Distinct spatial organization governs oral mucosal immunity.

Who am I? (re-)Defining fibroblast identity and immunological function in the age of bioinformatics.

Mucosal sites represent the primary routes of HIV transmission, yet the oral mucosa appears uniquely resistant to HIV-1 infection. Since the oral cavity is not conducive to either infection or transmission of HIV, characterizing the responsible resistance factors, particularly components of innate immunity, and their mechanisms of action may identify new opportunities for interference with viral acquisition at other mucosal sites. Although many of the innate immune factors described to date exhibit both antibacterial and antiviral activities, molecules that inhibit HIV-1 are largely HIV-1 specific. Nonetheless, if these oral innate factors can be identified, characterized, and harnessed, they may be useful in protection and/or intervention in acute HIV-1 infection. Endogenous molecules acting independently or synergistically, given their lack of substantive side effects, may represent an alternative novel approach for reducing HIV risk and suppressing infection. While HIV infection via breast milk does involve oral trafficking and is an important area of study, it will only be briefly discussed here. The major emphasis of this article is on the oral cavity and salivary substances that can influence HIV infection Epidemiology of oral transmission of HIV More than twenty-five years into the global HIV/AIDS epidemic, it is well established that in addition to blood, the reproductive and/or rectal mucosae represent the primary portals of HIV entry in adults. There is less definitive evidence regarding whether other mucosal sites serve as viral conduits and in particular, uncertainty remains as to the possibility of transmission of HIV-1 via the oral entrance to the gastrointestinal (GI) tract. This is not a trivial concern, since despite the successes of combination therapy for HIV/AIDS, infection rates remain high, compounded by persistence of latent viral reservoirs, antiretroviral drug resistance, and the lack of an effective vaccine or potential cure. Although it is often accepted that oral transmission is of little or no consequence, we have failed to capitalize on that information to identify the mechanism(s) of protection at the oral mucosal surface. These mechanisms could provide clues for novel strategies of prevention/intervention at other more vulnerable viral entry sites. Early in the epidemic, evidence of the presence of HIV-1 in the oral cavity suggested the possibility of transmissibility via this route [1, 2]. Whereas in some early studies [3–7], oral transmission could not be established, a few studies implicated this route as a potential, even likely mode of transmission (e.g., [8]). In larger cohorts of individuals and in well-designed studies to monitor oral transmission, the rarity of HIV-1 infection by this route was substantiated [9]. Nonetheless, potential shortcomings in these studies, including accurately documenting the definitive route of infection, underscore our lack of certainty regarding the relative risk of oral transmission. (Reviews: [10, 11]). The low frequency of documented oral transmission of HIV-1 could reflect low titers of infectious virus in saliva. While high levels of HIV-1 RNA and antigens can routinely be measured in oral fluids, infectious virus was not detected in early studies attempting to isolate infectious virus from saliva [12, 13] or more recently in large cohort studies, despite plasma viremia [14, 15].

