramount for comprehending the immune response in the tumor microenvironment (TME). TLS are comprised of a T-cellular compartment and a B-cellular compartment, the latter encompassing follicular dendritic cells (FDCs), high endothelial venules (HEVs), and lymphatic vessels. While T helper cells predominate in cancer TLS, the specific functions of their subpopulations remain inadequately understood. Notably, T follicular helper (Tfh) cells play a central role in the activation of CD8+ T cells, and both Tfh cells and Tfh-associated genes have been linked to enhanced CRC survival. In stage II CRC TLS, an escalation in the number of FoxP3+ T regulatory cells (Tregs) is regarded as a negative prognostic factor. Moreover, within TLS, T lymphocytes shield B lymphocytes from the immunosuppressive effects of the TME. B lymphocyte activation is succeeded by class recombination (CSR) and somatic hypermutation (SHM). Dendritic cells (DCs) constitute a vital cellular component of the TLS T compartment. During steady state and early stages of CRC, specialized antigen-presenting cells such as DCs migrate to regional lymph nodes through afferent lymphatics. They deliver MHC antigen-derived peptide complexes (tumor antigens) to naïve CD4+ and CD8+ T cells, which subsequently infiltrate the tumor site as antigen-specific T cells. Key DC markers studied in TLS include CD83 and DC-LAMP. Research has indicated that the DC-LAMP gene signature in tumor TLS reflects Th1 cell targeting, cytotoxicity, and T cell activation. This review comprehensively outlines the functions performed by distinct cell subsets within tertiary lymphoid structures (TLS) in tumors.

Nasopharyngeal carcinoma (NPC) is a type of malignant tumors commonly found in Southeast Asia and China, with insidious onset and clinical symptoms. N6-methyladenosine (m6A) modification significantly contributes to tumorigenesis and progression by altering RNA secondary structure and influencing RNA-protein binding at the transcriptome level. However, the mechanism and role of abnormal m6A modification in nasopharyngeal carcinoma remain unclear.

Nasopharyngeal Carcinoma tissues from 3 patients and non-cancerous nasopharyngeal tissues from 3 individuals, all from Fujian Cancer Hospital, were sequenced for m6A methylation. These were combined with transcriptome sequencing data from 192 nasopharyngeal cancer tissues. Genes linked to prognosis were discovered using differential analysis and univariate Cox regression. Subsequently, a prognostic model associated with m6A was developed through the application of LASSO regression analysis. The model's accuracy was verified using both internal transcriptome databases and external databases. An extensive evaluation of the tumor's immune microenvironment and signaling pathways was performed, analyzing both transcriptomic and single-cell data.

The m6A methylation sequencing analysis revealed 194 genes with varying expression levels, many of which are predominantly associated with immune pathways. By integrating transcriptome sequencing data, 19 m6A-modified genes were found to be upregulated in tumor tissues, leading to the development of a three-gene (EME1, WNT4, SHISA2) risk prognosis model. The group with lower risk exhibited notable enrichment in pathways related to immunity, displaying traits like enhanced survival rates, stronger immune profiles, and increased responsiveness to immunotherapy when compared to the higher-risk group. Single-cell analysis revealed that malignant cells exhibited the highest risk score levels compared to immune cells, with a high-risk score indicating worse biological behavior. The three hub genes demonstrated significant correlation with m6A modification regulators, and MeRIP-RT-PCR confirmed the occurrence of m6A methylation in these genes within nasopharyngeal carcinoma cells.

A prognostic model for nasopharyngeal carcinoma risk based on m6A modification genes was developed, and its prognostic value was confirmed through self-assessment data. The study highlighted the crucial impact of m6A modification on the immune landscape of nasopharyngeal cancer.

Tertiary lymphoid structures (TLS) are ectopic clusters of immune cells found in non-lymphoid tissues, particularly within the tumor microenvironment (TME). These structures resemble secondary lymphoid organs and have been identified in various solid tumors, including colorectal cancer (CRC), where they are associated with favorable prognosis. The role of TLS in modulating the immune response within the TME and their impact on cancer prognosis has garnered increasing attention in recent years.

This review aims to summarize the current understanding of TLS in CRC, focusing on their formation, function, and potential as prognostic markers and therapeutic targets. We explore the mechanisms by which TLS influence the immune response within the TME and their correlation with clinical outcomes in CRC patients.

We conducted a comprehensive review of recent studies that investigated the presence and role of TLS in CRC. The review includes data from histopathological analyses, immunohistochemical studies, and clinical trials, examining the association between TLS density, composition, and CRC prognosis. Additionally, we explored emerging therapeutic strategies targeting TLS formation and function within the TME.

