High Endothelial Venules Research Articles

Research trends, hotspots and future directions of tertiary lymphoid structures in cancer: a comprehensive informatics analysis and visualization study.

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.

Histopathological Correlation Between High Endothelial Venule Neogenesis and the Tumor Microenvironment in Lung Adenocarcinoma.

The dynamic interplay between cancer cells and the microenvironment involves a wide range of intricate relationships that evolve during different stages of tumor progression. Recent attention has focused on high endothelial venules (HEVs), specialized endothelial cells in tumors with a unique cuboidal shape similar to those in lymph nodes. Previous animal studies have shown that normalization of tumor angiogenesis through anti-VEGFR2 therapy promotes HEV formation. However, few reports exist regarding the relationship between HEVs and preexisting blood vessels or interstitial fibers. In this study, we histologically examined whether tumor vascular structure correlates with HEV neogenesis. A total of 109 patients with pathological stage I lung adenocarcinoma who had undergone curative lung resection at our Institute between 2012 and 2016 were included. HEVs were identified by anti-peripheral node addressin (PNAd) staining. Immunostaining and Elastica-Masson-Goldner staining were performed on tumor sections and quantified. PNAd-positive cells were identified in 102 (93.6%) patients. Nearly all PNAd-positive cells were located within or near immune cell clusters. We investigated the correlation between microvessel structures or interstitial fibers and the number/density of PNAd-positive vessels, but no significant correlation was found. Since PNAd-positive cells were concentrated in immune cell aggregates, we focused our analysis specifically on these regions. Immune cell aggregates with abundant PNAd-positive vessels had a greater microvessel density along with by rich collagen fiber production, and displayed a more mature morphological phenotype of HEVs. The generation of PNAd-positive cells in tumors is governed by an angiogenetic mechanism distinct from that of broader tumor microenvironment. Furthermore, the accumulation of immune cells is associated with increased HEV maturation.

Lymphoid Aggregates in Canine Cutaneous and Subcutaneous Sarcomas: Immunohistochemical and Gene Expression Evidence for Tertiary Lymphoid Structures.

Canine cutaneous/subcutaneous soft-tissue sarcomas (STS) are diversely derived mesenchymal neoplasms with a risk of recurrence and/or metastasis depending on the extent of surgical excision and histologic grade. Lymphoid aggregates (LAs) are often described in these tumours but not characterised. In humans, LA characterised as tertiary lymphoid structures (TLSs) improve the prognosis of many tumours, including sarcomas. We sought to determine if LA meeting a size criterion (> 700 cells) in canine sarcomas met the criteria of TLS and the overall prevalence of LA of any size. RNA expression in large LAs versus aggregate-adjacent sarcoma tissue (AAS) was measured in laser capture microdissected tissue and compared to curl-derived RNA from aggregate-free sarcomas and lymph nodes. CD3, CD20, MUM-1 and PNAd expressions were measured using immunohistochemistry. CD20 and CD3 mRNA were more highly expressed in LA versus AAS (13.8 fold, p = 0.0003 and 2.3 fold, p = 0.043). This was supported by the IHC findings. The large LAs were also enriched in chemokine RNA expression characteristic of TLS (CXCR5 5.8 fold, p < 00001, CCL19 3.68 fold, p = 0.0209, CCL21 6.87 fold, p = 0.0209 and CXCL13 2.68 fold, p = 0.0924). Plasma cells and high endothelial venules were identified in LA containing tumours but not in control tissue. Large LAs were present in 12% of tumours, and LA of any size in 30%. We conclude that large LAs in canine STS are consistent with TLS.

Dutasteride, a 5 alpha reductase inhibitor, could be associated with the exacerbation of inflammation in patients with benign prostatic hyperplasia.

