Formation Of Tertiary Lymphoid Structures Research Articles

Tertiary lymphoid structures as a potential prognostic biomarker for combined hepatocellular-cholangiocarcinoma.

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA), as a rare primary hepatic tumor, is challenging to accurately assess in terms of the clinical outcomes and prognostic risk factors in patients. This study aimed to clarify the function of tertiary lymphoid structure (TLS) status in predicting the outcome of cHCC-CCA and to preliminarily explore the possible mechanism of TLS formation. The TLSs, with different spatial distributions and densities, of 137 cHCC-CCA were quantified, and their association with prognosis was assessed by Cox regression and Kaplan-Meier analyses. We further validated TLS possible efficacy in predicting immunotherapy responsiveness in two cHCC-CCA case reports. TLS composition and its relationship to CXCL12 expression were analysed by fluorescent multiplex immunohistochemistry. A high intratumoural TLS score was correlated with prolonged survival, whereas a high TLS density in adjacent tissue indicated a worse prognosis in cHCC-CCA. Mature TLSs were related to favorable outcomes and showed more CD8 + T cells infiltrating tumor tissues. We further divided the cHCC-CCA patients into four immune grades by combining the peri-TLS and intra-TLS, and these grades were an independent prognostic factor. In addition, our reported cases suggested a potential value of TLS in predicting immunotherapy response in cHCC-CCA patients. Our findings suggested that CXCL12 expression in cHCC-CCA tissue was significantly correlated with TLS presence. The spatial distribution and density of TLSs revealing the characteristics of the cHCC-CCA immune microenvironment, significantly correlated with prognosis and provided a potential immunotherapy response biomarker for cHCC-CCA.

TCL1A-expressing B cells are critical for tertiary lymphoid structure formation and the prognosis of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a malignant tumor with a poor prognosis. Traditional treatments have limited effectiveness. Regulation of the immune response represents a promising new approach for OSCC treatment. B cells are among the most abundant immune cells in OSCC. However, the role of B cells in OSCC treatment has not been fully elucidated. Single-cell RNA sequencing analysis of 13 tissues and 8 adjacent normal tissues from OSCC patients was performed to explore differences in B-cell gene expression between OSCC tissues and normal tissues. We further investigated the relationship between differentially expressed genes and the immune response to OSCC. We utilized tissue microarray data for 146 OSCC clinical samples and RNA sequencing data of 359 OSCC samples from The Cancer Genome Atlas (TCGA) to investigate the role of T-cell leukemia 1A (TCL1A) in OSCC prognosis. Multiplex immunohistochemistry (mIHC) was employed to investigate the spatial distribution of TCL1A in OSCC tissues. We then investigated the effect of TCL1A on B-cell proliferation and trogocytosis. Finally, lentiviral transduction was performed to induce TCL1A overexpression in B lymphoblastoid cell lines (BLCLs) to verify the function of TCL1A. Our findings revealed that TCL1A was predominantly expressed in B cells and was associated with a better prognosis in OSCC patients. Additionally, we found that TCL1A-expressing B cells are located at the periphery of lymphatic follicles and are associated with tertiary lymphoid structures (TLS) formation in OSCC. Mechanistically, upregulation of TCL1A promoted the trogocytosis of B cells on dendritic cells by mediating the upregulation of CR2, thereby improving antigen-presenting ability. Moreover, the upregulation of TCL1A expression promoted the proliferation of B cells. This study revealed the role of B-cell TCL1A expression in TLS formation and its effect on OSCC prognosis. These findings highlight TCL1A as a novel target for OSCC immunotherapy.

Coordinated chemokine expression defines macrophage subsets across tissues.

Lung-resident macrophages, which include alveolar macrophages and interstitial macrophages (IMs), exhibit a high degree of diversity, generally attributed to different activation states, and often complicated by the influx of monocytes into the pool of tissue-resident macrophages. To gain a deeper insight into the functional diversity of IMs, here we perform comprehensive transcriptional profiling of resident IMs and reveal ten distinct chemokine-expressing IM subsets at steady state and during inflammation. Similar IM subsets that exhibited coordinated chemokine signatures and differentially expressed genes were observed across various tissues and species, indicating conserved specialized functional roles. Other macrophage types shared specific IM chemokine profiles, while also presenting their own unique chemokine signatures. Depletion of CD206hi IMs in Pf4creR26EYFP+DTR and Pf4creR26EYFPCx3cr1DTR mice led to diminished inflammatory cell recruitment, reduced tertiary lymphoid structure formation and fewer germinal center B cells in models of allergen- and infection-driven inflammation. These observations highlight the specialized roles of IMs, defined by their coordinated chemokine production, in regulating immune cell influx and organizing tertiary lymphoid tissue architecture.