The presence of TLS in CRC is generally associated with an improved prognosis, particularly in early-stage disease. TLS formation is driven by chronic inflammation and is characterized by the organization of B and T cell zones, high endothelial venules (HEVs), and follicular dendritic cells (FDCs). The density and maturity of TLS are linked to better patient outcomes, including reduced recurrence rates and increased survival. Furthermore, the interplay between TLS and immune checkpoint inhibitors (ICIs) suggests potential therapeutic implications for enhancing anti-tumor immunity in CRC.

TLS represent a significant prognostic marker in CRC, with their presence correlating with favorable clinical outcomes. Ongoing research is required to fully understand the mechanisms by which TLS modulate the immune response within the TME and to develop effective therapies that harness their potential. The integration of TLS-focused strategies in CRC treatment could lead to improved patient management and outcomes.

Papillary thyroid carcinoma (PTC) is the most prevalent form of thyroid cancer, with a disease recurrence rate of around 20%. Lymphoid formations, which occur in nonlymphoid tissues during chronic inflammatory, infectious, and immune responses, have been linked with tumor suppression. Lymphoid aggregates potentially enhance the body's antitumor response, offering an avenue for attracting tumor-infiltrating lymphocytes and fostering their coordination. Increasing evidence highlights the role of lymphoid aggregate density in managing tumor invasion and metastasis, with a favorable impact noted on overall and disease-free survival (DFS) across various cancer types. In this study, we present a machine vision model to predict recurrence in different histologic subtypes of PTC using measurements related to peritumoral lymphoid aggregate density. We demonstrated that quantifying peritumoral lymphocytic presence not only is associated with better prognosis but also, along with tumor-infiltrating lymphocytes within the tumor, adds additional prognostic value in the absence of well-known second mutations including TERT. Annotations of peritumoral lymphoid aggregates on 171 well-differentiated PTCs in the Cancer Genome Atlas Thyroid Carcinoma (TCGA-THCA) data set were used to train a deep-learning model to predict regions of lymphoid aggregates across the entire tissue. The fractional area of the tissue regions covered by these lymphocytes was dichotomized to determine the following 2 risk groups: a significant and low density of peritumoral lymphocytes. DFS prognosticated using these risk groups via the Kaplan-Meier analysis revealed a hazard ratio (HR) of 2.51 (95% CI: 2.36, 2.66), tested on 170 new patients also from the TCGA-THCA data set. The prognostic performance of peritumoral lymphocyte aggregate density was compared against the univariate Kaplan-Meier analysis of DFS using the fractional area of intratumoral lymphocytes within the primary tumor with an HR of 2.04 (95% CI: 1.89, 2.19). Combining the lymphocyte features in and around the tumor yielded a statistically significant improvement in prognostic performance (HR, 3.17 [95% CI: 3.02, 3.32]) on training and were independently evaluated against 62 patients outside TCGA-THCA with an HR of 2.44 (95% CI: 2.19, 2.69). Multivariable Cox regression analysis on the validation set revealed that the density of peritumoral and intratumoral lymphocytes was prognostic independent of histologic subtype with a concordance index of 0.815.

Many studies have reported the presence of tertiary lymphoid structures (TLSs) in cancer, but the research progress of TLSs in cancer has not been systematically analyzed. Therefore, we analyzed the global scientific knowledge in the field using informatics methods. The results showed that TLSs in cancer have received increasing attention since the 21st century, with an annual publication growth rate of 27.86%. Unsupervised hierarchical clustering based on machine learning further categorized the research features into four clusters, with the cluster related to immunotherapy being considered an emerging cluster. TLSs and immunotherapy were identified as the top two hotspots with the highest occurrence frequency and total link strength. The Walktrap algorithm indicated that "TLSs, carcinoma, prognostic value" and "high endothelial venules, germinal-centers, node-like structures" are important to TLSs but remain underexplored, representing promising research directions. These findings suggest that cancer-related TLSs have brought new insights into antitumor immunity, and targeting TLSs has the potential to transform the landscape of antitumor immunotherapy.

Spatial barcoding-based transcriptomic (ST) data require deconvolution for cellular-level downstream analysis. Here we present SDePER, a hybrid machine learning and regression method to deconvolve ST data using reference single-cell RNA sequencing (scRNA-seq) data. SDePER tackles platform effects between ST and scRNA-seq data, ensuring a linear relationship between them while addressing sparsity and spatial correlations in cell types across capture spots. SDePER estimates cell-type proportions, enabling enhanced resolution tissue mapping by imputing cell-type compositions and gene expressions at unmeasured locations. Applications to simulated data and four real datasets showed SDePER's superior accuracy and robustness over existing methods.

