Neoadjuvant Immunotherapy Promotes the Formation of Mature Tertiary Lymphoid Structures in a Remodeled Pancreatic Tumor Microenvironment.

<div>Abstract<p>Pancreatic ductal adenocarcinoma (PDAC) is a rapidly progressing cancer that responds poorly to immunotherapies. Intratumoral tertiary lymphoid structures (TLS) have been associated with rare long-term PDAC survivors, but the role of TLS in PDAC and their spatial relationships within the context of the broader tumor microenvironment remain unknown. In this study, we report the generation of a spatial multiomic atlas of PDAC tumors and tumor-adjacent lymph nodes from patients treated with combination neoadjuvant immunotherapies. Using machine learning–enabled hematoxylin and eosin image classification models, imaging mass cytometry, and unsupervised gene expression matrix factorization methods for spatial transcriptomics, we characterized cellular states within and adjacent to TLS spanning distinct spatial niches and pathologic responses. Unsupervised learning identified TLS-specific spatial gene expression signatures that are significantly associated with improved survival in patients with PDAC. We identified spatial features of pathologic immune responses, including intratumoral TLS–associated B-cell maturation colocalizing with IgG dissemination and extracellular matrix remodeling. Our findings offer insights into the cellular and molecular landscape of TLS in PDACs during immunotherapy treatment.</p></div>

Background: In pancreatic ductal adenocarcinoma (PDAC), rare long-term survivors correlate with high intratumoral tertiary lymphoid structure (TLS) density. This finding prompted our investigation of clinically viable strategies to induce TLS in patients with immune-excluded tumors. We previously reported the induction of intratumoral TLS following administration of a neoadjuvant GM-CSF-secreting allogeneic vaccine (GVAX) to PDAC patients (NCT00727441). However, no clinical benefit was observed, likely owing to immune tolerance mechanisms governing the PDAC tumor microenvironment (TME). We previously observed upregulation of both the PD-1 and 4-1BB pathways with GVAX, and thus in a subsequent neoadjuvant trial combined GVAX with PD-1 blockade and 4-1BB agonism (NCT02451982) which was associated with pathologic responses. We hypothesized this combination strategy induced TLS of higher maturity and anti-tumor activity compared to GVAX alone. Methods: To explore how this therapeutic strategy affected TLS morphology and intercellular crosstalk, we leveraged the Visium spatial transcriptomics platform and a 35-marker customized TLS panel for imaging mass cytometry. We generated cellular and molecular maps of the TME after neoadjuvant treatment in 26 PDAC patients (GVAX n=19, GVAX+aPD1 n=2, GVAX+aPD1+a41BB n=5). To compare TLS maturation in parallel with secondary lymphoid organ-mediated tumor immunity, we also profiled tumor-adjacent lymph nodes. We applied unsupervised learning with non-negative matrix factorization (NMF) and trained AI-enabled image classification models to characterize cellular states within tissue structures of interest. Results: We identified spatial gene expression NMF patterns in PDAC TLS spanning across distinct morphologies and neoadjuvant treatment arms. Intratumoral TLS after GVAX were found to propagate activated B cells expressing immunoglobulins that infiltrated into malignant cellular niches. TLS NMF patterns were also associated with autoimmune disease signatures, such as diabetes, in a subset of patients. We scored TLS using tumor-draining lymph nodes as a reference and found increased maturation of TLS after PD-1 blockade, while addition of 4-1BB agonism significantly boosted the cytotoxic NK/T cell compartment compared to GVAX alone. Conclusions: We present machine learning approaches for spatial multi-omics analysis to characterize the TLS-enriched TME. We mined genome-wide spatial TLS gene expression patterns elucidating the spatial dynamics of humoral immunity of rare immunotherapy pathologic responders in PDAC. Altogether our findings shed light on the plasticity of TLS in neoadjuvant immunotherapy and suggest future immunotherapy approaches should target both humoral and cytotoxic NK/T cell compartments to augment responses in solid tumors. Citation Format: Dimitrios N. Sidiropoulos, Sarah M. Shin, Alexander Girgis, Daniel H. Shu, Janelle Montagne, Atul Deshpande, Jeanette A. Johnson, Lucie Dequiedt, Victoria Jacobs, Aleksandra Ogurtsova, Guanglan Mo, Xuan Yuan, Genevieve Stein-O’Brien, Mark Yarchoan, Qingfeng Zhu, Ashley Kiemen, Elizabeth M. Jaffee, Lei Zheng, Won Jin Ho, Robert Anders, Elana J. Fertig, Luciane T. Kagohara. Machine learning integrating spatial omics uncovers humoral immunity patterns in intratumoral tertiary lymphoid structures in pancreatic cancer pathologic responders [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 1159.