Establishment of homotrimer collagen type I signature and its association with clinical manifestation and tertiary lymphoid structures formation in liver cancer

Establishment of homotrimer collagen type I signature and its association with clinical manifestation and tertiary lymphoid structures formation in liver cancer

CCL9/CCR1 axis-driven chemotactic nanovesicles for attenuating metastasis of SMAD4-deficient colorectal cancer by trapping TGF-β

SMAD4 deficiency in colorectal cancer (CRC) is highly correlated with liver metastasis and high mortality, yet there are few effective precision therapies available. Here, we show that CCR1+-granulocytic myeloid-derived suppressor cells (G-MDSCs) are highly infiltrated in SMAD4-deficient CRC via CCL15/CCR1 and CCL9/CCR1 axis in clinical specimens and mouse models, respectively. The excessive TGF-β, secreted by tumor-infiltrated CCR1+-G-MDSCs, suppresses the immune response of cytotoxic T lymphocytes (CTLs), thus facilitating metastasis. Hereby, we develop engineered nanovesicles displaying CCR1 and TGFBR2 molecules (C/T-NVs) to chemotactically target the tumor driven by CCL9/CCR1 axis and trap TGF-β through TGF-β-TGFBR2 specific binding. Chemotactic C/T-NVs counteract CCR1+-G-MDSC infiltration through competitive responding CCL9/CCR1 axis. C/T-NVs-induced intratumoral TGF-β exhaustion alleviates the TGF-β-suppressed immune response of CTLs. Collectively, C/T-NVs attenuate liver metastasis of SMAD4-deficient CRC. In further exploration, high expression of programmed cell death ligand-1 (PD-L1) is observed in clinical specimens of SMAD4-deficient CRC. Combining C/T-NVs with anti-PD-L1 antibody (aPD-L1) induces tertiary lymphoid structure formation with sustained activation of CTLs, CXCL13+-CD4+ T, CXCR5+-CD20+ B cells, and enhanced secretion of cytotoxic cytokine interleukin-21 and IFN-γ around tumors, thus eradicating metastatic foci. Our strategy elicits pleiotropic antimetastatic immunity, paving the way for nanovesicle-mediated precision immunotherapy in SMAD4-deficient CRC.

Lymphatic vessels in the age of cancer immunotherapy.

Lymphatic transport maintains homeostatic health and is necessary for immune surveillance, and yet lymphatic growth is often associated with solid tumour development and dissemination. Although tumour-associated lymphatic remodelling and growth were initially presumed tosimply expand a passive route for regional metastasis, emerging research puts lymphatic vessels and their active transport at the interface of metastasis, tumour-associated inflammation and systemic immune surveillance. Here, we discuss active mechanisms through which lymphatic vessels shape their transport function to influence peripheral tissue immunity and the current understanding of how tumour-associated lymphatic vessels may both augment and disrupt antitumour immune surveillance. We end by looking forward to emerging areas of interest in the field of cancer immunotherapy in which lymphatic vessels and their transport function are likely key players: the formation of tertiary lymphoid structures, immune surveillance in the central nervous system, the microbiome, obesity and ageing. The lessons learnt support a working framework that defines the lymphatic system as a key determinant of both local and systemic inflammatory networks and thereby a crucial player in the response to cancer immunotherapy.

Abstract LB065: Tumor-infiltrating lymphocytes (TIL) engineered with regulatable membrane-bound IL15 (mbIL15) and LIGHT (TNFSF14) show enhanced efficacy in fibroblast-containing cold tumors

