Immune Aggregates Research Articles

Tertiary lymphoid structures as local perpetuators of organ-specific immune injury: implication for lupus nephritis.

In response to inflammatory stimuli in conditions such as autoimmune disorders, infections and cancers, immune cells organize in nonlymphoid tissues, which resemble secondary lymphoid organs. Such immune cell clusters are called tertiary lymphoid structures (TLS). Here, we describe the potential role of TLS in the pathogenesis of autoimmune disease, focusing on lupus nephritis, a condition that incurs major morbidity and mortality. In the kidneys of patients and animals with lupus nephritis, the presence of immune cell aggregates with similar cell composition, structure, and gene signature as lymph nodes and of lymphoid tissue-inducer and -organizer cells, along with evidence of communication between stromal and immune cells are indicative of the formation of TLS. TLS formation in kidneys affected by lupus may be instigated by local increases in lymphorganogenic chemokines such as CXCL13, and in molecules associated with leukocyte migration and vascularization. Importantly, the presence of TLS in kidneys is associated with severe tubulointerstitial inflammation, higher disease activity and chronicity indices, and poor response to treatment in patients with lupus nephritis. TLS may contribute to the pathogenesis of lupus nephritis by increasing local IFN-I production, facilitating the recruitment and supporting survival of autoreactive B cells, maintaining local production of systemic autoantibodies such as anti-dsDNA and anti-Sm/RNP autoantibodies, and initiating epitope spreading to local autoantigens. Resolution of TLS, along with improvement in lupus, by treating animals with soluble BAFF receptor, docosahexaenoic acid, complement inhibitor C4BP(β-), S1P1 receptor modulator Cenerimod, dexamethasone, and anti-CXCL13 further emphasizes a role of TLS in the pathogenesis of lupus. However, the mechanisms underlying TLS formation and their roles in the pathogenesis of lupus nephritis are not fully comprehended. Furthermore, the lack of non-invasive methods to visualize/quantify TLS in kidneys is also a major hurdle; however, recent success in visualizing TLS in lupus-prone mice by photon emission computed tomography provides hope for early detection and manipulation of TLS.

Meningeal inflammation as a driver of cortical grey matter pathology and clinical progression in multiple sclerosis.

Growing evidence from cerebrospinal fluid samples and post-mortem brain tissue from individuals with multiple sclerosis (MS) and rodent models indicates that the meninges have a key role in the inflammatory and neurodegenerative mechanisms underlying progressive MS pathology. The subarachnoid space and associated perivascular spaces between the membranes of the meninges are the access points for entry of lymphocytes, monocytes and macrophages into the brain parenchyma, and the main route for diffusion of inflammatory and cytotoxic molecules from the cerebrospinal fluid into the brain tissue. In addition, the meningeal spaces act as an exit route for CNS-derived antigens, immune cells and metabolites. A number of studies have demonstrated an association between chronic meningeal inflammation and a more severe clinical course of MS, suggesting that the build-up of immune cell aggregates in the meninges represents a rational target for therapeutic intervention. Therefore, understanding the precise cell and molecular mechanisms, timing and anatomical features involved in the compartmentalization of inflammation within the meningeal spaces in MS is vital. Here, we present a detailed review and discussion of the cellular, molecular and radiological evidence for a role of meningeal inflammation in MS, alongside the clinical and therapeutic implications.

Integrative analysis of tertiary lymphoid structures and immune microenvironment in patients with esophageal carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common upper gastrointestinal malignancies worldwide. Tertiary lymphoid structures (TLS) are tumor-infiltrating immune cells aggregates coupled with stromal cells which are similar to secondary lymphoid organs. The objective of this study is to explore the predictive effects of two common genes associated with TLS models on prognosis and immunotherapy effects in ESCC patients. Clinical information for ESCC patients in the TCGA(The Cancer Genome Altas) cohort and GSE 53625 were collected. All of the samples were classified as either high score group or low score group based on two TLS signatures, and the association between TLS signatures and survival, clinical indicators, genomic burden, stemness indices analysis, tumor microenvironment and immunotherapy response were performed. Furthermore, the mature TLS was also assessed in ESCC tissue microarray. In our study, we quantified the score of TLS_9 and TLS_12, respectively, reflecting the different statuses of TLS (TLS_9 = B and T cells in TLSs; TLS_12 = neogenesis of TLSs). Subsequently, we explored the effect of TLS score on ESCC tumor microenvironment quantified by multiple algorithms. We found that a correlation analysis indicated that TLS_9 and TLS_12 were all positively correlated with CD8+ T cell, NK cells, CD4+ T cells, M1 macrophages and so on. Meanwhile, some cells present a different correlation pattern of TLS_9 and TLS_12, including activated CD4+ memory T cells and Tgd cells. Immune-related analysis revealed that the TLS_12 and TLS_9 scores were all positively correlated with immune dysfunction, yet negatively correlated with immune exclusion. Following this, the biological roles of TLS_9 and TLS_12 scores were investigated. Also, we noticed that the TLS score could significantly affect the CAFs infiltration and be associated with the genomic burden and tumor stemness. In addition, we explored the prognostic value of mature TLS through tissue microarray (TMA). Our result displayed ESCC patients with the presence of mature TLS had a better prognosis than ESCC patients without it. Our study indicated that ESCC patients with the presence of TLS had better outcomes and an inflamed immune microenvironment. In addition, both TLS-9 and TLS-12 gene signatures could be used as potential biomarkers for the immunotherapy of ESCC patients.