α-1 blockers and dutasteride are widely used as agents to treat benign prostatic hyperplasia (BPH); the impact of these drugs on prostatic inflammation is still unclear. Herein, we investigated the impact of α-1 blockers and dutasteride treatment of BPH in terms of the degree of prostatic inflammation. Tissue specimens were obtained from 143 BPH patients who were administered α-1 blockers up until their operation. Thirty-three of the patients had also been treated with dutasteride before the procedure. The degree of prostatic inflammation was quantified histologically by the ratio of high endothelial venule (HEV)-like vessels. We divided this retrospective cohort into α-1 blocker monotherapy and combination therapy (α-1 blockers + dutasteride) groups and evaluated clinical parameters of the two groups in relation to the degree of chronic prostatic inflammation. At the same time, we assessed factors exacerbating chronic prostatic inflammation. Comparison of the monotherapy and combination therapy groups showed no significant differences in the parameters of the urodynamic study or degree of chronic prostatic inflammation, whereas the IPSS total score, voiding subscore, nocturia, intermittency, weak stream, and straining were significantly lower in the combination than the monotherapy group. The duration of α-1 blockers administration was not correlated with the ratio of HEV-like vessels, while that of dutasteride was strongly correlated (correlation coefficient = 0.595; p < 0.001). Multiple regression analysis demonstrated that the duration of dutasteride administration was a key factor exacerbating the degree of chronic prostatic inflammation. The present study showed that despite their ameliorating effect on prostatic hyperplasia, dutasteride contributed significantly to chronic prostatic inflammation.

Mucosal Addressin Cell Adhesion Molecule-1 Mediates T Cell Migration into Pancreas-Draining Lymph Nodes for Initiation of the Autoimmune Response in Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease that is caused by autoreactive T cell-mediated destruction of insulin-producing β cells in the pancreatic islets. Although naive autoreactive T cells are initially primed by islet antigens in pancreas-draining lymph nodes (pan-LNs), the adhesion molecules that recruit T cells into pan-LNs are unknown. We show that high endothelial venules in pan-LNs of young nonobese diabetic mice have a unique adhesion molecule profile that includes strong expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Anti-MAdCAM-1 antibody blocked more than 80% of the migration of naive autoreactive CD4+ T cells from blood vessels into pan-LNs. Transient blockade of MAdCAM-1 in young nonobese diabetic mice led to increased numbers of autoreactive regulatory CD4+ T cells in pan-LNs and pancreas and to long-lasting protection from T1D. These results indicate the importance of MAdCAM-1 in the development of T1D and suggest MAdCAM-1 as a potential therapeutic target for treating T1D.

High M2/M1 Macrophage Ratio Observed in Nasal Polyps Formed in Allergic Fungal Rhinosinusitis.

Allergic fungal rhinosinusitis (AFRS) shares similarities with eosinophilic chronic rhinosinusitis (ECRS), both characterized by intractable nasal polyps. The key distinction lies in the presence of fungal infection within the nasal cavity. While ECRS nasal polyps are known for significant infiltration of M2 macrophages and eosinophils, as well as an increase in high endothelial venule (HEV)-like vessels, these features are less commonly reported in AFRS. This study compared clinicopathological findings between AFRS (n=10), ECRS (n=12), and non-ECRS (n=10) patients' nasal polyps using immunohistochemical analysis for CD163 and CD68 to assess the M2/M1 macrophage ratio, and peripheral lymph node addressin (PNAd) and CD34 to evaluate the proportion of HEV-like vessels. AFRS showed a significantly higher number of CD163-positive M2 macrophages and an increased M2/M1 ratio compared with ECRS. However, the percentage of HEV-like vessels and the number of eosinophils infiltrating the nasal polyps were similar in both AFRS and ECRS. The observed increase in M2 macrophages in AFRS nasal polyps is presumed to be induced by fungal infection in the nasal cavity, in comparison with ECRS. These results highlight the distinctive immunological profiles of AFRS and ECRS, emphasizing the role of macrophage polarization in their pathogenesis.

Tertiary lymphoid structures in endometriosis

To determine whether tertiary lymphoid structures (TLSs), which reflect organized immune cell aggregates present in non-lymphoid tissues, are consistent features of endometriosis lesions. Detailed histopathological analysis of endometrial and lesion tissue from endometriosis patients and controls was performed. Multiplex immunofluorescence on select samples was then conducted to identify canonical cell populations present within TLSs: CD3+ and CD8+ T-cells, CD79a+ B-cells, CD208+ dendritic cells, CD21+ follicular dendritic cells (fDC), and PNAd+ high endothelial venules (HEVs). Histologically confirmed endometriosis patients (N=113; 44.3± 6.0) and control individuals (N=110; 44.6±7.1). Not applicable. Detection of TLSs as characterized by the presence of all canonical cell types that constitute TLS and structure morphology. Of the selected samples (N=18; 6 ectopic/eutopic/control), mature TLSs were identified in 3 ectopic tissue samples present on the ovary and fallopian tube, with immature TLSs (lacking fDC networks and HEVs) present throughout eutopic and control endometrial samples. These findings demonstrate the presence of TLSs across various endometriosis phenotypes, prompting further research into their significance within disease pathophysiology and the prognostic implications for patients.