Abstract Introduction: Adoptive cellular therapies (ACT) have encountered challenges in solid tumors due in part to the immunosuppressive tumor microenvironment (TME). We have developed OBX-115, TIL engineered to express mbIL15 regulatable using the cytoDRiVE® platform, which allows for TIL expansion, persistence, and anti-tumor efficacy under control of the FDA-approved small-molecule ligand, acetazolamide (ACZ), eliminating the need for co-administration of IL2 (NCT05470283). LIGHT, a tumor necrosis factor family member, interacts with lymphotoxin beta receptor (LTbR) and herpes virus entry mediator (HVEM) found on various TME cell types, including stromal cells such as cancer associated fibroblasts (CAF). In preclinical studies, LIGHT expression within a tumor has been linked to the formation of tertiary lymphoid structures and vascular normalization (Ramachandran Cancer Cell 2023), both associated with better clinical outcomes (Sautès-Fridman Nat Rev Cancer 2019). We hypothesized that engineering TIL with regulatable mbIL15 and LIGHT expression could enhance their efficacy by modifying the TME. Methods: TIL from colorectal (CRC) and head and neck squamous cell carcinoma (HNSCC) were transduced with retroviral vectors to express regulatable mbIL15 and LIGHT. ACZ-induced surface expression of mbIL15 and LIGHT in expanded TIL was examined using flow cytometry. Functional signaling of LIGHT was assessed through co-culture with Jurkat-HVEM-NF-kappaB reporter cells and LTbR+ HUVEC cells. In vitro, engineered TIL were tested in stromal-rich tumor models (CRC and HNSCC) by co-culturing with autologous patient-derived tumor/CAF hybrid spheroids. In vivo, antigen-independent TIL persistence was assessed in NSG mice without exogenous IL2. Syngeneic studies were performed to assess the efficacy of adoptively transferred mbIL15 and LIGHT-engineered Pmel cells (CD8+ T cells transgenic for a gp100-specific T cell receptor) in a subcutaneous cold tumor model (B16-F10). Results: Engineered TIL were successfully expanded without exogenous IL2. ACZ-dependent mbIL15 and LIGHT expression were confirmed, validating co-regulation and functionality in vitro. TIL engineered with mbIL15 and LIGHT displayed significantly increased cytotoxicity against autologous tumor/CAF spheroids compared with TIL expressing mbIL15 alone (p<0.005) in CRC and HNSCC tumor/CAF hybrid models. TIL with mbIL15 and LIGHT expanded in vivo and persisted for ≥42 days without exogenous IL2 support. Moreover, Pmel cells engineered with mbIL15 and LIGHT demonstrated durable anti-tumor efficacy in B16-F10 tumor-bearing mice, which was greater than Pmel cells engineered with mbIL15 alone (p<0.01). Conclusions: These preclinical results suggest that TIL engineered with regulatable mbIL15 and LIGHT using the cytoDRiVE platform have the potential to address the high unmet clinical need in cold tumors with suppressive TME, which are currently not amenable to ACT. Citation Format: Balazs Koscso, Zheng Ao, Carmela Passaro, Nirzari Shah, Ngoc Ly, Patricia Timpug, Bulent A. Aksoy, Dexue Sun, Dan Jun Li, Kerri-Lynn Sheahan, Violet Young, Theresa Ross, Benjamin Primack, Meghan Langley, Jeremy Tchaicha, Dhruv K. Sethi, Jan ter Meulen, Michelle Ols. Tumor-infiltrating lymphocytes (TIL) engineered with regulatable membrane-bound IL15 (mbIL15) and LIGHT (TNFSF14) show enhanced efficacy in fibroblast-containing cold 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 LB065.

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.

Neoadjuvant chemotherapy is associated with suppression of the B cell-centered immune landscape in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at advanced stages and associated with early distant metastasis and poor survival. Besides clinical factors, the tumor microenvironment (TME) emerged as a crucial determinant of patient survival and therapy response in many tumors, including PDAC. Thus, the presence of tumor-infiltrating lymphocytes and the formation of tertiary lymphoid structures (TLS) is associated with longer survival in PDAC. Although neoadjuvant therapy (NeoTx) has improved the management of locally advanced tumors, detailed insight into its effect on various TME components is limited. While a remodeling towards a proinflammatory state was reported for PDAC-infiltrating T cells, the effect of NeoTx on B cell subsets, including plasma cells, and TLS formation is widely unclear. We thus investigated the frequency, composition, and spatial distribution of PDAC-infiltrating B cells in primary resected (PR) versus neoadjuvant-treated patients using a novel multiplex immunohistochemistry panel. The NeoTx group displayed significantly lower frequencies of pan B cells, GC B cells, plasmablasts, and plasma cells, accompanied by a reduced abundance of TLS. This finding was supported by bulk RNA-sequencing analysis of an independent fresh frozen tissue cohort, which revealed that major B cell pathways were downregulated in the NeoTx group. We further observed that plasma cells frequently formed aggregates that localized close to TLS and that TLS+ patients displayed significantly higher plasma cell frequencies compared to TLS- patients in the PR group. Additionally, high densities of CD20+ intratumoral B cells were significantly associated with longer overall survival in the PR group. While CD20+ B cells held no prognostic value for NeoTx patients, an increased frequency of proliferating CD20+Ki67+ B cells emerged as an independent prognostic factor for longer survival in the NeoTx group. These results indicate that NeoTx differentially affects PDAC-infiltrating immune cells and may have detrimental effects on the existing B cell landscape and the formation of TLS. Gaining further insight into the underlying molecular mechanisms is crucial to overcome the intrinsic immunotherapy resistance of PDAC and develop novel strategies to improve the long-term outcome of PDAC patients.

Identification of a risk score model based on tertiary lymphoid structure-related genes for predicting immunotherapy efficacy in non-small cell lung cancer.