Premalignancy induced B cell-centered immune aggregates for immune-prevention against triple negative breast cancer

Abstract Immune surveillance is believed to be a critical mechanism to eradicate premalignant mutant cells to prevent cancer. However, the cellular organization and mechanisms for immune surveillance remain largely unknown because premalignant mutant cells are “invisible” in patient samples and conventional animal models. To gain a glimpse into this process, our lab developed a genetically engineered mouse model recapitulating human basal-like breast cancer through unequivocal labeling of premalignant p53-Brca1 mutant cells with GFP. Using this model, we discovered prominent immune aggregates in close proximity to mutant ducts, which eventually disperse at the tumor stage. These immune aggregates manifest tertiary lymphoid structure (TLS)-like organization, enriched with activated B cells co-clustered with T cells and CD11b+ myeloid-derived cells. To fully characterize these TLS-like aggregates, we are profiling the dynamic changes in immune composition and activation status from premalignancy to malignancy. In parallel, to investigate the molecular mechanism behind their organization, we are studying the relevant chemokines, like CXCL13, and corresponding source cells. Ultimately, we plan to perform functional experiments to determine if these TLS-like structures are responsible for controlling the malignant progression of breast cancer. Our work provides direct evidence of how immune response against premalignant cells before breast cancer forms. Supported by grants from NIH (R01CA256199-01A1)

Venous plexus-associated lymphoid hubs support meningeal humoral immunity

Abstract There is increasing interest in how immune cells in the meninges, the membranes that surround the brain and spinal cord, contribute to homeostasis and disease in the central nervous system. The outer layer of meninges, the dura mater, contains both innate and adaptive immune cells and functions as a site for B cell development. In this study, we identified previously undescribed lymphoid structures surrounding fenestrated venous plexi in the dura mater. We found the most elaborate immune organization, including lymphatic vessels, surrounding the rostral-rhinal confluence of sinuses. We termed this structure that interfaced with the skull bone marrow and a comparable venous plexus at the skull base, the rostral-rhinal venolymphatic hub. This hub emerged during development in mice at P8/9 before formation of the superior sagittal sinus. Single cell RNA sequencing demonstrated that rostral rhinal hub hosts a diverse array of resident innate and adaptive immune cells during steady state. Immune aggregates were also present in this structure during homeostasis and expanded with age or following challenge with systemic or nasal antigens. Following intranasal VSV infection, the rostral-rhinal hub supported local germinal center (GC) reactions consisting of T follicular helper cells as well as GC B cells that underwent proliferation, somatic hypermutation, class switch, and conversion into plasma cells locally. These data demonstrate lymphoid architecture around vascular plexi in the dura mater that can sample antigens and rapidly elaborate local humoral immune responses to protect the meninges and underlying brain parenchyma from pathogens. This research was supported by the intramural program of the National Institute of Neurological Disorders and Stroke, NIH

Gene co-expression regulatory analysis associated with tertiary lymphoid structures and endometrial cancer progression