Clarification of a unique mucosal vaccination route for improved systemic and mucosal immune response in broiler

There are often outbreaks of infectious diseases on farms, which not only disrupt production but also cause significant economic losses. Vaccines are given to prevent the spread of these infectious diseases, but they produce only systemic antibodies or antibodies in the mucosa of a particular system. So, a new mucosal vaccination route is needed where the vaccine will produce antibodies in the systemic circulation as well as in the mucosa of many systems. In our study, the cloaca was targeted because it is associated with the mucosa of many systems. Whole-mount and routine histological staining show both lymphatic nodules and diffuse lymphatic tissues in the lamina propria of cloaca. These lymphatic tissues are made up of Bu-1+ B-cells, CD3+ T-cells, and KUL01+ macrophages and monocytes. So, this is a new mucosa-associated lymphoid tissue, named cloaca-associated lymphoid tissue (CALT). The CALT contains antigen-presenting cells (dendritic cells, macrophages, B cells, MHC II molecules, and T cells) and is equipped with blood vessels and high endothelial venules, which indicate its functional status. More antibodies were produced in the treatment group compared to the vehicle control group after administration of the infectious bursal disease (IBD) and the Newcastle disease (ND) vaccine through cloaca. In addition, the cloaca-associated route produces a higher number of antibodies than the other traditional routes, which reveals the uniqueness of this route. Cloacal-vaccinated chickens showed less damage to the follicle and epithelium of the bursa of Fabricius compared to other groups, indicating its lower cytotoxic effect. Therefore, the cloaca-associated mucosal vaccination route produces more antibodies than other mucosal vaccination routes, which will protect the chickens on the farm to a greater extent.

Immune priming and induction of tertiary lymphoid structures in a cord-blood humanized mouse model of gastrointestinal stromal tumor

ABSTRACT Gastrointestinal stromal tumors (GISTs) harbor diverse immune cell populations but so far immunotherapy in patients has been disappointing. Here, we established cord blood humanized mouse models of localized and disseminated GIST to explore the remodeling of the tumor environment for improved immunotherapy. Specifically, we assessed the ability of a cancer vascular targeting peptide (VTP) to bind to mouse and patient GIST angiogenic blood vessels and deliver the TNF superfamily member LIGHT (TNFS14) into tumors. LIGHT-VTP treatment of GIST in humanized mice improved vascular function and tumor oxygenation, which correlated with an overall increase in intratumoral human effector T cells. Concomitant with LIGHT-mediated vascular remodeling, we observed intratumoral high endothelial venules (HEVs) and tertiary lymphoid structures (TLS), which resemble spontaneous TLS found in GIST patients. Thus, by overcoming the limitations of immunodeficient xenograft models, we demonstrate the therapeutic feasibility of vascular targeting and immune priming in human GIST. Since TLS positively correlate with patient prognosis and improved response to immune checkpoint inhibition, vascular LIGHT targeting in GIST is a highly translatable approach to improve immunotherapeutic outcomes.

LTβR Agonism Promotes Anti-Tumor Immune Responses via Modulation of the Tumor Microenvironment.