Tertiary lymphoid structures (TLSs) affect the prognosis and efficacy of immunotherapy in patients with non-small cell lung cancer (NSCLC), but the underlying mechanisms are not well understood. TLSs were identified and categorized online from the Cancer Digital Slide Archive (CDSA). Overall survival (OS) and disease-free survival (DFS) were analyzed. GSE111414 and GSE136961 datasets were downloaded from the GEO database. GSVA, GO and KEGG were used to explore the signaling pathways. Immune cell infiltration was analyzed by xCell, ssGSEA and MCP-counter. The analysis of WGCNA, Lasso and multivariate cox regression were conducted to develop a gene risk score model based on the SU2C-MARK cohort. TLS-positive was a protective factor for OS according to multivariate cox regression analysis (p = 0.029). Both the TLS-positive and TLS-mature groups exhibited genes enrichment in immune activation pathways. The TLS-mature group showed more activated dendritic cell infiltration than the TLS-immature group. We screened TLS-related genes using WGCNA. Lasso and multivariate cox regression analysis were used to construct a five-genes (RGS8, RUF4, HLA-DQB2, THEMIS, and TRBV12-5) risk score model, the progression free survival (PFS) and OS of patients in the low-risk group were markedly superior to those in the high-risk group (p < 0.0001; p = 0.0015, respectively). Calibration and ROC curves indicated that the combined model with gene risk score and clinical features could predict the PFS of patients who have received immunotherapy more accurately than a single clinical factor. Our data suggested a pivotal role of TLSs formation in survival outcome and immunotherapy response of NSCLC patients. Tumors with mature TLS formation showed more activated immune microenvironment. In addition, the model constructed by TLS-related genes could predict the response to immunotherapy and is meaningful for clinical decision-making.

Quantification and molecular correlates of tertiary lymphoid structures in primary prostate cancer.

To morphologically describe tertiary lymphoid structures (TLS) in prostatectomy specimens and correlate them with clinical and transcriptomic features. A total of 72 consecutive cases of entirely submitted radical prostatectomy (RP) patients tested with the Decipher Genomic Classifier were included in the study. Images were manually annotated using QuPath tools to denote tumor regions and each cluster of TLS. Clusters of lymphocytes that were surrounded on all four sides by tumor were defined as intra-tumor TLS (IT-TLS). Clusters of lymphocytes at the leading edge of carcinoma with either the prostatic pseudocapsule or benign parenchyma at one end were defined as peri-tumor TLS (PT-TLS). A classification algorithm to distinguish lymphocytes from non-lymphocytic cells using a supervised machine learning model was used. The associations between TLS formation and 265 gene expression-based signatures were examined. The magnitude of total TLS correlations with primary tumor gene expression signatures was moderate (~0.35-0.5) with several HLA, T-cell and B-cell Cluster signatures, showing positive correlation with various metrics for quantification of TLS. On the other hand, immune suppressive signatures (Treg, MDSC) were negatively correlated. While signatures for macrophages, NK cells and other immune cell types were uncorrelated for the most part. PT-TLS was associated with MHC signatures while IT TLS correlated with MHC andT-cell signatures. Clusters of inflammatory cells in the RP specimen can be divided spatially into PT TLS and IT-TLS, each with its unique molecular correlates of tumor immune microenvironment. The presence of TLS is positively correlated with MHC signatures, T- cell and B-cell cluster signatures but, negatively correlated with immune suppressive signatures. A subset of prostate cancer demonstrate a robust inflammatory response, and warrant further characterization in larger cohorts.

Abstract 7464: Tertiary lymphoid structures in pancreatic cancer: Biomarkers of immunogenic patients or direct contributors to anti-tumor immunity

Abstract 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.

Deep learning on tertiary lymphoid structures in hematoxylin-eosin predicts cancer prognosis and immunotherapy response

Tertiary lymphoid structures (TLSs) have been associated with favorable immunotherapy responses and prognosis in various cancers. Despite their significance, their quantification using multiplex immunohistochemistry (mIHC) staining of T and B lymphocytes remains labor-intensive, limiting its clinical utility. To address this challenge, we curated a dataset from matched mIHC and H&E whole-slide images (WSIs) and developed a deep learning model for automated segmentation of TLSs. The model achieved Dice coefficients of 0.91 on the internal test set and 0.866 on the external validation set, along with intersection over union (IoU) scores of 0.819 and 0.787, respectively. The TLS ratio, defined as the segmented TLS area over the total tissue area, correlated with B lymphocyte levels and the expression of CXCL13, a chemokine associated with TLS formation, in 6140 patients spanning 16 tumor types from The Cancer Genome Atlas (TCGA). The prognostic models for overall survival indicated that the inclusion of the TLS ratio with TNM staging significantly enhanced the models’ discriminative ability, outperforming the traditional models that solely incorporated TNM staging, in 10 out of 15 TCGA tumor types. Furthermore, when applied to biopsied treatment-naïve tumor samples, higher TLS ratios predicted a positive immunotherapy response across multiple cohorts, including specific therapies for esophageal squamous cell carcinoma, non-small cell lung cancer, and stomach adenocarcinoma. In conclusion, our deep learning-based approach offers an automated and reproducible method for TLS segmentation and quantification, highlighting its potential in predicting immunotherapy response and informing cancer prognosis.