Abstract Endometrial cancer (EC) is the most common cancer diagnosis of the reproductive organs among women in the United States. Previous studies have shown the chemokine CXCL13 was correlated with forming B-cell and other immune cell aggregates in the presence of high endothelial venules (HEV). These ectopic lymphoid formations are referred to as tertiary lymphoid structures (TLS). Some studies have shown that TLS are associated with a reduced risk of recurrence and improved immune response in cancers, while others have described negative cancer outcomes associated with TLS. Few studies have focused on the role of TLS in EC and the pathways involved in tumorigenesis. In this project, we identify chemokine-mediated gene networks associated with TLS formation and EC pathogenicity. We obtained RNA-Seq EC samples (n=643), which included Type I Uterine Corpus Endometrial Cancer (UCEC, n=587) and Type II Uterine Carcinosarcoma (UCS, n= 56) from the Cancer Genome Atlas (TCGA). Hierarchically clustered genes were correlated, functionally annotated for mRNA, and evaluated using a weighted gene network co-expression analysis (WGCNA). Functional enrichment analysis, such as Gene Ontology (GO) and the database for annotation, visualization, and integrated discovery (DAVID), were used to identify and visualize biological functions associated with the hub genes overrepresented in significant modules. Modules associated with metastasis and survival are representative of novel and known pathways. The analysis reveals that CXCL13 may be involved in cell cycle progression and apoptosis pathways. This study provides novel insights into the biological pathways associated with TLS, EC tumorigenesis, and patient survival risks. Supported by Frederick National Laboratory for Cancer Research subcontract IDIQ NCI (75N91019D00024) and MSM/TU/UABCCC Partnership NCI (U54CA118638)

Unique structure of ozoralizumab, a trivalent anti-TNFα NANOBODY® compound, offers the potential advantage of mitigating the risk of immune complex-induced inflammation.

Biologics have become an important component of treatment strategies for a variety of diseases, but the immunogenicity of large immune complexes (ICs) and aggregates of biologics may increase risk of adverse events is a concern for biologics and it remains unclear whether large ICs consisting of intrinsic antigen and therapeutic antibodies are actually involved in acute local inflammation such as injection site reaction (ISR). Ozoralizumab is a trivalent, bispecific NANOBODY® compound that differs structurally from IgGs. Treatment with ozoralizumab has been shown to provide beneficial effects in the treatment of rheumatoid arthritis (RA) comparable to those obtained with other TNFα inhibitors. Very few ISRs (2%) have been reported after ozoralizumab administration, and the drug has been shown to have acceptable safety and tolerability. In this study, in order to elucidate the mechanism underlying the reduced incidence of ISRs associated with ozoralizumab administration, we investigated the stoichiometry of two TNFα inhibitors (ozoralizumab and adalimumab, an anti-TNFα IgG) ICs and the induction by these drugs of Fcγ receptor (FcγR)-mediated immune responses on neutrophils. Ozoralizumab-TNFα ICs are smaller than adalimumab-TNFα ICs and lack an Fc portion, thus mitigating FcγR-mediated immune responses on neutrophils. We also developed a model of anti-TNFα antibody-TNFα IC-induced subcutaneous inflammation and found that ozoralizumab-TNFα ICs do not induce any significant inflammation at injection sites. The results of our studies suggest that ozoralizumab is a promising candidate for the treatment of RA that entails a lower risk of the IC-mediated immune cell activation that leads to unwanted immune responses.

Abstract 4322: AI assisted detection and characterization of tertiary lymphoid structures in patients with colorectal cancer

Abstract Tertiary Lymphoid Structures (TLSs) are ectopic immune cell aggregates that form in response to abnormal physiology and inflammation. TLS presence in the tumor environment has been associated with positive clinical outcomes and response to immune checkpoint inhibition therapy. Using a fluorescent multiplex panel consisting of Pan-cytokeratin (PanCK), Programmed Death Ligand-1 (PD-L1), cluster of differentiation (CD) 8, CD3, and CD68 we developed a HALO-based artificial intelligence (AI) classifier to detect TLSs in colorectal cancer (CRC) tissue. In conjunction with cell-type and spatial analyses, we used the AI TLS classifier to characterize several features of the TLS and peri-TLS environment. We trained the AI classifier using 51 examples of TLSs from 8 patients, and applied it to images of 21 CRC patient tissues. TLSs were detected in 9/21 patient samples. We then used cellular detection and spatial analysis to characterize features of the TLS, including the germinal center (GC), T cell zone and peri-TLS area. We detected a higher cell density (Mean=12250 ±2039 SD; cells/mm2) within the interior of AI-detected GCs relative to the 1st 200 μm outside of the AI-defined GC border (8005±1870). In addition, we examined the spatial distribution of CD3+ and CD8+ T cells relative to the TLS GC. We found elevated CD3+ cell density in the interior of the GC (5784±1649) and within 200 μm of the GC border (5126±2239) which decreased at distances >200 μm from the GC border. We found CD8+ density to be elevated within the 1st 200 μm of the GC border (1903±1182) and declined 400 μm away from the GC border (963±828). The spatial distribution of T cells surrounding an area of high cell density is indicative of a T cell zone surrounding germinal center. Further analysis of the AI-detected TLS GCs indicated that PD-L1+ cell density was highest within the germinal center (355± 534;9) relative to the exterior of the germinal center (1st 150μm outside germinal center:276± 441). We created an AI classifier that can detect TLS(s) within patient tissue in the absence of a B-cell marker and confirmed that our AI classifier was detecting TLSs by characterizing canonical features of TLSs. Further exploration of the TLS and peri-TLS area may offer insights into immune-tumor interaction within the tumor microenvironment, and paired detection of TLSs in a clinical cohort with outcome data may predict patient response to immune checkpoint inhibitor therapy. Citation Format: Samantha Burg, Nathaniel Hart, Astin Powers, Wenjun Zhang. AI assisted detection and characterization of tertiary lymphoid structures in patients with colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4322.