4. Gastrointestinal mucosal immunity

BackgroundTertiary Lymphoid Structures (TLS) are highly organized ectopic lymphoid structures found in inflamed or tumor tissues, acting as sites of lymphoid recruitment and immune activation. A high TLS density within the tumor is commonly associated with an increased prognostic effect of TILs and with an improved disease free survival and overall survival for patients.1 However, the existence of conflicting studies suggest that multiple TLS features should be taken into account when assessing their prognostic value, such as their location, cellular composition, maturation stage and spatial organisation, as those may affect their functionalities.2MethodsWith the aim of gaining insights into TLS biology and evaluating the prognostic role of TLS in Non-Small Cell Lung Carcinoma according to their multiple features, we developed a TLS multiplex immunofluorescent (mIF) panel that includes T cells (CD3, CD8), B cells (CD20), Follicular Dendritic cells (CD21, CD23) and mature dendritic cells (DC-LAMP) markers. We deployed this panel across a cohort of primary tumors from NSCLC patients (n=408) and established a mIF image analysis workstream to assess the status and spatial location of each cell within the tissue. A H&E staining of the same tissue section was performed to evaluate mIF spatial data in relation to the tumor context. Additional multi-omics assessments were conducted across the same cohort including; whole exome sequencing, NanoString transcriptomics, and immunohistochemistry (e.g. PD-L1, FOXP3, NKp46, LKB1, CTLA4). We have leveraged clinical metadata, including demographics (e.g. age, sex, smoking status) and clinical risk factors (e.g. stage, grade, Standard of Care treatment) with clinical follow up (e.g. OS, PFS) for prevalence analysis, novel biomarker identification, and survival association.ResultsAssessment of the prevalence of each cell phenotype within the tumor tissue and TLS, the cell-cell interactions, the distance between each cell type, and the distance of non-TLS immune cells to the closest TLS will be described, demonstrating the different types of lymphoid aggregates and TLS and their functional status. An integrative analysis combining spatial biology data with multi-omics and clinical data will be presented evaluating the prognostic value of TLS composition, maturation status and spatial organization, in correlation with additional biomarkers and clinical characteristics.ConclusionsThis exploratory study using cutting-edge technologies enables us to better understand how TLS orchestrate an organised anti-tumour response, defining TLS spatial biomarker signatures, TLS gene signatures, and TLS features associated with patient outcomes to evaluate in the clinic.ReferencesMarie-Caroline Dieu-Nosjean, Jérémy Goc, Nicolas A Giraldo, Catherine Sautès-Fridman, Wolf Herman Fridman. Tertiary lymphoid structures in cancer and beyond. Trends Immunol 2014;35(11):571–580.Catherine Sautès-Fridman, Florent Petitprez, Julien Calderaro, Wolf Herman Fridman. Tertiary lymphoid structures in the era of cancer immunotherapy. Nat Rev Cancer 2019;19(6):307–325.Ethics ApprovalThe study was approved by AstraZeneca.

The maintenance of barrier function at exposed surfaces of the mammalian body is essential for limiting exposure to environmental stimuli, preventing systemic dissemination of commensal and pathogenic microbes and retaining normal homeostasis of the entire body. Indeed, dysregulated barrier function is associated with many infectious and inflammatory diseases, including psoriasis, influenza, inflammatory bowel disease and human immunodeficiency virus, which collectively afflict millions of people worldwide. Studies have shown that interleukin 22 (IL-22) is expressed at barrier surfaces and that its expression is dysregulated in certain human diseases, which suggests a critical role in the maintenance of normal barrier homeostasis. Consistent with that, studies of mouse model systems have identified a critical role for signaling by IL-22 through its receptor (IL-22R) in the promotion of antimicrobial immunity, inflammation and tissue repair at barrier surfaces. In this review we will discuss how the expression of IL-22 and IL-22R is regulated, the functions of the IL-22-IL-22R pathway in regulating immunity, inflammation and tissue homeostasis, and the therapeutic potential of targeting this pathway in human disease.

Lymphotoxin signalling in tertiary lymphoid structures and immunotherapy.

Immunity at the Barriers

Pancreatic ductal adenocarcinoma (PDAC) is a cancer with low survival rates and currently no immunotherapy options. However, many tumors from PDAC patients can be heavily infiltrated with T and B cells associated with favorable survival outcomes indicating anti-tumor immunity is functional in certain patients. In some of these lymphocyte-inflamed PDAC tumors, spontaneous organization of tertiary lymphoid structures (TLS) is evident upon surgical resection and these patients have a clear survival advantage. TLS are predictive of immune checkpoint blockade response in some cancer types, yet PDAC patients, with or without TLS, are still insensitive to these strategies. To address this paradox, we evaluated surgically resected PDAC tumors from previously untreated patients and discovered that tumors with TLS (TLS+) recruited T and B cells with specific anti-tumor and memory phenotypes suggesting immunosurveillance of disease was enhanced. Furthermore, a subset of TLS+ patients contained germinal centers and improved humoral immune function corresponding to an increased neoantigen burden and long-term survival. When assessing gene expression differences between TLS+ and TLS- patients, operable inflammatory and immunosuppressive pathways emerged that could be therapeutically exploited to activate TLS neogenesis. To address these mechanistic questions, we have developed a method for inducing TLS formation in implantable mouse PDAC tumors in the orthotopic setting utilizing an antibody that agonizes the lymphotoxin beta receptor (LTBR) in mice bearing established PDAC tumors. These TLS are complexed with T cells, B cells, CXCL13+ cells, and PNAd+ HEV associated with reduced tumor growth, presence of lymphocyte-recruiting cancer associated fibroblasts and increases in anti-tumor T and B cell phenotypes. Interestingly, some mouse PDAC cell lines are resistant to TLS formation by LTBR agonism while others are susceptible offering a model to study patient heterogeneity. We will use this TLS+ PDAC mouse model to address how TLS may directly improve antigen-specific T cell and B cell immunity and elucidate the pathways that regulate TLS formation. These data will offer new strategies to overcome immunotherapy resistance in cancer patients. Citation Format: Shrijan Khanal, Olivia Harder, Elijah Kirschstein, Morgan Mack, Carl Ware, Michael Gough, Kristina Young, Andrew J. Gunderson. Tertiary lymphoid structures in pancreatic cancer: Biomarkers of immunogenic patients or direct contributors to anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7464.

