Dynamic remodeling of tertiary lymphoid structures in response to cancer therapy: a recent review

Blockade of the inhibitory PD1 pathway on CD8+ and CD4+ tumor infiltrating lymphocytes (TILs) has revolutionized standard of care for cancer patients. However, these approaches only benefit approximately 20% of cancer patients, thus a more comprehensive understanding of the immune response is paramount for the development of new therapeutic approaches and enhancement of the anti-tumor immune response. The presence of tertiary lymphoid structures (TLS) correlates with enhanced anti-tumor immunity and improved prognosis in several solid tumors. These ectopic lymphoid aggregates can exhibit features like secondary lymphoid organs (SLOs), including high endothelial venules, a T cell zone with mature dendritic cells (DCs), and a germinal center (GC) with follicular DCs and B cells, that facilitate the induction of immune responses in situ. Furthermore, the presence of TLS correlate with superior responses to immunotherapy and mature, GC-containing TLS correlate with increased T cell function in human tumors. However, current immunotherapies do not target TLS despite their predominance in the tumor microenvironment (TME) and key role in the adaptive immune response. Developing therapies that adequately induce TLS is limited by a lack of objective, bioinformatic analyses to elucidate the cellular and locational heterogeneity of TLS. TLS differences in composition are thought to define maturation levels i.e. early TLS have B and T cell aggregates but lack follicular DC and GC appear as the TLS mature. However, cellular composition is just one aspect of TLS heterogeneity. To successfully increase mature TLS in cancer patients for maximal humoral immunity, we must first understand the complete transcriptomic and proteomic profile of TLS in cancer patients. We have utilized the innovative Nanostring GeoMx platform to spatially interrogate the transcriptomics and proteomics associated with TLS. By pairing this technology with high level multispectral immunofluorescence via the Vectra Polaris system, we can link TLS heterogeneity to TLS position within the tumor. Further, we can analyze the complete activation profile of B and T cells to understand how TLS heterogeneity affects lymphocyte function. Our data have indicated that TLS can be linked to increased CD8+ T cell infiltrate and function. In fact, when mature TLS are not present, CD8+ T cell and Treg interactions increase, ultimately creating a more immunosuppressive TME. Lastly, utilizing a novel, robust bioinformatic pipeline, we have identified new genes that will aid in an objective definition of TLS. Specifically, CD27 is increased within TLS that are proximal to the tumor compared to those in the normal adjacent tissue. These studies will revolutionize the way in which TLS are defined within the TME, ultimately offering comprehensive gene and protein analysis of TLS that includes spatial geography of the structures and an unbiased, objective definition of TLS for future clinical utility and immunotherapeutic targeting. Citation Format: Dongyan Liu, Xiang Li, Rajesh Acharya, Ernest M. Meyer, Shelley Reynolds, Ayana Ruffin, Robert L. Ferris, Dario A.A. Vignali, Riyue Bao, Tullia C. Bruno. Utilizing spatial transcriptomics to elucidate tertiary lymphoid structure heterogeneity in human cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-083.

Tertiary lymphoid structures (TLS), formerly recognized as Crohn's-like structures, serve as crucial biomarkers for evaluating the progression of colorectal cancer (CRC). Understanding their spatial distribution, cellular composition, and interactions within CRC is paramount for comprehending the immune response in the tumor microenvironment (TME). TLS are comprised of a T-cellular compartment and a B-cellular compartment, the latter encompassing follicular dendritic cells (FDCs), high endothelial venules (HEVs), and lymphatic vessels. While T helper cells predominate in cancer TLS, the specific functions of their subpopulations remain inadequately understood. Notably, T follicular helper (Tfh) cells play a central role in the activation of CD8+ T cells, and both Tfh cells and Tfh-associated genes have been linked to enhanced CRC survival. In stage II CRC TLS, an escalation in the number of FoxP3+ T regulatory cells (Tregs) is regarded as a negative prognostic factor. Moreover, within TLS, T lymphocytes shield B lymphocytes from the immunosuppressive effects of the TME. B lymphocyte activation is succeeded by class recombination (CSR) and somatic hypermutation (SHM). Dendritic cells (DCs) constitute a vital cellular component of the TLS T compartment. During steady state and early stages of CRC, specialized antigen-presenting cells such as DCs migrate to regional lymph nodes through afferent lymphatics. They deliver MHC antigen-derived peptide complexes (tumor antigens) to naïve CD4+ and CD8+ T cells, which subsequently infiltrate the tumor site as antigen-specific T cells. Key DC markers studied in TLS include CD83 and DC-LAMP. Research has indicated that the DC-LAMP gene signature in tumor TLS reflects Th1 cell targeting, cytotoxicity, and T cell activation. This review comprehensively outlines the functions performed by distinct cell subsets within tertiary lymphoid structures (TLS) in tumors.