B cells and tertiary lymphoid structures are associated with survival in papillary thyroid cancer.

The function of B cells in papillary thyroid cancer (PTC) is controversial. The role of B-cell-related tertiary lymphoid structures (TLSs) is still unclear. Whether B cells exert their anti-tumor effect through forming TLS in PTC needs further investigation. We detected the percentage of B cells in PTC tissues by multi-parameter flow cytometry. Paraffin-embedded tumor tissues of 125 PTC patients were collected and stained with Haematoxylin-Eosin (H&E) for inflammatory infiltration analysis in combination with clinical features. Multiplexed immunohistochemistry (mIHC) was performed to verify the TLSs in above inflammatory infiltration. Correlation of B cells and TLSs with prognosis was analyzed using the TCGA database. We observed that PTC patients with higher expression of B lineage cell genes had improved survival and the percentage of B cells in the PTC tumor tissues was variable. Moreover, PTC tumor tissues with more B cells were surrounded by immune cell aggregates of varying sizes. We furtherly confirmed the immune cell aggregates as TLSs with different maturation stages. By analyzing PTC data from TCGA database, we found the maturation stages of TLSs were associated with genders and clinical stages among PTC patients. Moreover, patients with high TLSs survived longer and had a better prognosis. B cells are associated with the existence of TLSs which have different maturation stages in PTC. Both B cells and TLSs are associated with the survival rate of PTC. These observations indicate that the anti-tumor effects of B cells in PTC are associated with TLSs formation.

Spatial mapping reveals granuloma diversity and histopathological superstructure in human tuberculosis.

The hallmark of tuberculosis (TB) is the formation of immune cell-enriched aggregates called granulomas. While granulomas are pathologically diverse, their tissue-wide heterogeneity has not been spatially resolved at the single-cell level in human tissues. By spatially mapping individual immune cells in every lesion across entire tissue sections, we report that in addition to necrotizing granulomas, the human TB lung contains abundant non-necrotizing leukocyte aggregates surrounding areas of necrotizing tissue. These cellular lesions were more diverse in composition than necrotizing lesions and could be stratified into four general classes based on cellular composition and spatial distribution of B cells and macrophages. The cellular composition of non-necrotizing structures also correlates with their proximity to necrotizing lesions, indicating these are foci of distinct immune reactions adjacent to necrotizing granulomas. Together, we show that during TB, diseased lung tissue develops a histopathological superstructure comprising at least four different types of non-necrotizing cellular aggregates organized as satellites of necrotizing granulomas.

Inflammatory monocytes promote granuloma control of Yersinia infection.

Granulomas are organized immune cell aggregates formed in response to chronic infection or antigen persistence. The bacterial pathogen Yersinia pseudotuberculosis (Yp) blocks innate inflammatory signalling and immune defence, inducing neutrophil-rich pyogranulomas (PGs) within lymphoid tissues. Here we uncover that Yp also triggers PG formation within the murine intestinal mucosa. Mice lacking circulating monocytes fail to form defined PGs, have defects in neutrophil activation and succumb to Yp infection. Yersinia lacking virulence factors that target actin polymerization to block phagocytosis and reactive oxygen burst do not induce PGs, indicating that intestinal PGs form in response to Yp disruption of cytoskeletal dynamics. Notably, mutation of the virulence factor YopH restores PG formation and control of Yp in mice lacking circulating monocytes, demonstrating that monocytes override YopH-dependent blockade of innate immune defence. This work reveals an unappreciated site of Yersinia intestinal invasion and defines host and pathogen drivers of intestinal granuloma formation.

Characteristics of immune-infiltrating cells in the tumor microenvironment of appendiceal cancer with peritoneal disease.