Biological functions of melatonin in relation to pathogenesis of oral lichen planus

Tertiary lymphoid structures (TLSs) are ectopic immune cell aggregations that develop in peripheral tissues in response to a wide range of chronic inflammatory conditions, including infection, autoimmune disease, and cancer. In the tumor microenvironment (TME), the structures of TLSs, including B-cell- and T-cell-enriched areas indicate that the TLSs might be the local site during the initiation and maintenance of humoral and cellular immune responses against cancers. Numerous studies have evaluated the expression of TLSs in different cancer patients and their association with prognoses of cancer patients. It was shown that well-developed TLSs characterized by mature B cells synthesized tumor specific antibodies, which were considered as specific markers for a good prognosis. However, there are still some immunosuppressive factors existing in the TLSs that may affect anti-tumor responses. These factors include dysfunctional B cells, regulatory T cells, and T follicular regulatory cells. The complexity and heterogeneity of the TLS composition may affect the function and activity of TLSs; it is therefore essential to fully understand the function and influencing factors in TLSs. It has been reported that checkpoint inhibitors and vaccines are currently being developed to reprogram the TME by establishing mature TLSs to improve cancer immunotherapies. In this review, we focused on recent advances in TLSs in human solid tumors, including structural characteristics and classes, antitumor mechanisms, immunosuppressive factors, and TLS-based therapeutic approaches.

Tumor-infiltrating lymphocytes (TIL) have been associated with good clinical outcomes in numerous large clinical trials for HER2-positive (HER2+) and triple-negative (TN) breast cancer (BC). Our previous work in human demonstrated that TIL can organize in tertiary lymphoid structures (TLS) with the B-cell chemoattractant, CXCL13, playing a key role in their formation as well as having a strong association with positive clinical outcomes. The present study investigated how TLS functionally contribute to immune responses in human BC. We prospectively collected fresh primary human BC tissues and prepared enzyme-free homogenates to produce TIL suspensions and tumor supernatants for flow cytometry and cytokine/chemokine/immunoglobulin (Ig) quantification, respectively. Matching formalin-fixed, paraffin-embedded tumor tissues were used for spatial analysis by dual immunohistochemistry and immunofluorescent confocal microscopy. We show that CD3+CD4+, CD3+CD8+ and CD19+ TIL in human BC contain subpopulations expressing CXCR5 (the CXCL13 receptor) and are co-localized in TLS. An in vitro functional assay activating Tfh cells with allogeneic B cells from human spleen evaluated the functionality of human BC Tfh TIL (CD45+CD3+CD4+CXCR5+). These experiments reveal that ICOS+PD-1hi Tfh TIL from TN and HER2+, but not luminal, BC possess helper activities for B cell IgG production. This observation is supported by a correlation between ICOS+PD-1hi Tfh TIL densities and IgG concentrations in primary human BC supernatants. A comparison of ICOS+PD-1hi and ICOS-PD-1- Tfh TIL confirms the activated, functional nature of these specialized TIL, which are characterized by high IL-21, IL-10 and CXCL13 mRNA expression. High IFNγ mRNA expression was detected in ICOS+PD-1hi Tfh TIL, suggesting their functional Th1 orientation. We also identified follicular regulatory T cells (Tfr), located in TLS that express CXCR5, CD25, demethylated Foxp3 and GARP, a marker of TGFβ activity. Tfr regulation of Tfh functions were evaluated by determining the ratio between functional Tfh TIL and GARP+ Tfr TIL. This analysis reveals a strong correlation between the Tfh/Tfr ratio and IgG production in primary tumor supernatants from TN and HER2+ human BC. In addition, strong correlations were observed with IgA and IgM production in HER2+ human BC. Our data show that Tfh TIL in human BC are major contributors to TLS activity, with the ratio between functional Tfh TIL and activated Tfr TIL directly influencing Ig production by B cells. Effective antitumor immunity in BC is therefore not only characterized by TIL density and organization but also by the balance between effector and regulatory TIL. These findings constitute important elements to consider when choosing patients for immunotherapy. Citation Format: Grégory Noël, Mireille Langouo, Soizic Garaud, Anaïs Boisson, Hugues Duvillier, Gert Van den Eynden, Denis Larsimont, Karen Willard-Gallo. The balance between activated follicular helper T cells and follicular regulatory T cells within tertiary lymphoid structures guides antitumor immune responses in human breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A40.