Background: The host immune system is one of the key players of anti-tumor functions. High numbers of tumor infiltrating lymphocytes has been showed to be a predictor of favorable clinical outcome in several solid cancers. The role of B cells in tumor microenvironment is still unclear. Recent studies have reported a correlation between the densities of B cells and the favorable clinical outcome of cancer patients. Moreover, some studies considered the organization of tumor infiltrating B cells as criteria in addition to the cell density. Tertiary lymphoid structures (TLSs) are defined as transient ectopic lymphoid organizations that can develop in non-lymphoid tissues at the site of chronic inflammation, and are anatomically and functionally similar to secondary lymphoid organs. In the present study, we investigated the association between tumor infiltrating B cells and clinicopathological features in gastric cancer. Materials and Methods: Tumor blocks were obtained from 226 patients with stage Ib to stage IV gastric cancer who had undergone initial surgical resection. The density of CD20+ B cells within the tumor and invasive margin area was assessed by immunohistochemistry. We also evaluated CD3+ T cells, CD21+ follicular dendritic cells (FDCs), Bcl6 germinal center B cells, and PNAd+ high endothelial venules (HEVs) to show the presence of TLSs in gastric cancer. Results: Tumor infiltrating B cells mostly organized clusters surrounded by CD3+ T cells. B cell area contained FDCs and some clusters had Bcl6+ B cells. There were HEVs around follicles. We observed most of TLSs at the tumor invasive margin. Kaplan-Meier survival analysis showed that high number of CD20+ B cells was associated with significantly better survival (P<0.0001). Univariate analysis demonstrated a correlation between CD20 high and longer OS, and multivariate analysis also showed that CD20 high was one of the independent predictors of OS. Conclusion: We demonstrated that B cells mostly infiltrated as TLSs and were associated with better prognosis in patients with gastric cancer. Our results suggested that B cells, perhaps as the structure of TLSs, might play an important role for immune response against gastric cancer. Citation Format: Chie Sakimura, Hiroaki Tanaka, Tatsuro Tamura, Go Ohira, Masatsune Shibutani, Sadaaki Yamazoe, Katsunobu Sakurai, Hisashi Nagahara, Kenjiro Kimura, Takahiro Toyokawa, Ryosuke Amano, Naoshi Kubo, Kazuya Muguruma, Kiyoshi Maeda, Masaichi Ohira, Kosei Hirakawa. Intratumoral tertiary lymphoid structures are associated with favorable prognosis of patients with gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1468.

Brain metastasis is the cause of death of more than 40% of all cancer patients and is five times more prevalent than primary brain tumors. Melanoma, lung, and breast cancers are the three most common cancers metastasizing to the brain. Currently, radiation and chemotherapy are the gold standard for the treatment of brain metastasis. However, despite the efficacy of current T cell-based immunotherapies in primary cancers, recent clinical trials have demonstrated little to no benefit in brain metastasis patients. Thus, we predict that tertiary lymphoid structures (TLS) could provide the cellular niches for increased T cell influx and activity. TLS are ectopic lymphoid structures consisting of clusters of B and CD4+ T cells with the presence of high endothelial venules (HEVs) and follicular dendritic cells (FDCs) as hallmarks of TLS formation. B cells and TLS correlate with superior response to immunotherapies and greater overall survival in solid tumors. Despite the positive prognostics of TLS and B cells in solid primary tumors, they are critically understudied in brain metastasis. However, the expression of CXCL13, a key initiating factor of TLS, has been shown to correlate with greater overall survival in melanoma brain metastasis patients. In this study, we hypothesize that B cell infiltration and TLS signatures in brain metastases would correlate with improved anti-tumor immunity and better overall survival. Utilizing multispectral imaging, we demonstrate that despite lacking TLS with canonical hallmarks (HEVs and FDCs), brain metastasis patients have specialized lymphoid structures consisting of proliferating B and T cells, potentially early TLS. We observed that the presence of these lymphoid structures correlates with increased CD8+ T cell infiltration. More specifically, CD8+ T cells are more likely to localize intratumorally in melanoma brain metastasis and in non-tumor regions in LBM patients. Additionally, we identified more active lymphoid aggregates by Ki67 staining in melanoma-brain metastasis patients relative to lung-brain metastasis, suggesting that the primary tumor could influence the type of lymphoid structures in the brain. We also found a correlation between TLS at the primary tumor site and increased B cell infiltration and early TLS formation at the metastatic site in lung cancer patients. Utilizing spatial proteomics and transcriptomics, we aim to carry out an in-depth analysis of the immune activation of the lymphoid structures and key molecular pathways in the tumors of brain metastasis. In conclusion, uncovering differences in B cells and lymphoid aggregates will set the ground truth for structure formation in brain metastases, which will help elucidate key immune targets related to TLS function in these immunologically unique tumors. Citation Format: Noor Nader, Elaine Byrnes, Sheryl Kunning, Thomas Pearce, Gabriel Sica, Timothy Burns, Laura Stabile, Xiaoran Zhang, Tullia Bruno. Lymphoid aggregates dictate immune activity in melanoma and lung brain metastases [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B005.