Pancreatic ductal adenocarcinoma (PDAC) is notorious for its resistance to various treatment modalities. The genetic heterogeneity of PDAC, coupled with the presence of a desmoplastic stroma within the tumor microenvironment (TME), contributes to an unfavorable prognosis. The mechanisms and consequences of interactions among different cell types, along with spatial variations influencing cellular function, potentially play a role in the pathogenesis of PDAC. Understanding the diverse compositions of the TME and elucidating the functions of microscopic neighborhoods may contribute to understanding the immune microenvironment status in pancreatic cancer. As we delve into the spatial biology of the microscopic neighborhoods within the TME, aiding in deciphering the factors that orchestrate this intricate ecosystem. This overview delineates the fundamental constituents and the structural arrangement of the PDAC microenvironment, highlighting their impact on cancer cell biology.

Tertiary lymphoid structures (TLS) can reflect cancer prognosis and clinical outcomes in various tumour tissues. Tumour-associated macrophages (TAMs) are indispensable components of the tumour microenvironment and play crucial roles in tumour development and immunotherapy. TAMs are associated with TLS induction via the modulation of the T cell response, which is a major component of the TLS. Despite their important roles in cancer immunology, the subtypes of TAMs that influence TLS and their correlation with prognosis are not completely understood. Here, we provide novel insights into the role of TAMs in regulating TLS formation. Furthermore, we discuss the prognostic value of these TAM subtypes and TLS, as well as the current antitumour therapies for inducing TLS. This study highlights an entirely new field of TLS regulation that may lead to the development of an innovative perspective on immunotherapy for cancer treatment.

The presence of tertiary lymphoid structures (TLSs) associated with distinct treatment efficacy and clinical prognosis has been identified in various cancer types. However, the mechanistic roles and clinical implications of TLSs in genitourinary (GU) cancers remain incompletely explored. Despite their potential role as predictive markers described in numerous studies, it is essential to comprehensively evaluate the characteristics of TLSs, including drivers of formation, structural foundation, cellular compositions, maturation stages, molecular features, and specific functionality to maximize their positive impacts on tumor-specific immunity. The unique contributions of these structures to cancer progression and biology have fueled interest in these structures as mediators of antitumor immunity. Emerging data are trying to explore the effects of therapeutic interventions targeting TLSs. Therefore, a better understanding of the molecular and phenotypic heterogeneity of TLSs may facilitate the development of TLSs-targeting therapeutic strategies to obtain optimal clinical benefits for GU cancers in the setting of immunotherapy. In this review, the authors focus on the phenotypic and functional heterogeneity of TLSs in cancer progression, current therapeutic interventions targeting TLSs and the clinical implications and therapeutic potential of TLSs in GU cancers.

After birth, the development of secondary lymphoid tissues (SLTs) in the colon is dependent on the expression of the aryl hydrocarbon receptor (AhR) in immune cells as a response to the availability of AhR ligands. However, little is known about how AhR activity from intestinal epithelial cells (IECs) may influence the development of tertiary lymphoid tissues (TLTs). As organized structures that develop at sites of inflammation or infection during adulthood, TLTs serve as localized centers of adaptive immune responses, and their presence has been associated with the resolution of inflammation and tumorigenesis in the colon. Here, we investigated the effect of the conditional loss of AhR activity in IECs in the formation and immune cell composition of TLTs in a model of acute inflammation. In females, loss of AhR activity in IECs reduced the formation of TLTs without significantly changing disease outcomes or immune cell composition within TLTs. In males lacking AhR expression in IECs, increased disease activity index, lower expression of functional-IEC genes, increased number of TLTs, increased T-cell density, and lower B- to T-cell ratio were observed. These findings may represent an unfavorable prognosis when exposed to dextran sodium sulfate (DSS)-induced epithelial damage compared with females. Sex and loss of IEC AhR also resulted in changes in microbial populations in the gut. Collectively, these data suggest that the formation of TLTs in the colon is influenced by sex and AhR expression in IECs.NEW & NOTEWORTHY This is the first research of its kind to demonstrate a clear connection between biological sex and the development of tertiary lymphoid tissues (TLT) in the colon. In addition, the research finds that in a preclinical model of inflammatory bowel disease, the expression of the aryl hydrocarbon receptor (AhR) influences the development of these structures in a sex-specific manner.

Pancreatic ductal adenocarcinoma (PDAC) is known to have a heterogeneous desmoplastic tumour microenvironment (TME) with a large number of immunosuppressive cells. Recently, high B-cell infiltration in PDAC has received growing interest as a potential therapeutic target.

Our literature review summarises the characteristics of tumour-associated tertiary lymphoid structures (TLSs) and highlight the key studies exploring the clinical outcomes of TLSs in PDAC patients and the direct effect on the TME.