217 Background: Appendiceal cancers (AC) are rare, associated with peritoneal metastases, and respond poorly to current systemic treatments. There are limited studies on the characteristics and prognostic impact of infiltrating immune cells within the tumour microenvironment (TME) of AC with peritoneal metastases. Infiltrating immune cells are associated with survival outcomes and predict the effectiveness of immunotherapies in solid tumours. Different definitions have been developed to describe infiltrating immune cells and associated structures such as tertiary lymphoid aggregates (TLS). The aim of our study was to describe the immune TME in patients with AC peritoneal metastases to understand mechanisms of sensitivity and resistance to chemoimmunotherapies. Methods: Fresh tissue was collected during cytoreductive surgery from consenting patients with AC with peritoneal disease (Jul 2020-Feb 2022). Uncultured Day 0 tissues were fixed in formalin prior to paraffin embedding (FFPE). Haematoxylin and eosin (H&E) staining was performed on sections of FFPE tissue. Sections were evaluated by light microscopy for the presence of immune cells based on morphological appearance. Patterns of infiltrating immune cells were classified based on location (peritumoural, intratumoural, perimucinous, stromal), and aggregation (none, immature, mature). Immune cells forming aggregates were counted and the diameter of aggregates measured using QuPath. Results: We evaluated 25 patients with AC peritoneal disease during the study period. Immune cells were detected in tissues from all except 3 patients; 16 cases (78 immune areas) were low grade (LG) disease of primary appendiceal mucinous neoplasms, and 6 cases (29 immune areas) were high grade (HG) from primary appendiceal adenocarcinomas (Table). Immune cell aggregates had a median diameter of 209 mm (75-491) and median cell count of 892 cells (46-6490). Conclusions: This study represents the first formal description and characterisation of infiltrating immune cells in appendiceal cancer peritoneal metastases. Perimucinous immune cells were not observed in HG disease despite presence of mucin. Intratumoural immune cells were not observed in LG disease, consistent with these tumours being more resistant to chemotherapy. We plan ongoing work correlating the immune features with clinical outcomes alongside immunohistochemical and transcriptomic characterisation of the aggregates. [Table: see text]

De-stressing plaques attenuates atherosclerosis progression

Atherogenesis is accompanied by formation of immune cell aggregates, so-called artery tertiary lymphoid organs (ATLOs), in the outermost layer of diseased arteries. In a recent study, Mohanta, Habenicht, and colleagues revealed that there are distinct interactions between ATLOs and the autonomous nervous system, which are critically implicated in atherosclerosis progression.

Stromal and Immune Cell Dynamics in Tumor Associated Tertiary Lymphoid Structures and Anti-Tumor Immune Responses.

Tertiary lymphoid structures (TLS) are ectopic lymphoid organs that have been observed in chronic inflammatory conditions including cancer, where they are thought to exert a positive effect on prognosis. Both immune and non-immune cells participate in the genesis of TLS by establishing complex cross-talks requiring both soluble factors and cell-to-cell contact. Several immune cell types, including T follicular helper cells (Tfh), regulatory T cells (Tregs), and myeloid cells, may accumulate in TLS, possibly promoting or inhibiting their development. In this manuscript, we propose to review the available evidence regarding specific aspects of the TLS formation in solid cancers, including 1) the role of stromal cell composition and architecture in the recruitment of specific immune subpopulations and the formation of immune cell aggregates; 2) the contribution of the myeloid compartment (macrophages and neutrophils) to the development of antibody responses and the TLS formation; 3) the immunological and metabolic mechanisms dictating recruitment, expansion and plasticity of Tregs into T follicular regulatory cells, which are potentially sensitive to immunotherapeutic strategies directed to costimulatory receptors or checkpoint molecules.

Lymphotoxin-alpha expression in the meninges causes lymphoid tissue formation and neurodegeneration.