The presence of high endothelial venules (HEV) and tertiary lymphoid structures (TLS) in solid tumors is correlated with favorable prognosis and better responses to immune-checkpoint blockade (ICB) in many cancer types. Elucidation of the molecular mechanisms underlying intratumoral HEV and TLS formation and their contribution to anti-tumor responses may facilitate development of improved treatment strategies. Lymphotoxin beta receptor (LTβR) signaling is a critical regulator of lymph node organogenesis and can cooperate with antiangiogenic and ICB treatment to augment tumor-associated HEV formation. Here, we demonstrated that LTβR signaling modulates the tumor microenvironment via multiple mechanisms to promote anti-tumor T cell responses. Systemic activation of the LTβR pathway via agonistic antibody treatment induced tumor-specific HEV formation, upregulated the expression of TLS-related chemokines, and enhanced dendritic cell (DC) and T cell infiltration and activation in syngeneic tumor models. In vitro studies confirmed direct effects of LTβR agonism on DC activation and maturation and associated DC-mediated T cell activation. Single agent LTβR agonist treatment inhibited syngeneic tumor growth in a CD8+ T cell- and HEV-dependent manner, and the LTβR agonist enhanced anti-tumor effects of anti-PD-1 and CAR T cell therapies. An in vivo tumor screen for TLS-inducing cytokines revealed that the combination of LTβR agonism and lymphotoxin alpha (LT⍺) expression promoted robust intratumoral TLS induction and enhanced tumor responses to anti-CTLA-4 treatment. Collectively, this study highlights crucial functions of LTβR signaling in modulating the tumor microenvironment and could inform future HEV/TLS-based strategies for cancer treatments.

Sphingosine-1-Phosphate Receptor 4 links neutrophils and early local inflammation to lymphocyte recruitment into the draining lymph node to facilitate robust germinal center formation.

The successful development of germinal centers (GC) relies heavily on innate mechanisms to amplify the initial inflammatory cascade. In addition to their role in antigen presentation, innate cells are essential for the redirection of circulating lymphocytes toward the draining lymph node (dLN) to maximize antigen surveillance. Sphingosine-1-Phosphate (S1P) and its receptors (S1PR1-5) affect various aspects of immunity; however, the role of S1PR4 in regulating an immune response is not well understood. Here we use a footpad model of localized TH1 inflammation to carefully monitor changes in leukocyte populations within the blood, the immunized tissue, and the dLN. Within hours of immunization, neutrophils failed to adequately mobilize and infiltrate into the footpad tissue of S1PR4-/- mice, thereby diminishing the local vascular changes thought to be necessary for redirecting circulating cells toward the inflamed region. Neutrophil depletion with anti-Ly6G antibodies significantly reduced early tissue edema as well as the redirection and initial accumulation of naïve lymphocytes in dLN of WT mice, while the effects were less prominent or absent in S1PR4-/- dLN. Adoptive transfer experiments further demonstrated that the lymphocyte homing deficiencies in vivo were not intrinsic to the donor S1PR4-/- lymphocytes, but were instead attributed to differences within the S1PR4-deficient host. Reduced cell recruitment in S1PR4-/- mice would seed the dLN with fewer antigen-respondent lymphocytes and indeed, dLN hypertrophy at the peak of the immune response was severely diminished, with attenuated GC and activation pathways in these mice. Histological examination of the S1PR4-/- dLN also revealed an underdeveloped vascular network with reduced expression of the leukocyte tethering ligand, PNAd, within high endothelial venule regions, suggesting inadequate growth of the dLN meant to support a robust GC response. Thus, our study reveals that S1PR4 may link early immune modulation by neutrophils to the initial recruitment of circulating lymphocytes and downstream expansion and maturation of the dLN, thereby contributing to optimal GC development during an adaptive response.

Zonation, ligand and dose dependence of S1PR1 signalling in blood and lymphatic vasculature.

Circulating levels of sphingosine 1-phosphate (S1P), an HDL-associated ligand for endothelial cell (EC) protective S1P receptor-1 (S1PR1), are reduced in disease states associated with endothelial dysfunction. Yet as S1PR1 has high affinity for S1P and can be activated by ligand-independent mechanisms and EC-autonomous S1P production, it is unclear if relative reductions in circulating S1P impact endothelial function. It is also unclear how EC S1PR1 insufficiency, whether induced by ligand deficiency or by S1PR1-directed immunosuppressive therapy, affects different vascular subsets. We here fine-map the zonation of S1PR1 signalling in the murine blood and lymphatic vasculature, superimpose cell type-specific and relative deficiencies in S1P production to define ligand source- and dose-dependence, and correlate receptor engagement to essential functions. In naïve blood vessels, despite broad expression, EC S1PR1 engagement was restricted to resistance-size arteries, lung capillaries and high-endothelial venules (HEV). Similar zonation was observed for albumin extravasation in EC S1PR1 deficient mice, and brain extravasation was reproduced with arterial EC-selective S1pr1 deletion. In lymphatic EC, S1PR1 engagement was high in collecting vessels and lymph nodes and low in terminal capillaries that drain tissue fluids. While EC S1P production sustained S1PR1 signaling in lymphatics and HEV, hematopoietic cells provided ∼90% of plasma S1P and sustained signaling in resistance arteries and lung capillaries. S1PR1 signaling and endothelial function were both surprisingly sensitive to reductions in plasma S1P with apparent saturation around 50% of normal levels. S1PR1 engagement did not depend on sex or age, but modestly increased in arteries in hypertension and diabetes. Sphingosine kinase (Sphk)-2 deficiency also increased S1PR1 engagement selectively in arteries, which could be attributed to Sphk1-dependent S1P release from perivascular macrophages. This study highlights vessel subtype-specific S1PR1 functions and mechanisms of engagement and supports the relevance of S1P as circulating biomarker for endothelial function.