The location, density and maturity stages of TLSs within tumours play a key role in determining the prognosis and is a new emerging target in cancer immunotherapy.

TLS development is imperative to improve the prognosis of PDAC patients. In the future, studying the genetics and immune characteristics of tumour infiltrating B cells and TLSs may lead towards enhancing adaptive immunity in PDAC and designing personalised therapies.

Immune checkpoint blockade (ICB) therapy provides remarkable clinical benefits for multiple cancer types. However, the overall response rate to ICB therapy remains low in esophageal squamous cell carcinoma (ESCC). This study aimed to identify biomarkers of ICB therapy for ESCC and interrogate its potential clinical relevance.

We investigated gene expression in 42 treatment-naïve ESCC tumor tissues and identified differentially expressed genes, tumor-infiltrating lymphocytes and immune-related genes signatures associated with differential immunotherapy responses. We systematically assessed the tumor microenvironment using the NanoString GeoMx digital spatial profiler, single-cell RNA-seq and multiplex immunohistochemistry in ESCC. Finally, we evaluated the associations between HLA-A-positive tertiary lymphoid structures (TLSs) and patients' responses to ICB in 60 ESCC patients.

Tumor infiltrating B lymphocytes and several immune-related gene signatures, such as the antigen presenting machinery (APM) signature, are significantly elevated in ICB treatment responders. Multiplex immunohistochemistry identified the presence of HLA-A+ TLSs and showed that TLS-resident cells increasingly express HLA-A as TLSs mature. Most TLS-resident HLA-A+ cells are tumor-infiltrating T (TIL-T) or tumor-infiltrating B (TIL-B) lymphocytes. Digital spatial profiling of spatially distinct TIL-T lymphocytes and single-cell RNA-seq data from 60 ESCC tumor tissues revealed that CXCL13-expressing exhausted TIL-Ts inside TLSs are reactivated with elevated expression of the APM signature as TLSs mature. Finally, we demonstrated that HLA-A+ TLSs and their major cellular components, TIL-Ts and TIL-Bs, are associated with a clinical benefit from ICB treatment for ESCC.

HLA-A+ TLSs are present in ESCC tumor tissues. TLS-resident TIL-Ts with elevated expression of the APM signature may be reactivated. HLA-A+ TLSs and their major cellular components, TIL-Ts and TIL-Bs, may serve as biomarkers for ICB-treated ESCC patients.

Tertiary lymphoid structures (TLS) is a particular component of tumor microenvironment (TME). However, its biological mechanisms in colorectal cancer (CRC) have not yet been understood. We desired to reveal the TLS gene signature in CRC and evaluate its role in prognosis and immunotherapy response.

The data was sourced from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. Based on TLS-related genes (TRGs), the TLS related subclusters were identified through unsupervised clustering. The TME between subclusters were evaluated by CIBERSORT and xCell. Subsequently, developing a risk model and conducting external validation. Integrating risk score and clinical characteristics to create a comprehensive nomogram. Further analyses were conducted to screen TLS-related hub genes and explore the relationship between hub genes, TME, and biological processes, using random forest analysis, enrichment and variation analysis, and competing endogenous RNA (ceRNA) network analysis. Multiple immunofluorescence (mIF) and immunohistochemistry (IHC) were employed to characterize the existence of TLS and the expression of hub gene.

Two subclusters that enriched or depleted in TLS were identified. The two subclusters had distinct prognoses, clinical characteristics, and tumor immune infiltration. We established a TLS-related prognostic risk model including 14 genes and validated its predictive power in two external datasets. The model's AUC values for 1-, 3-, and 5-year overall survival (OS) were 0.704, 0.737, and 0.746. The low-risk group had a superior survival rate, more abundant infiltration of immune cells, lower tumor immune dysfunction and exclusion (TIDE) score, and exhibited better immunotherapy efficacy. In addition, we selected the top important features within the model: VSIG4, SELL and PRRX1. Enrichment analysis showed that the hub genes significantly affected signaling pathways related to TLS and tumor progression. The ceRNA network: PRRX1-miRNA (hsa-miR-20a-5p, hsa-miR-485-5p) -lncRNA has been discovered. Finally, IHC and mIF results confirmed that the expression level of PRRX1 was markedly elevated in the TLS- CRC group.

We conducted a study to thoroughly describe TLS gene signature in CRC. The TLS-related risk model was applicable for prognostic prediction and assessment of immunotherapy efficacy. The TLS-hub gene PRRX1, which had the potential to function as an immunomodulatory factor of TLS, could be a therapeutic target for CRC.