Organized meningeal immune cell infiltrates are suggested to play an important role in cortical grey matter pathology in the multiple sclerosis brain, but the mechanisms involved are as yet unresolved. Lymphotoxin-alpha plays a key role in lymphoid organ development and cellular cytotoxicity in the immune system and its expression is increased in the CSF of naïve and progressive multiple sclerosis patients and post-mortem meningeal tissue. Here we show that persistently increased levels of lymphotoxin-alpha in the cerebral meninges can give rise to lymphoid-like structures and underlying multiple sclerosis-like cortical pathology. Stereotaxic injections of recombinant lymphotoxin-alpha into the rat meninges led to acute meningeal inflammation and subpial demyelination that resolved after 28 days, with demyelination being dependent on prior subclinical immunization with myelin oligodendrocyte glycoprotein. Injection of a lymphotoxin-alpha lentiviral vector into the cortical meningeal space, to produce chronic localized overexpression of the cytokine, induced extensive lymphoid-like immune cell aggregates, maintained over 3 months, including T-cell rich zones containing podoplanin + fibroblastic reticular stromal cells and B-cell rich zones with a network of follicular dendritic cells, together with expression of lymphoid chemokines and their receptors. Extensive microglial and astroglial activation, subpial demyelination and marked neuronal loss occurred in the underlying cortical parenchyma. Whereas subpial demyelination was partially dependent on previous myelin oligodendrocyte glycoprotein immunization, the neuronal loss was present irrespective of immunization. Conditioned medium from LTα treated microglia was able to induce a reactive phenotype in astrocytes. Our results show that chronic lymphotoxin-alpha overexpression alone is sufficient to induce formation of meningeal lymphoid-like structures and subsequent neurodegeneration, similar to that seen in the progressive multiple sclerosis brain.

Abstract 3820: Spatial transcriptomics of advanced hepatocellular carcinomas distinguishes intercellular interactions in responders and non-responders to cabozantinib and nivolumab neoadjuvant therapy

Abstract The recent development of genome-wide spatial transcriptomics (ST) approaches enable near single-cell gene expression profiling to infer cellular composition and intercellular interactions that drive cancer development and responses to therapy. This study applied ST on 10 surgical biospecimens from a clinical trial with neoadjuvant therapy with cabozantinib (multi-kinase inhibitor) and nivolumab (anti-PD-1 monoclonal antibody) with advanced hepatocellular carcinoma (HCC). Within our cohort, 6 of the samples were obtained from non-responders and 4 with demonstrated pathological response were previously associated with immune infiltration using spatial proteomics technologies. Our analysis with ST was performed to determine the specific pathways that drive immune infiltration in responders and to map intercellular interactions relevant for response and resistance to the combined therapy.Analysis of these data uncovered three main differences between responders and non-responders. First, to better understand the tumor mechanisms of response and resistance, we performed differential expression and pathway analysis only in the subset of tumor clusters from responders versus non-responders. In responders, we observed enrichment for pathways associated with immune response (TNF-alpha, IFN-gamma, T cell differentiation), while in non-responders the deregulated pathways are associated with cell growth, transcriptional activity and hypoxia (Myc, E2F, oxidative phosphorylation). Second, the intercellular interaction analyses indicate that CD8-HLA interactions are more abundant in responders, while interactions activating VEGFR, the main target of cabozantinib, are enriched in non-responders. The interaction profiles are evidence that in responders the tumor cells express tumor specific antigens that are recognized by the cytotoxic cells which activity is enhanced by nivolumab. In non-responders, the activation of the VEGF pathway is an indication that the tumor cells have developed mechanism of resistance to cabozantinib. Third, responding tumors have higher densities of immune and stromal cells, and the immune cells are enriched with aggregates composed of both B and T cells. The regions surrounded by these immune aggregates are transcriptionally distinct from regions enriched for stromal cells, suggesting that tumor gene expression profile drives immune infiltration.Overall, the ST analysis of neoadjuvant HCC treated samples detects tumor induced immune cell immune infiltration in responders compared to non-responders with enrichment of cytotoxic interactions to eliminate the tumor cells. It also identifies intercellular interactions suggestive of resistance to anti-VEGF blockade. Citation Format: Luciane T. Kagohara, Shuming Zhang, Long Yuan, Qingfeng Zhu, Robert Anders, Daniel Shu, Aleksander S. Popel, Elizabeth M. Jaffee, Mark Yarchoan, Elana J. Fertig. Spatial transcriptomics of advanced hepatocellular carcinomas distinguishes intercellular interactions in responders and non-responders to cabozantinib and nivolumab neoadjuvant therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3820.