Intratumoral high endothelial venules in solid tumors: a pooled study.

We performed this pooled analysis for the first time to comprehensively explore the prognostic value of tumor-associated high endothelial venules (TA-HEVs) and determine their relationships with clinicopathological features in solid tumors. Four online databases, including PubMed, Web of Science, Embase, and Cochrane Library, were comprehensively searched to identify studies assessing the effect of TA-HEVs on prognosis or clinicopathological features. Hazard ratios (HRs) with 95% confidence intervals (CIs) were applied to evaluate survival outcomes, including overall survival (OS), disease-free survival (DFS), progression-free survival (PFS), and cancer-specific survival (CSS). The association between TA-HEV status and clinicopathological characteristics was assessed by odds ratios (ORs) combined with 95% CIs. Subgroup analysis was conducted to explore sources of heterogeneity. The sensitivity analysis was performed to evaluate the stability of our findings. Meanwhile, Funnel plots were employed to visually evaluate potential publication bias, and both Begg's and Egger's tests were adopted to quantitatively determine publication bias. A total of 13 retrospective cohort studies, involving 1,933 patients were finally included in this meta-analysis. Effect-size pooling analysis showed that the positivity of TA-HEVs was related to improved OS (pooled HR: 0.75, 95% CI: 0.62-0.93, P<0.01), and DFS (pooled HR = 0.54, 95% CI = 0.41-0.72, P< 0.01). However, TA-HEV positivity in solid tumors was not linked to PFS (pooled HR = 0.75, 95% CI 0.34-1.64, P = 0.47) or CSS (pooled HR: 0.58, 95% CI: 0.04-7.58, P= 0.68). Further subgroup analysis demonstrated that ethnicity and source of HR were the main factors contributing to heterogeneity. Moreover, TA-HEVs were inversely associated with lymph node metastasis and distant metastasis, but were positively related to worse tumor differentiation. However, TA-HEVs were not significantly correlated with sex, LVI, clinical stage, and depth of invasion. Sensitivity analysis suggested that the pooled results were stable and reliable, with no significant publication bias in all included articles. This is the first comprehensive analysis of the prognostic value of TA-HEVs in solid tumors using existing literature. Overall, our study demonstrated a significant correlation between TA-HEVs and prognosis as well as clinicopathological features. TA-HEVs may serve as novel immune-related biomarkers for clinical assessments and prognosis prediction in solid tumors. https://www.crd.york.ac.uk/prospero/display_record.php, identifier CRD42023394998.

Clinicopathological characteristics of angioimmunoblastic T-cell lymphoma with B-cell proliferation or neoplasms