Immunotherapy profoundly changed the landscape of cancer therapy by providing long-lasting responses in subsets of patients and is now the standard of care in several solid tumor types. However, immunotherapy activity beyond conventional immune checkpoint inhibition is plateauing, and biomarkers are overall lacking to guide treatment selection. Most studies have focused on T cell engagement and response, but there is a growing evidence that B cells may be key players in the establishment of an organized immune response, notably through tertiary lymphoid structures. Mechanisms of B cell response include antibody-dependent cellular cytotoxicity and phagocytosis, promotion of CD4+ and CD8+ T cell activation, maintenance of antitumor immune memory. In several solid tumor types, higher levels of B cells, specific B cell subpopulations, or the presence of tertiary lymphoid structures have been associated with improved outcomes on immune checkpoint inhibitors. The fate of B cell subpopulations may be widely influenced by the cytokine milieu, with versatile roles for B-specific cytokines B cell activating factor and B cell attracting chemokine-1/CXCL13, and a master regulatory role for IL-10. Roles of B cell-specific immune checkpoints such as TIM-1 are emerging and could represent potential therapeutic targets. Overall, the expanding field of B cells in solid tumors of holds promise for the improvement of current immunotherapy strategies and patient selection.

The presence of tertiary lymphoid structures (TLSs) on pancreatic pathological images is an important prognostic indicator of pancreatic tumors. Therefore, TLSs detection on pancreatic pathological images plays a crucial role in diagnosis and treatment for patients with pancreatic tumors. However, fully supervised detection algorithms based on deep learning usually require a large number of manual annotations, which is time-consuming and labor-intensive. In this paper, we aim to detect the TLSs in a manner of few-shot learning by proposing a weakly supervised segmentation network. We firstly obtain the lymphocyte density maps by combining a pretrained model for nuclei segmentation and a domain adversarial network for lymphocyte nuclei recognition. Then, we establish a cross-scale attention guidance mechanism by jointly learning the coarse-scale features from the original histopathology images and fine-scale features from our designed lymphocyte density attention. A noise-sensitive constraint is introduced by an embedding signed distance function loss in the training procedure to reduce tiny prediction errors. Experimental results on two collected datasets demonstrate that our proposed method significantly outperforms the state-of-the-art segmentation-based algorithms in terms of TLSs detection accuracy. Additionally, we apply our method to study the congruent relationship between the density of TLSs and peripancreatic vascular invasion and obtain some clinically statistical results.

Tertiary lymphoid structures (TLS) are lymph node-like aggregates that can form in association with chronic inflammation or cancer. Mature TLS are organized into B and T cell zones, and are not encapsulated but include all cell types necessary for eliciting an adaptive immune response. TLS have been observed in various cancer types and are generally associated with a positive prognosis as well as increased sensitivity to cancer immunotherapy. However, a comprehensive understanding of the roles of TLS in eliciting anti-tumor immunity as well as the mechanisms involved in their formation and function is still lacking. Further studies in orthotopic, immunocompetent cancer models are necessary to evaluate the influence of TLS on cancer therapies, and to develop new treatments that promote their formation in cancer. Here, we review key insights obtained from functional murine studies, discuss appropriate models that can be used to study cancer-associated TLS, and suggest guidelines on how to identify TLS and distinguish them from other antigen-presenting niches.

Lung adenocarcinoma (LUAD) seriously threatens people's health worldwide. Programmed cell death (PCD) plays a critical role in regulating LUAD growth and metastasis as well as in therapeutic response. However, currently, there is a lack of integrative analysis of PCD-related signatures of LUAD for accurate prediction of prognosis and therapeutic response.

The bulk transcriptome and clinical information of LUAD were obtained from TCGA and GEO databases. A total of 1382 genes involved in regulating 13 various PCD patterns (apoptosis, necroptosis, pyroptosis, ferroptosis, cuproptosis, netotic cell death, entotic cell death, lysosome-dependent cell death, parthanatos, autophagy-dependent cell death, oxeiptosis, alkaliptosis and disulfidptosis) were included in the study. Weighted gene co-expression network analysis (WGCNA) and differential expression analysis were performed to identify PCD-associated differential expression genes (DEGs). An unsupervised consensus clustering algorithm was used to explore the potential subtypes of LUAD based on the expression profiles of PCD-associated DEGs. Univariate Cox regression analysis, Least Absolute Shrinkage and Selection Operator (LASSO) regression, Random Forest (RF) analysis and stepwise multivariate Cox analysis were performed to construct a prognostic gene signature. The "oncoPredict" algorithm was utilized for drug-sensitive analysis. GSVA and GSEA were utilized to perform function enrichment analysis. MCPcounter, quanTIseq, Xcell and ssGSEA algorithms were used for tumor immune microenvironment analysis. A nomogram incorporating PCDI and clinicopathological characteristics was established to predict the prognosis of LUAD patients.