Abstract 2661: Identification of a novel morphology of tertiary lymphoid structure in patients with hepatocellular carcinoma treated with neoadjuvant immune checkpoint blockade

Abstract Background: Tertiary lymphoid structures (TLS) are ectopic lymphoid follicles that arise in non-lymphoid tissue. TLS may contribute to response to immune checkpoint blockade (ICB) in solid tumors, but understanding of the life cycle of these structures, particularly the circumstances of their resolution and the functional contribution of this stage to the adaptive immune response, remains incomplete. Methods: We employed a multi-omics approach to evaluate TLS in the tumors of patients with hepatocellular carcinoma (HCC) treated with neoadjuvant ICB (n = 19) and untreated controls (n = 14). TLS density was assessed by immunohistochemistry and correlated with pathologic response, relapse free survival, and overall survival. Imaging mass cytometry was used to characterize distinct TLS morphologies in areas of tumor regression bed and viable tumor. Individual TLS from treated tumors were microdissected and characterized by T cell receptor (TCR) and immunoglobulin heavy chain (IGH) sequencing, and TCR clonotypes identified in TLS were further characterized by comparison against matched single cell RNA and TCR sequencing of peripheral blood and TIL. Results: Intratumoral TLS density was significantly increased in HCC tumors treated with neoadjuvant ICB compared to untreated controls (p = 0.05). Tumors with major or complete pathologic response to treatment had significantly higher intratumoral TLS density compared to tumors with partial pathologic response (p = 0.000246) and non-response (p = 0.0129). In the treatment group, patients with tumors in the top tertile of intratumoral TLS density had significantly longer relapse free survival (p = 0.021 by log-rank test). In the regression bed of treated tumors, an involuted TLS morphology was identified which was notable for dispersion of the B cell germinal center, retention of a T cell zone enriched for T-DC interactions, and increased expression of markers of T cell memory. In a subset of patients, immune repertoire sequencing demonstrated increased TCRβ clonality and BCR somatic hypermutation at involuted TLS. Highly expanded T cell clonotypes identified in TLS were found in the GZMK+ and GZMB+ CD8 T effector memory clusters in matched single cell data. Conclusions: These data suggest that in HCC tumors treated with neoadjuvant ICB, treatment may induce formation of intratumoral TLS, which are associated with superior pathologic response and relapse free survival. An involuted TLS morphology is identified in tumor regression bed with features consistent with dissolution of the B cell germinal center, persistent antigen presentation to T cells, and increased T cell memory formation. Taken together, these findings suggest a potential role for late-stage TLS in supporting consolidation of the intratumoral T cell repertoire after elimination of viable tumor. Citation Format: Daniel H. Shu, Won Jin Ho, Luciane T. Kagohara, Alexander Girgis, Sarah M. Shin, Ludmila Danilova, Jae W. Lee, Dimitrios N. N. Sidiropoulos, Sarah Mitchell, Kabeer Munjal, Kathryn Howe, Kayla J. Bendinelli, Hanfei Qi, Guanglan Mo, Janelle Montagne, Tamara Y. Lopez-Vidal, Qingfeng Zhu, Amanda L. Huff, Xuan Yuan, Alexei Hernandez, Erin M. Coyne, Neeha Zaidi, Daniel J. Zabransky, Logan L. Engle, Aleksandra Ogurtsova, Marina Baretti, Daniel Laheru, Jennifer N. Durham, Hao Wang, Robert Anders, Elizabeth M. Jaffee, Elana J. Fertig, Mark Yarchoan. Identification of a novel morphology of tertiary lymphoid structure in patients with hepatocellular carcinoma treated with neoadjuvant immune checkpoint blockade [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 2661.