<h3>Introduction/Background*</h3> Tertiary lymphoid structures (TLS) are ectopic lymphoid tissues that form in and around cancers. TLS consist of a germinal centre (GC) with proliferating B-cells and follicular dendritic cells (FDCs), as well as a peripheral T-cell zone. Local and systemic B- and T-cell responses are thought to be initiated and maintained at the TLS. Here, we analysed whether TLS were associated with anti-tumour immunity and a reduced risk of recurrence in endometrial cancer (EC). <h3>Methodology</h3> TLS were quantified by an expert pathologist (TB) on H&amp;E-stained tumour slides of the cancer genome atlas uterine cancer cohort (TCGA UCEC), and by immuno-histochemistry (IHC) on tumour slides from the PORTEC-3 trial biobank. Time to recurrence analysis were performed according to Kaplan-Meier’s method, using log-rank tests and Cox’ proportional hazards models, including prespecified multivariable analysis with clinicopathological and molecular risk factors. <h3>Result(s)*</h3> Differential gene expression analysis of TLS-positive and TLS-negative cases from TCGA UCEC identified, among others, L1-cell adhesion molecule (L1CAM) (figure 1A). IHC of PORTEC-3 cases revealed expression of L1CAM in TLS GCs, where it co-localised with CD21 on FDCs. TLS were observed in the myometrial wall distal and proximal to the tumour (figure 1B). Tumour tissues of 377 PORTEC-3 participants could be included for analysis of L1CAM-defined TLS. L1CAM-defined TLS were identified independent of L1CAM expression in tumour cells and across all molecular subgroups, though enriched in mismatch repair deficient (MMRd) and polymerase-epsilon mutant (<i>POLE</i>mut) EC (figure 2). TLS were significantly more frequent in MMRd EC with secondary <i>TP53</i> mutations (p=0.008). Intra-tumoural CD8⁺-cell densities were significantly higher in TLS-positive cases. Five-year risk of recurrence was 7.2% (95%CI 0.9-13.1) in EC patients with TLS (n=70), and 27.8% (95%CI 22.6-32.7, p-value &lt;0.0001) in EC patients without TLS (n=307). This favourable prognostic impact was independent of clinicopathological and molecular factors (adjusted HR 0.32 95%CI 0.14-0.74, p=0.0077). <h3>Conclusion*</h3> L1CAM identifies tertiary lymphoid structures with germinal centres. Our data suggest a pivotal role of TLS in the risk of recurrence of EC. L1CAM IHC is simple, available across many study cohorts and could be readily implemented as biomarker of TLS in future trials and clinical care.

Cancer immunotherapies transition endothelial cells into HEVs that generate TCF1+ T lymphocyte niches through a feed-forward loop.

e15083 Background: The tumor immune infiltrate and organized lymphocytic aggregates within the tumor microenvironment, known as tertiary lymphoid structures (TLS), play a critical role in cancer. We hypothesize that the maturation stage of TLS harbors prognostic information on recurrence risk in patients (pts) with non-metastatic colorectal cancer (nmCRC). Methods: In a comprehensive immunofluorescence and clinical analysis of 111 pts with UICC stage II &amp; III nmCRC (median age: 65 yrs; female: n = 53 (48%); stage III: n = 69 (62%)), we quantified the number and maturation status of tumor-associated TLS in baseline surgical specimens:[1] Early TLS (E-TLS, composed of dense lymphocytic aggregates without follicular dendritic cells (FDCs), [2] Primary follicle-like TLS (PFL-TLS, having FDCs but no germinal center (GC) reaction), and [3] Secondary follicle-like TLS (SFL-TLS, having an active GC reaction). The 3-year incidence of recurrence was the primary endpoint of this study, which occurred in 19 pts (3-year recurrence risk = 18.3%). Results: Most TLS formed in tissue adjacent to the tumor. The median number of TLS/mm of tumor perimeter was 1.0 [25th-75th percentile: 0.5-1.7]. The average proportions of different TLS maturation stages were 56% of E-TLS [40-78], 20% of PFL-TLS [6-37], and 16% of SFL-TLS [0-32]. A structural equation model was fitted to summarize the TLS counts and maturation stages into a TLS maturation immunoscore for predicting recurrence. 3-year recurrence risks were 31.7% (95%CI: 17.2-47.3), 15.9% (5.7-30.5), and 9.4% (2.4-22.4) in pts in the 1st, 2nd, and 3rd tertile of the score (Gray’s test p = 0.05). A higher score was significantly associated with a lower recurrence risk (Hazard ratio (HR) for 10 units increase = 0.76, 95%CI: 0.59-0.97, p = 0.03), and this association prevailed in multivariable analysis adjusting for age, ECOG performance status, stage, and adjuvant chemotherapy (Adjusted HR = 0.73, 0.54-0.99, p = 0.05). Conclusions: Tumors of nmCRC pts with a very low risk of recurrence are characterized by an increased fraction of mature TLS comprising FDCs and GCs. If confirmed prospectively, adjuvant chemotherapy may be avoided in nmCRC pts with a high TLS maturation score.