Objective: To investigate the clinical, pathological and immunophenotypic features, and differential diagnosis of angioimmunoblastic T-cell lymphoma (AITL) with B-cell proliferation or neoplasms. Methods: Eight qualified cases were collected from the Department of Pathology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China from January 2019 to July 2023. One case was diagnosed with AITL and diffuse large B-cell lymphoma (DLBCL) and the other seven cases were diagnosed with AITL and B-cell proliferation. Clinical characteristics and pathological morphology were summarized. Immunohistochemical analysis, fluorescence in situ hybridization and gene rearrangement detection were performed. Results: The patients' average age was 58 years. Five of them were male. Biopsies of the enlarged cervical lymph nodes showed structural destruction and exhibited various histologic patterns. Some cases revealed Burkitt-like morphology, a moderate tumor volume and slightly irregular nuclei. Some cases showed prominent nucleoli. High endothelial venules and expanded follicular dendritic cells were detected. Tumor cells derived from T-follicular helper (TFH) cells were positive for two or more TFH biomarkers. Nodular or diffuse patchy proliferation of B cells was noted around the tumor tissue, which was initially considered as B-cell lymphoma. All of the 8 cases showed monoclonal rearrangements of the T-cell receptor genes while 5 of them also showed clonal rearrangements of the Ig genes. Seven of the 8 cases were subject to the detection of C-MYC gene breakage and were all negative. EBV-positive cells were seen in 6 cases. Neoplastic B cells were positive for C-MYC (>40%), while proliferative B cells were negative for C-MYC (<40%). Conclusions: The histological morphology of AITL with B-cell proliferation or lymphoma may be different from AITL. An integrated analysis, incorporating clinical, morphologic, immunophenotypic, and molecular assessment, helps reach an accurate diagnosis. This group of cases demonstrated the clinical and pathological characteristics of AITL accompanied by B-cell proliferation and B-cell lymphoma. The findings suggest that C-MYC maybe a feasible indicator for distinguishing B-cell proliferation from B-cell lymphoma, and provide a simple and feasible immunohistochemical marker for the diagnosis and research of composite lymphoma.

#1449 Maturation of tertiary lymphoid structures in progressive kidney damage due to chronic phosphate load

Abstract Background and Aims We have recently shown that a high phosphate diet (HPD) in mice leads to an increase in FGF23 levels and causes progressive tubular damage and interstitial fibrosis with increased accumulation of macrophages and concomitant perivascular immune cell aggregates in the corticomedullary zone. Such tertiary lymphoid structures (TLS) can develop as a result of chronic inflammation and have already been described in lupus nephritis and IgA nephropathy, among others. In our model, the question arose to what extent damage to the proximal tubule is temporally associated with the development and maturation of TLS and which factors favor TLS development. Method Wild-type mice were fed HPD for 1, 2, 3, 4 and 6 months and the kidneys of each were compared with those of mice on a normal phosphate diet (NPD). To evaluate the effect of high FGF23 ± phosphate, Fgf23 was overexpressed in mice on NPD and HPD using an adeno-associated virus (AAV-Fgf23), and hypophosphatemic Hyp mice were used. Results An increased tubular damage score was seen after only two months of HPD. In parallel, perivascular immune cell clusters developed in the corticomedullary zone and in the renal cortex, which were characterized by aggregates of CD45R+ B cells and CD3+ T cells, as well as LYVE1+ lymphatic vessels after three months of HPD and were accompanied by increased expression of venous markers (Plvap, Aplnr, EphB4) and cell adhesion molecules (Sell, Vcam1, Madcam1). Further analyses showed a time-dependent induction of the immunofibroblast-derived chemokines Cxcl13 and Ccl19, as well as the lymphotoxins Lta and Ltb, which are important for the differentiation of immunofibroblasts into follicular dendritic cells (FDC) and follicular reticular cells (FRC). Already after four months of HPD, proliferating B-cell clusters with FDCs, FRCs located in T-cell clusters, podoplanin+ networks, high endothelial venules, plasma cells and increased IgD synthesis indicated fully mature TLS, while tubular damage and fibrosis continued to increase until six months of HPD. Compared to AAV-Fgf23 mice on NPD, only AAV-Fgf23 animals on HPD developed tubular damage with fibrosis and fully mature TLS. Hypophosphatemic Hyp mice with high FGF23 showed neither signs of tubular damage nor the development of TLS. Conclusion The development of renal TLS due to chronic high phosphate load coincides with the development of tubular damage, with earlier maturation of TLS and progression of tubular damage.

4-nitroquinoline 1-oxide-induced oral epithelial lesions exhibit time- and stage-dependent changes in the tumor immune microenvironment.

Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity and is associated with high morbidity due to local invasion and lymph node metastasis. Tumor infiltrating lymphocytes (TILs) are associated with good prognosis in oral cancer patients and dictate response to treatment. Ectopic sites for immune activation in tumors, known as tertiary lymphoid structures (TLS), and tumor-associated high-endothelial venules (TA-HEVs), which are specialized lymphocyte recruiting vessels, are associated with a favorable prognosis in OSCC. Why only some tumors support the development of TLS and HEVs is poorly understood. In the current study we explored the infiltration of lymphocyte subsets and the development of TLS and HEVs in oral epithelial lesions using the 4-nitroquinoline 1-oxide (4NQO)-induced mouse model of oral carcinogenesis. We found that the immune response to 4NQO-induced oral epithelial lesions was dominated by T cell subsets. The number of T cells (CD4+, FoxP3+, and CD8+), B cells (B220+) and PNAd+ HEVs increased from the earliest to the latest endpoints. All the immune markers increased with the severity of the dysplasia, while the number of HEVs and B cells further increased in SCCs. HEVs were present already in early-stage lesions, while TLS did not develop at any timepoint. This suggests that the 4NQO model is applicable to study the dynamics of the tumor immune microenvironment at early phases of oral cancer development, including the regulation of TA-HEVs in OTSCC.

Prognostic Effect of Tertiary Lymphoid Structures in Epstein–Barr Virus–Associated Gastric Carcinomas Measured by Digital Image Analysis

Epstein–Barr virus–associated gastric carcinoma (EBVaGC) is characterized by prominent tumor-infiltrating lymphocytes (TILs) and has a favorable prognosis. Tertiary lymphoid structures (TLS), characterized by ectopic aggregated lymphocytes with high-endothelial venules (HEV), are associated with favorable outcomes in various solid tumors. We hypothesized that EBVaGC, characterized by intense TILs, may be closely associated with TLS or HEV. To test this hypothesis, we digitally analyzed the TLS, HEV, and TILs in 73 surgically resected advanced EBVaGCs. For HEV, dual MECA-79 and CD31 dual immunohistochemistry were performed, and the ectopic expression of MECA-79 in tumor cells was measured. In 73 patients with EBVaGC, a high-TLS ratio was found in 29 (39.7%) cases, high-tumor-associated HEV density in 44 (60.3%) cases, and high-CD8+ TIL density in 38 (52.1%) cases. Ectopic tumor expression of MECA-79 was observed in 36 patients (49.3%) cases. A low-TLS ratio and tumor-associated HEV density were significantly associated with lymph node metastasis (P = .005 and .042, respectively). Ectopic MECA-79 expression was significantly associated with lymph node metastasis (P = .003). Patients with a low-TLS ratio (P = .038), low-HEV density (P = .042), and ectopic tumor MECA-79 expression (P = .032) had significantly worse prognoses. In conclusion, TLS ratio and HEV density affect the survival of patients with EBVaGC and may be related to the immune response that interrupts lymph node metastasis.

Abstract LB123: Conditionally active, therapeutic lymphotoxin beta receptor (LTBR) agonist bispecific antibodies for induction of tertiary lymphoid structures in the treatment of solid tumors

Abstract The presence of tertiary lymphoid structures (TLS) and associated high endothelial venules (HEV) in the tumor microenvironment strongly correlates with improved prognosis and treatment outcomes across a wide range of solid tumors. These clinical observations, now replicated repeatedly, highlight the therapeutic potential of induction of TLS and/or boosting TLS functions, which include acting as a point of entry and education for immune effector cells locally in the tumor. Lymphotoxin beta receptor (LTBR) signalling is essential for lymphoid structure development and the ectopic expression of its ligands, lymphotoxin αβ and LIGHT, is sufficient for TLS formation in vivo but challenging for therapeutic development. Here we present human bispecific antibodies (bsAbs) which conditionally agonize LTBR on co-engagement of fibroblast activation protein (FAP), a tumor microenvironment specific marker expressed by cancer associated fibroblasts. In vitro characterisation of these therapeutic bsAbs is presented, along with in vivo preclinical evaluation of surrogate bsAbs binding to mouse LTBR and mouse FAP in murine tumor models. Results - Therapeutic bsAbs were identified following an extensive screening campaign combining LTBR and FAP binding arms on an engineered human Fc with minimal FcyR binding. In vitro assays using primary cancer associated fibroblasts and LTBR positive cells demonstrated conditionally active therapeutic bsAbs potently activated LTBR in the presence of FAP expressing cells, whereas in the absence of FAP expressing cells no activity was observed. Surrogate bsAbs demonstrated monotherapy activity and led to tumor regressions in combination with anti-PDL1 therapy in an EMT6 mouse model, a syngeneic orthotopic model of breast cancer. Flow cytometry of endpoint tumors showed robust formation of HEVs and increased infiltration of T cells and B cells. In addition, we demonstrate that LTBR agonism, but not PD-L1 inhibition, leads to the formation of TLS structures containing organized lymphocyte aggregates with the appearance of germinal centres and accumulations of T and B cells in a mouse model of lung cancer. In conclusion, conditionally active FAP-LTBR bispecifics activate LTBR in the tumor microenvironment and induce HEV and TLS formation, leading to potent monotherapy activity in vivo, and to tumor regression in combination with PD-L1. These data support the development of FAP-LTBR bispecifics for the treatment of solid tumors as monotherapy and in combination with standard of care. Citation Format: Marta Lewandowska, Fevzi Demircioglu, Lucy Penfold, Rebecca B. Riddle, Sameer Sirohi, Floriane Laurent, Richard Brown, Sonia Bains, Dara Bevan, Emma Stanley, Jeanine Pignatelli, James W. Legg, Ray Jupp. Conditionally active, therapeutic lymphotoxin beta receptor (LTBR) agonist bispecific antibodies for induction of tertiary lymphoid structures in the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB123.