Abstract LB064: B cells synergize with T cell regulation at immune hotspots in lung squamous cell carcinoma

Abstract The interplay of immune cell subpopulations underpinning heterogeneous immune infiltration is poorly understood, hindering the establishment of robust prognostic markers and therapeutic targets. To study the prognostic implications and constitution of immune cell aggregates in lung squamous cell carcinoma, we integrated transcriptomic data and deep-learning-based spatial analysis. With the automatically identified tumor and immune cells on hematoxylin and eosin (H&E) diagnostic whole slide images, we were able to map regions with lymphoid aggregates (immune hotspots - IHs) and tumor aggregates (cancer hotspots) using the Getis-Ord hotspot analysis (Getis and Ord, 1992; Nawaz et al., 2015), in 462 TCGA samples. IHs were further categorized into intratumoral (intra-IH) and peritumoral (peri-IH) based on whether they overlapped with cancer hotspots. The intra-IH score, quantifying the fraction of IHs at the cancer interface, was associated with poor overall survival independently of age, stage, and packs per year (p < 0.01, Hazard Ratio (HR) = 2.1 [1.3-3.2]). The increased score was coupled with upregulated transcriptional signals of B cells and T regulatory cells, and expression of B cell-related genes CXCR5, MS4A1 (CD20), CD79b. To investigate cellular compositions of IHs, we selected 10 TRACERX patients with high intra-IH score based on H&E and performed immunohistochemistry on two serial sections followed by a deep learning model to locate subpopulations of T cells (CD4+FOXP3-, CD4+FOXP3+, CD8+) and B cells (CD20+CXCR5-, CD20+CXCR5+, and CD79b+). By spatially aligning these IHC images with H&E, we observed higher abundances of CD20+CXCR5+ and CD79b+ B cells at peri-IHs than intra-IHs (p<0.01; p<0.05), whereas none of the T cell subtypes showed a difference in localization. Tertiary lymphoid structures (TLSs) accounted for a minor proportion of peri-IHs (5.04 +- 4.38%) and intra-IHs (0.43 +- 0.61%). Furthermore, percentages of CD20+CXCR5+ B cells were significantly higher at peri-IHs than intra-IHs when TLSs were excluded (p=0.007), suggesting that the enrichment of CD20+CXCR5+ B cells at peri-IHs was not fully explained by the presence of TLSs. To test if IHs have a role in the regulation of anti-tumor activity of T cytotoxic cells, we further measured the ratio of CD8+ to CD4+FOXP3+ T cells infiltrating into the tumor nest. The ratio at tumor regions adjacent to intra-IH was lower than the rest of the tumor nest (p<0.001), suggesting a suppressive effect of intra-IH on tumor-infiltrating CD8+ T cells. Taken together, our results signified a protumor role of intratumoral lymphoid aggregates beyond TLSs, which may be attributed to the spatial interplay between CD8+ T cells, Tregs and B cells. Immune hotspots can serve as a prognostic indicator and potential therapeutic target in lung squamous cell carcinoma. Citation Format: Hanyun Zhang, Khalid AbdulJabbar, David A. Moore, Ayse Akarca, Katey Enfield, Mariam Jamal-Hanjani, Shan E Ahmed Raza, Selvaraju Veeriah, Dhruva Biswas, Roberto Salgado, Nicholas McGranahan, TRACERx Consortium, John Le Quesne, Charles Swanton, Teresa Marafioti, Yinyin Yuan. B cells synergize with T cell regulation at immune hotspots in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB064.

Abstract 627: Custom Vectra® Polaris™ fluorescent multiplex IHC panel identifies mature tertiary lymphoid structures in colorectal cancer

Abstract Introduction: Tertiary lymphoid structures (TLS) are promising prognostic indicators of positive outcomes for patients with solid tumors including colorectal cancer (CRC) [1]. Large-scale retrospective analysis shows patients with mature TLS in particular respond to PD-1/PD-L1 antibody treatment with improved objective response, progression-free and overall survival [2]. Since not all patients respond to PD-1/PD-L1 antibody treatment, identifying patients with mature TLS is clinically relevant as it enables selection of patients likely to respond to immune checkpoint blockade. Mature TLS are composed of T cells (CD3+), follicular B cells (CD20+), germinal center B cells (CD23+), and follicular dendritic cells (CD21+) [3]. The ability to identify and evaluate TLS is limited by detection methods which traditionally employ hematoxylin and eosin (H&E) staining for visual quantification of immune aggregates, potentially significantly underestimating their quantity [4]. Multiplexed fluorescent immunohistochemistry (IHC) detection assays have the capability to precisely quantify mature TLS within the TME. Colorectal cancer specimens expressing a dynamic range of TLS are stained with a novel custom Vectra® Polaris™ multiplex immunofluorescence panel detecting CD20, CD21, CD23, CD3 and pan-cytokeratin. Identification of mature TLS with CD3+CD20+CD21+ and CD3+CD20+CD23+ expression in the TME is reported. Results: Vectra® Polaris™ TLS multiplex immunofluorescence panel successfully identified mature TLS in the TME of colorectal cancer patient samples. TLS were quantified via custom analytics algorithms generated with Indica HALO software. Conclusion: Characteristics of TME immunity in colorectal cancer differentially impact an individual patient’s odds of survival [5]. The novel Vectra® Polaris™ multiplex assay in this study shows a detailed picture of mature TLS in patient cancer samples. Future applications of this panel include investigations of TLS associated with successful anti-tumor immune development and therapeutic response to treatment. [1] Oncoimmunology. 2020; 9(1): 1724763 [2] Nat Cancer. 2, 794-802 (2021) [3] Oncoimmunology. 2021; 10(1): 1900508. [4] Mod Pathol 2017 Sep;30(9):1204-1212. doi: 10.1038/modpathol.2017.43. [5] Cell. 2020 Sep 3;182(5):1341-1359.e19. doi: 10.1016/j.cell.2020.07.005 Citation Format: Sara G. Pollan, Arezoo Hanifi, James Hargrove, Erinn A. Parnell, Jessica Lin, Josette William Ragheb, Qingyan Au. Custom Vectra® Polaris™ fluorescent multiplex IHC panel identifies mature tertiary lymphoid structures in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 627.