Forty PCD-associated DEGs related to LUAD were obtained by WGCNA analysis and differential expression analysis, followed by unsupervised clustering to identify two LUAD molecular subtypes. A programmed cell death index (PCDI) with a five-gene signature was established by machine learning algorithms. LUAD patients were then divided into a high PCDI group and a low PCDI group using the median PCDI as a cutoff. Survival and therapeutic analysis revealed that the high PCDI group had a poor prognosis and was more sensitive to targeted drugs but less sensitive to immunotherapy compared to the low PCDI group. Further enrichment analysis showed that B cell-related pathways were significantly downregulated in the high PCDI group. Accordingly, the decreased tumor immune cell infiltration and the lower tumor tertiary lymphoid structure (TLS) scores were also found in the high PCDI group. Finally, a nomogram with reliable predictive performance PCDI was constructed by incorporating PCDI and clinicopathological characteristics, and a user-friendly online website was established for clinical reference ( https://nomogramiv.shinyapps.io/NomogramPCDI/ ).

We performed the first comprehensive analysis of the clinical relevance of genes regulating 13 PCD patterns in LUAD and identified two LUAD molecular subtypes with distinct PCD-related gene signature which indicated differential prognosis and treatment sensitivity. Our study provided a new index to predict the efficacy of therapeutic interventions and the prognosis of LUAD patients for guiding personalized treatments.

CD4 and CD8 T cells are key players in the immune response against both pathogenic infections and cancer. CD4 T cells provide help to CD8 T cells via multiple mechanisms, including licensing dendritic cells (DCs), co-stimulation, and cytokine production. During acute infection and vaccination, CD4 T cell help is important for the development of CD8 T cell memory. However, during chronic viral infection and cancer, CD4 helper T cells are critical for the sustained effector CD8 T cell response, through a variety of mechanisms. In this review, we focus on T cell responses in conditions of chronic Ag stimulation, such as chronic viral infection and cancer. In particular, we address the significant role of CD4 T cell help in promoting effector CD8 T cell responses, emerging techniques that can be utilized to further our understanding of how these interactions may take place in the context of tertiary lymphoid structures, and how this key information can be harnessed for therapeutic utility against cancer.

The active form of vitamin D is the hormonally active 1,25(OH)2D3 (Vit D) vitamin, which plays an important role in bone biology and host immunity. The vitamin D receptor (VDR) is a nuclear ligand-dependent transcription factor expressed by many cells. Ligation of VDR by VitD regulates a wide plethora of genes and physiologic functions through the formation of the complex Vit D-VDR signaling cascade. The influence of Vit D-VDR signaling in host immune response to microbial infection has been of interest to many researchers. This is particularly important in oral health and diseases, as oral mucosa is exposed to a complex microbiota, with certain species capable of causing disruption to immune homeostasis. In this review, we focus on the immune modulatory roles of Vit D in the bone degenerative oral disease, periodontitis.

Tertiary lymphoid structures (TLSs) are clusters of lymphoid cells with an organization that resembles that of secondary lymphoid organs. Both structures share common developmental characteristics, although TLSs usually appear in chronically inflamed non-lymphoid tissues, such as tumors. TLSs contain diverse types of immune cells, with varying degrees of spatial organization that represent different stages of maturation. These structures support both humoral and cellular immune responses, thus the correlation between the existence of TLS and clinical outcomes in cancer patients has been extensively studied. The finding that TLSs are associated with better prognosis in some types of cancer has led to the design of therapeutic strategies based on promoting the formation of these structures. Agents such as chemokines, cytokines, antibodies and cancer vaccines have been used in combination with traditional antitumor treatments to enhance TLS generation, with good results. The induction of TLS formation therefore represents a novel and promising avenue for the treatment of a number of tumor types.