Presence of immunoglobulin E-expressing antibody-secreting cells in the dermis close to bullous pemphigoid lesions.

Antibody-secreting cells (ASCs) produce immunoglobulin (Ig) G and IgE autoantibodies in secondary lymphoid organs. Evidence also suggests their existence in the skin in various chronic inflammatory conditions, and in association with CXCL12 and CXCL13, they regulate the recruitment/survival of ASCs and germinal center formation to generate ASCs, respectively. However, the presence of IgG and IgE in bullous pemphigoid (BP) lesions needs to be addressed. Here, we aimed to analyse BP skin for the presence of IgG and IgE and the factors contributing to their generation, recruitment, and persistence. Skin samples from 30 patients with BP were stained to identify ASCs and the immunoglobulin type they expressed. The presence of tertiary lymphoid organ (TLO) elements, which generate ASCs in non-lymphoid tissues, and the chemokines CXCL12 and CXCL13, which regulate the migration/persistence of ASCs in lymphoid tissues and formation of TLOs, respectively, were evaluated in BP skin. BP skin harboured ASCs expressing the two types of antibodies IgG and IgE. ASCs were found in high-grade cellular aggregates containing TLO elements: T cells, B cells, CXCL12+ cells, CXCL13+ cells and high endothelial venules. IgG+ ASCs were detected among these aggregates, whereas IgE+ ASCs were dispersed throughout the dermis. CXCL12+ fibroblast-like cells were located close to ASCs. The inflammatory microenvironment of BP lesions may contribute to the antibody load characteristic of the skin of patients with BP by providing a site for the presence of ASCs. CXCL13 and CXCL12 expression may contribute to the generation and recruitment/survival of ASCs, respectively.

Comprehensive analysis of human tissues reveals unique expression and localization patterns of HSF1 and HSF2.

Heat shock factors (HSFs) are the main transcriptional regulators of the evolutionarily conserved heat shock response. Beyond cell stress, several studies have demonstrated that HSFs also contribute to a vast variety of human pathologies, ranging from metabolic diseases to cancer and neurodegeneration. Despite their evident role in mitigating cellular perturbations, the functions of HSF1 and HSF2 in physiological proteostasis have remained inconclusive. Here, we analyzed a comprehensive selection of paraffin-embedded human tissue samples with immunohistochemistry. We demonstrate that both HSF1 and HSF2 display distinct expression and subcellular localization patterns in benign tissues. HSF1 localizes to the nucleus in all epithelial cell types, whereas nuclear expression of HSF2 was limited to only a few cell types, especially the spermatogonia and the urothelial umbrella cells. We observed a consistent and robust cytoplasmic expression of HSF2 across all studied smooth muscle and endothelial cells, including the smooth muscle cells surrounding the vasculature and the high endothelial venules in lymph nodes. Outstandingly, HSF2 localized specifically at cell-cell adhesion sites in a broad selection of tissue types, such as the cardiac muscle, liver, and epididymis. To the best of our knowledge, this is the first study to systematically describe the expression and localization patterns of HSF1 and HSF2 in benign human tissues. Thus, our work expands the biological landscape of these factors and creates the foundation for the identification of specific roles of HSF1 and HSF2 in normal physiological processes.