Abstract 1148: Multimodal spatial transcriptomics and proteomics analysis of the resectable NSCLC ecosystem following neoadjuvant chemoimmunotherapy

Abstract Neoadjuvant and perioperative immunotherapy with checkpoint inhibitors (ICIs), combined with chemotherapy (CT), have improved outcomes for patients with resectable non-small cell lung cancer (NSCLC). Pathologic tumor response has been utilized in clinical trials as a surrogate of clinical efficacy. Yet, over half of treated patients experience non-response, highlighting the need for novel markers to identify those who are most likely to derive therapeutic benefit. To dissect the spatial complexity of the tumor-associated immune landscape following neoadjuvant chemoimmunotherapy (CT+ICI), we selected patients (n =3) with a range of residual viable tumor (%RVT) - pathological complete response (pCR; 0% RVT), partial response (20% RVT), and no-response (100% RVT). We performed multimodal spatial transcriptomics (ST) and proteomics (using a 35-plex protein panel) on the same tissue sections using the Visium CytAssist from 10X Genomics. We also examined spatial patterns of 20-plex panel of immune and stromal markers at single-cell resolution in the same samples using the COMET Lunaphore platform. In the pCR patient, we studied two tumor areas to assess the impact of tumor response heterogeneity on the immune landscape. Our analysis identified distinct immune profiles across three patients with varying %RVT, reflecting the heterogeneity of the immune response. We found robust gene-protein correlation, such as MS4A1 gene expression and CD20 protein levels, in our multimodal spatial-omics analysis. Intriguingly, the arrangement of B cells and their interaction with surrounding plasma cells (PCs) varied notably across patients. Unlike in the patients with partial or no response, pronounced tertiary lymphoid structure (TLS) formation and signatures were observed in the pCR patient, with the CXCL13-CXCR5 axis overlapping with the TLS profile, suggesting a functional interplay that may be crucial in mediating response to therapy. Intriguingly, in the pCR patient, PCs uniquely arrayed around dense B cell aggregates, marked by MS4A1, CR2, FCER1, and CXCL13. PCs were not only less abundant in non-PCR patients but they also did not array around lymphoid aggregates. Our high-resolution and multimodal analysis identified spatially-resolved expression and patterns for B lineage cells that may be associated, and thus, underlie response of resectable NSCLC to neoadjuvant CT+ICI. Ongoing efforts on this expanding cohort will shed further light on transcriptional states and immunogenomic roles of B lineage cells in neoadjuvant CT+ICI-treated NSCLC. Citation Format: Zahraa Rahal, Tieling Zhou, Sujuan Yang, Alejandra Serrano, Jiping Feng, Ansam Sinjab, John T. Le, Xin Sun, Michael Wang, Wei Hu, Jianjun Zang, Tullia Bruno, Hai T. Tran, Stephen G. Swisher, Cheuk Hong Leung, Heather Y. Lin, J. Jack Lee, Jing Wang, John Victor Heymach, Don Lynn Gibbons, Ignacio Ivan Wistuba, Annikka Weissferdt, Jared K. Burks, Luisa Maren Solis Soto, Humam Kadara, Tina Cascone. Multimodal spatial transcriptomics and proteomics analysis of the resectable NSCLC ecosystem following neoadjuvant chemoimmunotherapy [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 1148.

Abstract 111: Establishing orthotopic renal cell carcinoma xenograft in PBMC-humanized immunodeficient NSG-MHC I/II double knock-out mice

Abstract Clear cell renal cell carcinoma (ccRCC), the predominant histological subtype of RCC, is highly correlated with immune cells, an association that is linked to a worse prognosis. Our study aimed to establish an orthotopic ccRCC mouse model reconstituted with human peripheral blood mononuclear cells (PBMCs) and implanted with human ccRCC SKRC-59 cells under the kidney capsule in immunodeficient NSG-MHC I/II double knock-out (DKO) mice. The study was conducted using two experimental designs to optimize humanization and tumor formation. In the first experimental design, the mice were engrafted with SKRC-59 cells under the left kidney capsule 24 hours post-irradiation on study day 0. The PBMCs were then injected intravenously on study day 9. The second experimental design involved irradiating mice on study day -1, followed by engraftment with PBMCs either perfectly (6/6) or partially (3/6) matched human leukocyte antigens (HLA) with the tumors. Subsequently, SKRC-59 cells were placed under the kidney capsule on study day 0. Daily post-operative observations were conducted for six days following surgery to ensure the well-being of the mice. Tumor burden was assessed twice weekly using an in vivo imaging system, along with monitoring body weights and clinical signs. The humanization rates were evaluated 7, 14, and 21 days post-PBMC engraftment, as well as at the end of the study, using peripheral blood. Approximately 5 to 6 weeks post-tumor implantation, the mice were euthanized, and both left and right kidneys were collected and weighed for further analysis. The humanization rates were significantly higher when the PBMCs were engrafted on the same day as irradiation compared to 10 days post-irradiation. The PBMC humanization, and HLA-type PBMC donors did not affect tumor burden. Additionally, we found that distant metastasis in the lungs developed in several mice. Using flow cytometry and immunohistochemistry (IHC) to analyze tumor infiltrating leukocytes (TILs), a predominant number of CD3+ T cells were found in human CD45+ TILs, indicating that this model could be useful for evaluating T cell-mediated immunotherapies. Moreover, lympho-myeloid aggression (LMA) was observed in this peripheral blood leukocytes (PBL) mouse model, showing early stage tertiary lymphoid structures (TLS), which potentially provides a powerful tool to recapitulate TLS formation and study associated mechanisms and therapies. Our study demonstrated the growth of ccRCC SKRC-59 tumors under the renal capsule and observed TILs and metastatic patterns in PBMC humanized DKO mice. This PBMC-humanized orthotopic ccRCC xenograft model can recapitulate tumor microenvironment (TME), and be potentially used to assess the safety and efficacy of various immunotherapeutics. Citation Format: Wonyoung Kang, Yufei Wang, Yasmin N. Laimon, Oanh Pham, Ben Matran, Lauren Bottoms, Sheng Khang, Hsien-Chi Yuan, Nithyassree Murugan, Aseman B. Sheshdeh, Li-chin Yao, Jiwon Yang, Mingshan Cheng, Sabina Signoretti, Wayne A. Marasco, James G. Keck. Establishing orthotopic renal cell carcinoma xenograft in PBMC-humanized immunodeficient NSG-MHC I/II double knock-out mice [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 111.