The oral cavity is inhabited by a wide spectrum of microbial species, and their colonization is mostly based on commensalism. These microbes are part of the normal oral flora, but there are also opportunistic species that can cause oral and systemic diseases. Although there is a strong exposure to various microorganisms, the oral mucosa reduces the colonization of microorganisms with high rotation and secretion of various types of cytokines and antimicrobial proteins such as defensins. In some circumstances, the imbalance between normal oral flora and pathogenic flora may lead to a change in the ratio of commensalism to parasitism. Healthy oral mucosa has many important functions. Thanks to its integrity, it is impermeable to most microorganisms and constitutes a mechanical barrier against their penetration into tissues. Our study aims to present the role and composition of the oral cavity microbiota as well as defense mechanisms within the oral mucosa which allow for maintaining a balance between such numerous species of microorganisms. We highlight the specific aspects of the oral mucosa protecting barrier and discuss up-to-date information on the immune cell system that ensures microbiota balance. This study presents the latest data on specific tissue stimuli in the regulation of the immune system with particular emphasis on the resistance of the gingival barrier. Despite advances in understanding the mechanisms regulating the balance on the microorganism/host axis, more research is still needed on how the combination of these diverse signals is involved in the regulation of immunity at the oral mucosa barrier.

Pancreatic adenocarcinoma (PAAD) is one of the most malignancies with a poor prognosis. The immune phenotype of pancreatic cancer is remarkably complex, presenting a significant challenge to the efficacy of immunotherapy. Tertiary lymphoid structures (TLSs) are active zones composed of various immune cells, which reflect immune responses against cancer. TLSs have emerged as critical biomarkers for predicting immunotherapy outcomes, potentially reflecting alterations in the metabolic characteristics of the host. Accumulating evidence indicates that 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) may shed light on cancer immunology. The tumor environment is known to be associated with the metabolic activity and antitumor immunity of immune cells. A total of 266 patients with PAAD who underwent 18F-FDG PET/CT followed by pancreatectomy were enrolled in this study, and the presence of TLSs was identified in 66 patients by HE staining, and fluorescent multiplex immunohistochemistry (mIHC) was employed to analyze the cellular composition of TLSs. Elevated maximum standard uptake values (SUVmax) showed significant correlation with TLS presence in PAAD patients. However, SUVmax value showed no correlation with overall survival in PAAD patients. Immunohistochemistry (IHC) staining demonstrated a statistically significant correlation between higher SUVmax levels and increased CD4 + and CD8 + tumor-infiltrating lymphocytes. In PAAD patients with TLS, CD4⁺ T-cell infiltration levels showed a positive correlation with SUVmax. In our study, a TLS signature containing 12-chemokine was proposed to determine high TLS score and low TLS score group. The high TLS score group exhibited increased infiltration of naïve/memory/activated B cells, central memory/activated CD4⁺/CD8⁺ T cells, Tregs, Th1/Th2/Th17 subsets, and neutrophils. Furthermore, GLUT3 and GLUT10 expression consistently correlated with TLS scores in both TCGA and CPTAC cohorts. GLUT3 and GLUT10 were found to be upregulated in the high TLS score group. A more inflamed immune infiltrative landscape was characterized in the high TLS score group with a high proportion of multiple immune cells. Increased FDG uptake is often induced by overexpression of glucose transporters (GLUT) and glucometabolic genes. Overexpression of GLUT3 and GLUT10 was found to be positively correlated with higher infiltration levels as well as immune activation of CD4+ and CD8+ T cells. To conclude, our study showed an association between SUVmax on 18F-FDG PET/CT and TLS presence in PAAD. FDG uptake may reflect local immune responses and glucose metabolism associated with TLS presence in PAAD. 18F-FDG PET/CT may serve as a non-invasive and aided tool for assessing the presence of TLS in tumor immune environment and predicting antitumor immunity in PAAD patients.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04205-x.