Background: Tertiary lymphoid structures (TLS) are ectopic immune cell aggregates in tumors that correlate with enhanced immunity and prognosis. Although TLS form via lymphotoxin (LT)-LTβR signaling, the TLS inducing molecules and cells remain unclear. Approach: To identify TLS inducing signals, we use pancreatic ductal adenocarcinoma (PDAC), a cold tumor where increased TLS density correlates with enhanced survival. To discover novel TLS inducers, we analyze bulk transcriptomes from human PDAC and 12 cold and hot cancers. We dissect the mechanisms in a Kras/p53-driven orthotopic PDAC mouse model, parabiosis, and inducible cell tracing. Results: In 328 analyzed human PDAC transcriptomes, we discover IL33–the alarmin released in inflamed tissues– among the most strongly correlated molecules with three distinct TLS signatures in PDAC (P<0.001) and 3887 human tumors (P<0.001). To test if this correlation is causative, we found anti-LTβR agonism induced intratumoral TLS density in wild-type but not Il33 -/- PDAC mice (P<0.001). To probe if IL33 served as an extracellular TLS trigger, we found recombinant IL33 (rIL33) boosted intratumoral TLS by up to 10-fold across six PDAC cell lines (P≤0.01–0.001) and reduced tumor growth. Thus, IL33 is a novel TLS-inducing alarmin. To identify the TLS inducer cells, we found that rIL33 dominantly expands intratumoral type 2 Innate Lymphoid cells (ILC2) expressing LT, suggesting ILC2 as TLS inducers. To test if ILC2 contribute to IL33-mediated TLS, we acutely depleted ILC2 using Nmur1 iCre-eGFP ROSA26 LSL-DTR mice and observed a ∼65% reduction in TLS during rIL33 treatment (P=0.006). Consistently, conditional LT depletion on ILC2 using Nmur1 iCre-eGFP Ltb fl/fl mice reduces TLS by ∼70% (P=0.006). Thus, IL33-activated ILC2s induce TLSs via LT. As rIL33 expands blood ILC2, we examined if ILC2 migrated to tumors. In parabiotic mice with PDACs in recipient pancreata, rIL33 treatment in donor mice induced hematogenous ILC2 migration to recipient PDACs (P=0.02). To trace the origin of intratumoral ILC2, we used photoconvertible KikGR mice. In rIL33-treated PDAC mice, we detected photoconverted ILC2 in tumors of gut-photoconverted but not control mice (P<0.001), demonstrating that ILC2 can migrate to PDACs through a gut-blood circuit. Furthermore, PDAC increases intrapancreatic ILC2 frequencies (P<0.001) and alters microbiota composition. Antibiotic-mediated depletion of the microbiota reduces blood ILC2 frequencies (P=0.01) and intratumoral TLS (P=0.02), suggesting that gut microbiota may regulate ILC2 migration to tumors. Finally, to exploit this lymphoneogenic pathway for immunotherapy, we demonstrated that engineered recombinant human IL33 expands intratumoral ILC2, TLS, and enhances anti-tumor activity in PDAC mice. Conclusions: In summary, we identify IL33 and ILC2 as novel molecule and cells that induce TLS in pancreatic cancer. As PDAC is a model cold cancer resistant to current immunotherapies, our findings position rIL33 as a promising immunotherapy to enhance TLS formation to treat cancer. Citation Format: Abderezak Zebboudj, Masataka Amisaki, Hiroshi Yano, Siqi Linsey Zhang, George Payne, Adrienne Kaya Chandra, Rebecca Yu, Pablo Guasp, Zachary M Sethna, Akihiro Ohmoto, Luis A. Rojas, Charlotte Cheng, Theresa Waters, Alexander Solovyov, Stephen Martis, Ashley Doane, Charlotte Reiche, Emmanuel Bruno, Martina Milighetti, Kevin Soares, Zagaa Odgerel, John Alec Moral, Julia Zaho, Mithat Gönen, Rui Gardner, Alexei V Tumanov, Abdul G Khan, Olivia Vergnolle, Elisabeth Nyakatura, Ivo C. Lorenz, Manuel Baca, Erin Patterson, Benjamin Greenbaum, David Artis, Taha Merghoub, Vinod P. Balachandran. Interleukin-33 activated ILC2s induce tertiary lymphoid structures in pancreatic cancer [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B079.

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.

Decision letter: Neutrophil-mediated fibroblast-tumor cell il-6/stat-3 signaling underlies the association between neutrophil-to-lymphocyte ratio dynamics and chemotherapy response in localized pancreatic cancer: A hybrid clinical-preclinical study

RORc-expressing immune cells negatively regulate tertiary lymphoid structure formation and support their pro-tumorigenic functions.