Abstract 3988: Deep spatial immunophenotyping of lymphoid aggregates in pancreatic cancer using multi-omic integration of ultra high-plex proteomics and transcriptomics

Abstract The most well-described mechanism of immune evasion in PDAC is exclusion of immune cells by a desmoplastic stroma, but such immune exclusion is not uniform. Through single-nucleus RNA-seq and whole-transcriptome digital spatial profiling of patient tumors, we previously reported marked heterogeneity in the immune composition of PDAC. Here, we developed a novel multi-omics workflow integrating single cell spatial proteomics (96-plex, Akoya Phenocycler®-Fusion) and transcriptomics (990-plex, Nanostring CosMx) in primary patient PDAC tumors to systematically study spatial relationships and functional states of malignant, stromal, and immune cells. Consecutive sections from four independent tumors enabled whole slide proteomic assessment of &amp;gt;3.5M cells, and transcriptomic assessment of &amp;gt;200K cells. Strikingly, we observed an abundance of immune cell aggregates in all tissue sections, up to 57 within a 1 cm2 tumor section. Aggregates were diverse in composition with representation of myeloid cells, CD8+ T cells, and stromal cells, including fibroblasts and nerves. B/T lymphoid aggregates were well vascularized with co-localized CD31+CD34+ endothelial cells and appeared at various phases of maturation towards putative tertiary lymphoid structures (TLSs) containing CD38+ B cells, CD4+ T cells, and CD21+ follicular DCs. These tissues were enriched for interferon stimulated genes in T cells and type 1 interferon response signatures in malignant cells and cancer associated fibroblasts (CAFs) forming inflammatory multicellular hubs that have been associated with improved response to immune checkpoint blockade. Surprisingly, B cells, in addition to macrophages and CD8+ T cells, were major producers of interferon gamma (B cells: 13.7%, macrophages: 36.6%, CD8+ T cells: 12.1% of IFNG+ cells). Finally, to gain insight into the necessary factors for TLS formation in PDAC, we quantified the expression of chemokines and adhesion molecules associated with TLS initiation. Activated dendritic cells expressed CCL19 and CCL21, endothelial cells expressed CCL21, and in concert with inflammatory CAFs (iCAFs), CXCL12. Malignant cells expressed CXCL13 and CCL20 and displayed high levels of cell adhesion molecules ICAM1, VCAM1, and ICAM2. Together, our data suggests that the functions of lymphoid tissue organizer cells can be substituted by endothelial cells, dendritic cells, iCAFs, and, surprisingly, malignant cells. Our spatial immunophenotyping study of pancreatic cancer thus highlights the marked immune heterogeneity of the disease and suggests a central role for malignant and stromal cells in mediating diverse mechanisms of immune evasion and engagement. Functional studies will be conducted to investigate these mechanisms of tumor-immune crosstalk and identify novel immunotherapeutic vulnerabilities for this deadly disease. Citation Format: Dennis Gong, Jingyi Cao, Peter Wang, Niyati Jhaveri, Yue Hou, Bassem B. Cheikh, HaYeun Ji, Mark Gregory, Jason Reeves, Michael Patrick, Carina Shiau, Jennifer Su, Jimmy A. Guo, Joseph M. Beechem, Martin Hemberg, William L. Hwang, Ryan Park. Deep spatial immunophenotyping of lymphoid aggregates in pancreatic cancer using multi-omic integration of ultra high-plex proteomics and transcriptomics [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 3988.