The keratinocytes actively participate in the cutaneous immune responses. Dysregulation and abnormal expression of inflammatory mediators or their receptors in keratinocytes are relevant to the pathogenesis of chronic inflammatory skin diseases. The mechanism of long-lasting inflammatory processes is related with the activation of nuclear factor (NF)-kappaB and mitogen-activated protein kinase (MAPK), which play a crucial role in the immune responses. There are potential interaction points between these two pathways. The aim of this study is to investigate the differences in expression levels and distributions of phosphorylated extracellular signal-regulated kinase (ERK)1/2, phosphorylated p38 MAPK and NF-kappaB p105/p50 in chronic inflammatory skin diseases. An immunohistochemical staining technique was employed to measure the expression of these molecules in 25 cases of lichen planus, 22 cases of psoriasis, 26 cases of chronic eczema, seven cases of prurigo and seven cases of normal skin. We observed that the expression of phosphorylated ERK1/2, phosphorylated p38 MAPK and NF-kappaB p105/p50 was significantly more augmented in the lesional epidermis of all the inflammatory skin diseases than those in normal skin (P < 0.05), and the number of positive keratinocytes was significantly more in lichen planus than that in other inflammatory diseases (P < 0.001). Moreover, the positive keratinocytes of these three molecules were more widely distributed in the entire layer of the epidermis in lichen planus than those in other diseases. We concluded that ERK1/2, p38 MAPK and NF-kappaB p105/p50 might play important roles in the pathophysiology of chronic inflammatory skin diseases.

Background: Oral mucosal immunity plays a central role in maintaining tissue homeostasis and orchestrating the phases of wound healing after dental procedures. Salivary immune markers, including secretory immunoglobulin A (sIgA) and pro-inflammatory cytokines, have been increasingly recognized as non-invasive indicators of immune status. Alterations in these markers may predict delayed wound healing, particularly in patients with systemic risk factors such as diabetes mellitus and smoking. Objectives: The present study was designed to clinically evaluate the association between oral mucosal immune responses and wound healing outcomes in dental patients. Specifically, the study aimed to determine whether salivary biomarkers could serve as predictors of post-operative healing efficiency. Methods: This prospective clinical study was conducted at Margalla Dental Hospital, Rawalpindi, Pakistan, from January 2022 to July 2023. A total of 120 patients undergoing extractions, periodontal flap surgeries, or single-tooth implant placements were enrolled. Unstimulated whole saliva samples were collected at baseline and day 7 to quantify sIgA, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) using ELISA. Wound healing was assessed on days 3, 7, and 14 using the Landry, Turnbull, and Howley Wound Healing Index (WHI). Statistical analyses included correlation and regression models to identify associations between biomarkers, systemic factors, and healing outcomes. Results: At day 7, 88 patients (73.3%) demonstrated good healing (WHI ≥ 4), while 32 patients (26.7%) exhibited delayed healing (WHI ≤ 3). Patients with good healing showed significantly higher sIgA concentrations and lower IL-6 and TNF-α levels compared to those with delayed healing (p &lt; 0.05). Diabetes mellitus, smoking, and flap-based procedures were strongly associated with delayed healing. Multivariate regression identified diabetes (OR: 3.2; 95% CI: 1.5–6.7), smoking (OR: 2.1; 95% CI: 1.1–4.2), and elevated baseline IL-6 (OR: 2.8; 95% CI: 1.3–5.9) as independent predictors of impaired healing outcomes. Conclusion: The study demonstrates that oral mucosal immunity significantly influences wound healing outcomes in dental patients. Higher salivary sIgA levels were associated with favorable healing, whereas elevated IL-6 and TNF-α predicted delayed recovery. Systemic conditions such as diabetes mellitus and smoking further increased the risk of impaired healing. Salivary biomarker assessment offers a promising, non-invasive tool for identifying high-risk patients and guiding personalized management in dental practice. Keywords: Oral mucosal immunity; wound healing; salivary biomarkers; cytokines; sIgA; diabetes mellitus; dental surgery.