Background and aimsThe presence of tertiary lymphoid structures (TLSs) may confer survival benefit to patients with pancreatic ductal adenocarcinoma (PDAC), in an otherwise immunologically inert malignancy. Yet, the precise role in PDAC has not been elucidated. Here, we aim to investigate the structure and role of TLSs in human and murine pancreatic cancer.MethodsMulticolor immunofluorescence and immunohistochemistry were used to fully characterize TLSs in human and murine (transgenic [KPC (KrasG12D, p53R172H, Pdx-1-Cre)] and orthotopic) pancreatic cancer. An orthotopic murine model was developed to study the development of TLSs and the effect of the combined chemotherapy and immunotherapy on tumor growth.ResultsMature, functional TLSs are not ubiquitous in human PDAC and KPC murine cancers and are absent in the orthotopic murine model. TLS formation can be induced in the orthotopic model of PDAC after intratumoral injection of lymphoid chemokines (CXCL13/CCL21). Coadministration of systemic chemotherapy (gemcitabine) and intratumoral lymphoid chemokines into orthotopic tumors altered immune cell infiltration ,facilitating TLS induction and potentiating antitumor activity of chemotherapy. This resulted in significant tumor reduction, an effect not achieved by either treatment alone. Antitumor activity seen after TLS induction is associated with B cell-mediated dendritic cell activation.ConclusionsThis study provides supportive evidence that TLS induction may potentiate the antitumor activity of chemotherapy in a murine model of PDAC. A detailed understanding of TLS kinetics and their induction, owing to multiple host and tumor factors, may help design personalized therapies harnessing the potential of immune-oncology.

Predictive power of tertiary lymphoid structure signature for neoadjuvant chemotherapy response and immunotherapy benefit in HER2-negative breast cancer.

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.

Pancreatic ductal adenocarcinoma (PDA) is usually unresponsive to immunotherapeutic approaches. However, tertiary lymphoid structures (TLS) are associated with favorable patient outcomes in PDA. A better understanding of the B cell infiltrate and biological features of TLS formation is needed to further explore their potential and improve patient management. We analyzed tumor tissues (n = 55) and corresponding blood samples (n = 51) from PDA patients by systematical immunohistochemistry and multiplex cytokine measurements. The tissue was compartmentalized in “tumor” and “stroma” and separately examined. Clinical patient information was used to perform survival analyses. We found that the mere number of B cells is not associated with patient survival, but formation of TLS in the peritumoral stroma is a prognostic favorable marker in PDA patients. TLS-positive tissues show a higher density of CD8+ T cells and CD20+ B cells and a higher IL2 level in the peritumoral stroma than tissues without TLS. Compartmental assessment shows that gradients of IL2 expression differ with regard to TLS formation: TLS presence is associated with higher IL2 levels in the stromal than in the tumoral compartment, while no difference is seen in patients without TLS. Focusing on the stroma-to-serum gradient, only patients without TLS show significantly higher IL2 levels in the serum than in stroma. Finally, low circulatory IL2 levels are associated with local TLS formation. Our findings highlight that TLS are prognostic favorable and associated with antitumoral features in the microenvironment of PDA. Also, we propose easily accessible serum IL2 levels as a potential marker for TLS prediction.

<div>Abstract<p><i>De novo</i> formation of tertiary lymphoid structures (TLS) has been described in lung cancers. Intratumoral TLS seem to be functional and are associated with a long-term survival for lung cancer patients, suggesting that they represent an activation site for tumor-specific T cells. Here, we characterized T-cell recruitment to TLS in human lung cancer to identify the adhesion molecules and chemoattractants orchestrating this migration. We found that most TLS T cells were CD62L+ and mainly of CD4+ memory phenotype, but naive T cells were highly enriched in these structures as compared with the rest of the tumor. A specific gene expression signature associated with T cell presence was identified in TLS, which included chemokines (CCL19, CCL21, CXCL13, CCL17, CCL22, and IL16), adhesion molecules (ICAM-2, ICAM-3, VCAM-1, and MAdCAM-1) and integrins (alphaL, alpha4, and alphaD). The presence of the corresponding receptors on TLS T cells was confirmed. Intratumoral PNAd+ high endothelial venules also were exclusively associated with TLS and colocalized with CD62L+ lymphocytes. Together, these data bring new insights into the T-cell recruitment to intratumoral TLS and suggest that blood T cell enter into TLS via high endothelial venules, which represent a new gateway for T cells to the tumor. Findings identify the molecules that mediate migration of tumor-specific T cells into TLS where T cell priming occurs, suggesting new strategies to enhance the efficacy of cancer immunotherapies. <i>Cancer Res; 71(20); 6391–9. ©2011 AACR</i>.</p></div>