Exosomes (exos) contain molecular cargo of therapeutic and diagnostic value for cancers and other inflammatory diseases, but their therapeutic potential for periodontitis (PD) remains unclear. Dendritic cells (DCs) are the directors of immune response and have been extensively used in immune therapy. We previously reported in a mouse model of PD that custom murine DC-derived exo subtypes could reprogram the immune response toward a bone-sparing or bone-loss phenotype, depending on immune profile. Further advancement of this technology requires the testing of human DC-based exos with human target cells. Our main objective in this study is to test the hypothesis that human monocyte-derived dendritic cell (MoDC)-derived exos constitute a well-tolerated and effective immune therapeutic approach to modulate human target DC and T cell immune responses in vitro. MoDC subtypes were generated with TGFb/IL-10 (regulatory (reg) MoDCs, CD86lowHLA-DRlowPDL1high), E. coli LPS (stimulatory (stim) MoDCs, CD86highHLA-DRhighPDL1low) and buffer (immature (i) MoDCs, CD86lowHLA-DRmedPDL1low). Exosomes were isolated from different MoDC subtypes and characterized. Once released from the secreting cell into the surrounding environment, exosomes protect their prepackaged molecular cargo and deliver it to bystander cells. This modulates the functions of these cells, depending on the cargo content. RegMoDCexos were internalized by recipient MoDCs and induced upregulation of PDL1 and downregulation of costimulatory molecules CD86, HLADR, and CD80, while stimMoDCexos had the opposite influence. RegMoDCexos induced CD25+Foxp3+ Tregs, which expressed CTLA4 and PD1 but not IL-17A. In contrast, T cells treated with stimMoDCexos induced IL-17A+ Th17 T cells, which were negative for immunoregulatory CTLA4 and PD1. T cells and DCs treated with iMoDCexos were immune 'neutral', equivalent to controls. In conclusion, human DC exos present an effective delivery system to modulate human DC and T cell immune responses in vitro. Thus, MoDC exos may present a viable immunotherapeutic agent for modulating immune response in the gingival tissue to inhibit bone loss in periodontal disease.

Colorectal carcinoma (CRC) represents a lethal disease with heterogeneous outcomes. Only patients with mismatch repair (MMR) deficient CRC showing microsatellite instability and hyper-mutated tumors can obtain clinical benefits from current immune checkpoint blockades; on the other hand, immune- or target-based therapeutic strategies are very limited for subjects with mismatch repair proficient CRC (CRC^pMMR). Here, we report a comprehensive typing of immune infiltrating cells in CRC^pMMR. We also tested the expression and interferon-γ-modulation of PD-L1/CD274. Relevant findings were subsequently validated by immunohistochemistry on fixed materials. CRC^pMMR contain a significantly increased fraction of CD163⁺ macrophages (TAMs) expressing TREM2 and CD66⁺ neutrophils (TANs) together with decrease in CD4^-CD8^-CD3⁺ double negative T lymphocytes (DNTs); no differences were revealed by the analysis of conventional and plasmacytoid dendritic cell populations. A fraction of tumor-infiltrating T-cells displays an exhausted phenotype, co-expressing PD-1 and TIM-3. Remarkably, expression of PD-L1 on fresh tumor cells and TAMs was undetectable even after in vitro stimulation with interferon-γ. These findings confirm the immune suppressive microenvironment of CRC^pMMR characterized by dense infiltration of TAMs, occurrence of TANs, lack of DNTs, T-cell exhaustion, and interferon-γ unresponsiveness by host and tumor cells. Appropriate bypass strategies should consider these combinations of immune escape mechanisms in CRC^pMMR.

Follicular regulatory T (Tfr) cells are a novel and unique subset of effector regulatory T (Treg) cells that are located in germinal centers (GCs). Tfr cells express transcription profiles that are characteristic of both follicular helper T (Tfh) cells and Treg cells and negatively regulate GC reactions, including Tfh cell activation and cytokine production, class switch recombination and B cell activation. Evidence also shows that Tfr cells have specific characteristics in different local immune microenvironments. This review focuses on the regulation of Tfr cell differentiation and function in unique local immune microenvironments, including the intestine and tumor.

Tumor-infiltrating lymphocytes (TILs) have long been recognized as playing an important role in tumor immune microenvironment. Lately, the Immunoscore (IS) has been proposed as a new method of quantifying the number of TILs in association with patient survival in several cancer types.

In 300 preoperatively untreated esophageal cancer (EC) patients who underwent curative resection at two different institutes, immunohistochemical staining using CD3 and CD8 antibodies was performed to evaluate IS, as objectively scored by auto-counted TILs in the tumor core and invasive margin. In addition, in pre-neoadjuvant chemotherapy (pre-NAC) endoscopic biopsies of a different cohort of 146 EC patients who received NAC, CD3, and CD8 were immunostained to evaluate TIL density.

In all cases, the IS-high (score 3-4) group tended to have better survival [5-year overall survival (OS) of the IS-high vs low group: 77.6 vs 65.8%, P = 0.0722] than the IS-low (score 1-2) group. This trend was more remarkable in cStage II-IV patients (70.2 vs 54.5%, P = 0.0208) and multivariate analysis of OS further identified IS (hazard ratio 2.07, P = 0.0043) to be an independent prognostic variable. In preNAC biopsies, NAC-responders had higher densities than non-responders of both CD3 + ( P = 0.0106) and CD8 + cells ( P = 0.0729) and, particularly CD3 + cell density was found to be an independent prognostic factor (hazard ratio 1.75, P = 0.0169).