Tertiary lymphoid structures in cancer: immune mechanisms and clinical implications.

Cancer is a major cause of death globally, and traditional treatments often have limited efficacy and adverse effects. Immunotherapy has shown promise in various malignancies but is less effective in tumors with low immunogenicity or immunosuppressive microenvironment, especially sarcomas. Tertiary lymphoid structures (TLSs) have been associated with a favorable response to immunotherapy and improved survival in cancer patients. However, the immunological mechanisms and clinical significance of TLS in malignant tumors are not fully understood. In this review, we elucidate the composition, neogenesis, and immune characteristics of TLS in tumors, as well as the inflammatory response in cancer development. An in-depth discussion of the unique immune characteristics of TLSs in lung cancer, breast cancer, melanoma, and soft tissue sarcomas will be presented. Additionally, the therapeutic implications of TLS, including its role as a marker of therapeutic response and prognosis, and strategies to promote TLS formation and maturation will be explored. Overall, we aim to provide a comprehensive understanding of the role of TLS in the tumor immune microenvironment and suggest potential interventions for cancer treatment.

Effects of immunogenic cell death-inducing chemotherapeutics on the immune cell activation and tertiary lymphoid structure formation in melanoma.

The infiltration and activation of immune cells in the tumor microenvironment (TIME) affect the prognosis of patients with cancer. Tertiary lymphoid structure (TLS) formation favors tumour- infiltrating-lymphocyte (TIL) recruitment and is regarded as an important indicator of good prognosis associated with immunotherapy in patients with tumors. Chemotherapy is currently one of the most commonly used clinical treatment methods. However, there have been no clear report to explore the effects of different types of chemotherapy on TLS formation in the TIME. This study examined the effects of immunogenic cell death (ICD)-inducing chemotherapeutics on immune cells, high-endothelial venules (HEV), and TLSs in mouse melanomas. Doxorubicin (an ICD inducer), gemcitabine (non-ICD inducer), and a combination of the two drugs was delivered intra-peritoneally to B16F1-loaded C57BL/6 mice. The infiltration of immune cells into tumor tissues was evaluated using flow cytometry. HEV and TLS formation was assessed using immunohistochemistry and multiple fluorescent immunohistochemical staining. Doxorubicin alone, gemcitabine alone, and the two-drug combination all slowed tumor growth, with the combined treatment demonstrating a more pronounced effect. Compared with the control group, the doxorubicin group showed a higher infiltration of CD8+ T cells and tissue-resident memory T cells (TRM) and an increase in the secretion of interferon-γ, granzyme B, and perforin in CD8+ T subsets and activation of B cells and dendritic cells. Doxorubicin alone and in combination with gemcitabine decreased regulatory T cells in the TIME. Moreover, doxorubicin treatment promoted the formation of HEV and TLS. Doxorubicin treatment also upregulated the expression of programmed cell death protein (PD)-1 in CD8+ T cells and programmed cell death protein ligand (PD-L)1 in tumor cells. These results indicate that doxorubicin with an ICD reaction promotes TLS formation and increases PD-1/PD-L1 expression in tumor tissues. The results demonstrate the development of a therapeutic avenue using combined immune checkpoint therapy.

Epigenetic targets to enhance antitumor immune response through the induction of tertiary lymphoid structures.

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates found in sites of chronic inflammation such as tumors and autoimmune diseases. The discovery that TLS formation at tumor sites correlated with good patient prognosis has triggered extensive research into various techniques to induce their formation at the tumor microenvironment (TME). One strategy is the exogenous induction of specific cytokines and chemokine expression in murine models. However, applying such systemic chemokine expression can result in significant toxicity and damage to healthy tissues. Also, the TLS formed from exogenous chemokine induction is heterogeneous and different from the ones associated with favorable prognosis. Therefore, there is a need to optimize additional approaches like immune cell engineering with lentiviral transduction to improve the TLS formation in vivo. Similarly, the genetic and epigenetic regulation of the different phases of TLS neogenesis are still unknown. Understanding these molecular regulations could help identify novel targets to induce tissue-specific TLS in the TME. This review offers a unique insight into the molecular checkpoints of the different stages and mechanisms involved in TLS formation. This review also highlights potential epigenetic targets to induce TLS neogenesis. The review further explores epigenetic therapies (epi-therapy) and ongoing clinical trials using epi-therapy in cancers. In addition, it builds upon the current knowledge of tools to generate TLS and TLS phenotyping biomarkers with predictive and prognostic clinical potential.