<div>Abstract<p><i>De novo</i> formation of tertiary lymphoid structures (TLS) has been described in lung cancers. Intratumoral TLS seem to be functional and are associated with a long-term survival for lung cancer patients, suggesting that they represent an activation site for tumor-specific T cells. Here, we characterized T-cell recruitment to TLS in human lung cancer to identify the adhesion molecules and chemoattractants orchestrating this migration. We found that most TLS T cells were CD62L+ and mainly of CD4+ memory phenotype, but naive T cells were highly enriched in these structures as compared with the rest of the tumor. A specific gene expression signature associated with T cell presence was identified in TLS, which included chemokines (CCL19, CCL21, CXCL13, CCL17, CCL22, and IL16), adhesion molecules (ICAM-2, ICAM-3, VCAM-1, and MAdCAM-1) and integrins (alphaL, alpha4, and alphaD). The presence of the corresponding receptors on TLS T cells was confirmed. Intratumoral PNAd+ high endothelial venules also were exclusively associated with TLS and colocalized with CD62L+ lymphocytes. Together, these data bring new insights into the T-cell recruitment to intratumoral TLS and suggest that blood T cell enter into TLS via high endothelial venules, which represent a new gateway for T cells to the tumor. Findings identify the molecules that mediate migration of tumor-specific T cells into TLS where T cell priming occurs, suggesting new strategies to enhance the efficacy of cancer immunotherapies. <i>Cancer Res; 71(20); 6391–9. ©2011 AACR</i>.</p></div>

De novo formation of tertiary lymphoid structures (TLS) has been described in lung cancers. Intratumoral TLS seem to be functional and are associated with a long-term survival for lung cancer patients, suggesting that they represent an activation site for tumor-specific T cells. Here, we characterized T-cell recruitment to TLS in human lung cancer to identify the adhesion molecules and chemoattractants orchestrating this migration. We found that most TLS T cells were CD62L+ and mainly of CD4+ memory phenotype, but naive T cells were highly enriched in these structures as compared with the rest of the tumor. A specific gene expression signature associated with T cell presence was identified in TLS, which included chemokines (CCL19, CCL21, CXCL13, CCL17, CCL22, and IL16), adhesion molecules (ICAM-2, ICAM-3, VCAM-1, and MAdCAM-1) and integrins (alphaL, alpha4, and alphaD). The presence of the corresponding receptors on TLS T cells was confirmed. Intratumoral PNAd+ high endothelial venules also were exclusively associated with TLS and colocalized with CD62L+ lymphocytes. Together, these data bring new insights into the T-cell recruitment to intratumoral TLS and suggest that blood T cell enter into TLS via high endothelial venules, which represent a new gateway for T cells to the tumor. Findings identify the molecules that mediate migration of tumor-specific T cells into TLS where T cell priming occurs, suggesting new strategies to enhance the efficacy of cancer immunotherapies.

Tertiary lymphoid structure, an ectopic lymphoid tissue induced under chronic inflammation, develops in various kidney diseases and is associated with poor prognosis. The immune system requires metabolic resources to support immune function and lymphocyte proliferation. Hence, dramatic metabolic alterations presumably occur during the formation of tertiary lymphoid structure. However, it remains unclear whether metabolic remodeling occurs during this formation and its underlying mechanism. In a murine model of renal tertiary lymphoid structures, we used imaging mass spectrometry and metabolome analysis to investigate the metabolic pathway that characterizes tertiary lymphoid structures. We also performed in situ hybridization with immunofluorescence and pharmacological inhibition to explore the expression and function of the key molecules governing the pivotal metabolic pathway. We analyzed urine samples from mice and humans to explore the metabolites estimating the presence of renal tertiary lymphoid structures. Significant glutathione accumulation and depletion of cysteine, which is essential for glutathione synthesis, was observed specifically within tertiary lymphoid structures. The kidneys with tertiary lymphoid structures exhibited higher glutathione concentrations than healthy kidneys. Tertiary lymphoid structures also showed significant accumulation of 4-HNE and 8-OHdG, markers of oxidative stress. Dendritic cells and fibroblasts within tertiary lymphoid structures expressed the cystine/glutamate transporter, that regulates glutathione synthesis, and supplied synthesized glutathione to lymphocytes, which lacked its expression. Pharmacological inhibition of the cystine/glutamate transporter prevented tertiary lymphoid structure formation in the kidney. Furthermore, enhanced glutathione synthesis within tertiary lymphoid structures was reflected in elevated urinary glutathione concentrations both in mice and humans, which effectively detected the presence of tertiary lymphoid structures in the kidney in IgA nephropathy patients. Glutathione significantly accumulated within tertiary lymphoid structures in the kidney. Inhibition of the cystine/glutamate transporter prevented the formation of tertiary lymphoid structures. Urinary glutathione served as a biomarker to detect tertiary lymphoid structures in the kidney.

BackgroundRecent evidence suggests that dietary fiber intake is associated with improved clinical and pathologic response to anti-PD-1 therapy,1 though this relationship is unclear in soft-tissue sarcoma. We evaluated the impact...