Radioresistant cells initiate lymphocyte-dependent lung inflammation and IFNγ-dependent mortality in STING gain-of-function mice

Pediatric patients with constitutively active mutations in the cytosolic double-stranded-DNA-sensing adaptor STING develop an autoinflammatory syndrome known as STING-associated vasculopathy with onset in infancy (SAVI). SAVI patients have elevated interferon-stimulated gene expression and suffer from interstitial lung disease (ILD) with lymphocyte predominate bronchus-associated lymphoid tissue (BALT). Mice harboring SAVI mutations (STING V154M [VM]) that recapitulate human disease also develop lymphocyte-rich BALT. Ablation of either T or B lymphocytes prolongs the survival of SAVI mice, but lung immune aggregates persist, indicating that T cells and B cells can independently be recruited as BALT. VM T cells produced IFNγ, and IFNγR deficiency prolonged the survival of SAVI mice; however, T-cell-dependent recruitment of infiltrating myeloid cells to the lung was IFNγ independent. Lethally irradiated VM recipients fully reconstituted with wild type bone-marrow-derived cells still developed ILD, pointing to a critical role for VM-expressing radioresistant parenchymal and/or stromal cells in the recruitment and activation of pathogenic lymphocytes. We identified lung endothelial cells as radioresistant cells that express STING. Transcriptional analysis of VM endothelial cells revealed up-regulation of chemokines, proinflammatory cytokines, and genes associated with antigen presentation. Together, our data show that VM-expressing radioresistant cells play a key role in the initiation of lung disease in VM mice and provide insights for the treatment of SAVI patients, with implications for ILD associated with other connective tissue disorders.

Morphological Modulation of Broiler Organs in Response to an Organic Acid–Phytogen Composite in Healthy Broilers

In this study, we investigated broiler growth traits, intestinal health and immune aggregates in response to supplementation of Origanum vulgare and Cinnamomum verum, citric acid and malic acid in drinking water at varying levels and durations on broiler. We randomly allocated 140 day-old broilers into four (4) groups, with each group having seven replicates (n = 5). A basal diet and water were provided to all the groups ad libitum in accordance with standard farming practices. Keeping group I as the control, group II was supplemented with 1 mL/3.3 L of water for 1–4 days, 1 mL/4 L from 15–18 days and 1 mL/4 L water from 26–29 days. Group III and IV were supplemented with 1 mL/4 L of water and 1 mL/6 L of water for 35 days, respectively. After the completion of the trial (day 35), two broilers per replicate (14 birds per group) were slaughtered to sample the visceral organs. The results demonstrated that the feed conversion ratio (FCR) and body weight gain improved (p ≤ 0.05) during the fourth and fifth week of the trial in group III in comparison to the control group. Although the small intestine villus height and villus-height-to-crypt-depth ratio improved (p ≤ 0.05) in group III, the muscularis externa thickness of the ileum was highest (p ≤ 0.05) in the control group. Similarly, the lymphatic follicular and nodular area of the immune organs improved (p ≤ 0.05) in group III. In all segments of the small intestine (duodenal, jejunal and ileal), the number of acidic and total goblet cells was highest (p ≤ 0.05) in group II. In conclusion, dietary Origanum vulgare, Cinnamomum verum, citric acid and malic acid at the dose of 1 mL/4 L of drinking water (group III) was the most effective in improving the growth performance, gut development and associated immune components in broilers.