The IS signature in surgical specimens and TIL density in preNAC- biopsies could be predictive markers of clinical outcomes in EC patients.

Tertiary lymphoid structures (TLSs) are ectopic lymphocyte aggregates that form at sites of chronic inflammation, including cancers, in non-lymphoid tissues. Although the formation of TLSs is similar to that of secondary lymphoid organs, the pathogenic factors leading to TLS formation in cancerous tissues and the mechanisms underlying the role of these structures in the intra-tumoral adaptive antitumor immune response are not fully understood. The presence of TLSs may impact patient prognosis and treatment outcomes. This review examines the current understanding of TLSs in cancers, including their composition and formation as well as their potential to predict prognosis and therapeutic efficacy. We also summarize strategies to induce TLS formation for cancer treatment.

Despite advances in therapy over the past decades, metastatic colorectal cancer (mCRC) remains a highly morbid disease. While the impact of MHC-I on immune infiltration in mCRC has been well studied, data on the consequences of MHC-II loss are lacking. Multiplex fluorescent immunohistochemistry (mfIHC) was performed on 149 patients undergoing curative intent resection for mCRC and stratified into high and low human leukocyte antigen isotype DR (HLA-DR) expressing tumors. Intratumoral HLA-DR expression was found in stromal bands, and its expression level was associated with different infiltrating immune cell makeup and distribution. Low HLA-DR expression was associated with increased intercellular distances and decreased population mixing of T helper cells and antigen-presenting cells (APC), suggestive of decreased interactions. This was associated with less co-localization of tumor cells and cytotoxic T lymphocytes (CTLs), which tended to be in a less activated state as determined by Ki67 and granzyme B expression. These findings suggest that low HLA-DR in the tumor microenvironment of mCRC may reflect a state of poor helper T-cell interactions with APCs and CTL-mediated anti-tumor activity. Efforts to restore/enhance MHC-II presentation may be a useful strategy to enhance checkpoint inhibition therapy in the future.

Intraductal papillary mucinous neoplasms (IPMN) are bona fide precursors to pancreatic ductal adenocarcinoma (PDAC). While genomic alterations during multistep IPMN progression have been well cataloged, the accompanying changes within the tumor immune microenvironment (TIME) have not been comprehensively studied. Herein, we investigated TIME-related alterations during IPMN progression, using multiplex immunofluorescence (mIF) coupled with high-resolution image analyses.

Two sets of formalin-fixed, paraffin-embedded tissue samples from surgically resected IPMNs were analyzed. The training set of 30 samples consisted of 11 low-grade IPMN (LG-IPMN), 17 high-grade IPMN (HG-IPMN), and 2 IPMN with PDAC, while a validation set of 93 samples comprised of 55 LG-IPMN and 38 HG-IPMN. The training set was analyzed with two panels of immuno-oncology-related biomarkers, while the validation set was analyzed with a subset of markers found significantly altered in the training set.

Cell types indicative of enhanced immune surveillance, including cytotoxic and memory T cells, and antigen-experienced T cells and B cells, were all found at higher densities within isolated LG-IPMNs compared with HG-IPMNs. Notably, the TIME of LG-IPMNs that had progressed at the time of surgical resection (progressor LGD) resembled that of the synchronous HG-IPMNs, underscoring that attenuated immune surveillance occurs even in LG-IPMNs destined for progression.

Our findings provide a basis for interception of cystic neoplasia to PDAC, through maintenance of sustained immune surveillance using vaccines and other prevention approaches.

Lymphoid follicles (LFs) are responsible for generation of adaptive immune responses in secondary lymphoid organs and form ectopically during chronic inflammation. A human model of ectopic LF formation will provide a tool to understand LF development and an alternative to non-human primates for preclinical evaluation of vaccines. Here, it is shown that primary human blood B- and T-lymphocytes autonomously assemble into ectopic LFs when cultured in a 3D extracellular matrix gel within one channel of a two-channel organ-on-a-chip microfluidic device. Superfusion via a parallel channel separated by a microporous membrane is required for LF formation and prevents lymphocyte autoactivation. These germinal center-like LFs contain B cells expressing Activation-Induced Cytidine Deaminase and exhibit plasma cell differentiation upon activation. To explore their utility for seasonal vaccine testing, autologous monocyte-derived dendritic cells are integrated into LF Chips. The human LF chips demonstrate improved antibody responses to split virion influenza vaccination compared to 2D cultures, which are enhanced by a squalene-in-water emulsion adjuvant, and this is accompanied by increases in LF size and number. When inoculated with commercial influenza vaccine, plasma cell formation and production of anti-hemagglutinin IgG are observed, as well as secretion of cytokines similar to vaccinated humans over clinically relevant timescales.