BackgroundOral squamous cell carcinomas are often heavily infiltrated by immune cells. The organization of B-cells, follicular dendritic cells, T-cells and high-endothelial venules into structures termed tertiary lymphoid structures have been detected in various types of cancer, where their presence is found to predict favourable outcome. The purpose of the present study was to evaluate the incidence of tertiary lymphoid structures in oral squamous cell carcinomas, and if present, analyse whether they were associated with clinical outcome.MethodsTumour samples from 80 patients with oral squamous cell carcinoma were immunohistochemically stained for B-cells, follicular dendritic cells, T-cells, germinal centre B-cells and high-endothelial venules. Some samples were sectioned at multiple levels to assess whether the presence of tertiary lymphoid structures varied within the tumour.ResultsTumour-associated tertiary lymphoid structures were detected in 21 % of the tumours and were associated with lower disease-specific death. The presence of tertiary lymphoid structures varied within different levels of a tissue block.ConclusionsTertiary lymphoid structure formation was found to be a positive prognostic factor for patients with oral squamous cell carcinoma. Increased knowledge about tertiary lymphoid structure formation in oral squamous cell carcinoma might help to develop and guide immune-modulatory cancer treatments.

B cells in tertiary lymphoid structures are associated with favorable prognosis in gastric cancer

<h3>Background</h3> Tertiary lymphoid structures (TLS) may develop in non-lymphoid tissues in response to a variety of different stimuli and can serve as foci for generating anti-tumor immunity.¹ TLS formation is...

Tertiary Lymphoid Structures in Rheumatoid Arthritis: NF-κB–Inducing Kinase–Positive Endothelial Cells as Central Players

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

3142 Background: Previous studies of non-small cell lung cancer (NSCLC) have shown that TLS can be predictive of therapy response and a positive prognostic factor for survival. Currently, TLS identification is manually performed by pathologists with limited morphological criteria. Standardizing TLS detection with an automated DIA workflow could guide clinical trials in precision medicine by improving patient stratification. Here, we investigate the reproducibility and sensitivity of our DIA platform for evaluating TLS in LUAD using digital histopathology and machine learning. Methods: TLS were assessed by 3 pathologists on whole slide images (WSI) in a validation cohort of 22 LUAD samples using current TLS characterization criteria of dense lymphoid structures, the presence/absence of a germinal center, and high endothelial venules (HEVs). The intraclass correlation coefficient (ICC) was used to measure reproducibility between pathologists. The BostonGene DIA platform was used to train models for automated TLS detection. Quantitative measurements of area, lymphocyte number, and density of each TLS were obtained. A prospective cohort of 8 samples was used to compare pathologist and DIA identification of TLS. Normalized numbers of TLS in the tumor area were used for cohort stratification for overall survival (OS) analysis using the Kaplan-Meier method in an independent clinical cohort of 104 TCGA-LUAD patients. Results: A panel of 3 pathologists identified 326 unique TLS from 22 samples. Between-pathologist detection of TLS, independent of germinal center or HEV criteria, resulted in good reproducibility with an ICC of 0.77. Our DIA platform exhibited excellent reproducibility with an ICC of 0.94 when compared to validated prospective cohort annotation. In total, 155 and 189 TLS were identified by pathologists and our DIA platform, respectively. The DIA platform demonstrated a markedly improved sensitivity of 0.91 for TLS identification. Furthermore, OS analysis revealed that a TLS density greater than 0.94 TLS per mm2 of tumor assessed by DIA is a statistically significant independent biomarker of better OS in the LUAD cohort from TCGA. Conclusions: These results demonstrate the BostonGene DIA platform detects TLS in LUAD, with improved reproducibility and sensitivity over previous methods. Additionally, the DIA platform showed a TLS density greater than 0.94 TLS per mm2 of tumor is a positive prognostic marker for OS in LUAD. Standardized TLS DIA identification can be exploited in digital pathology applications for future clinical trials, informing clinicians of predictive and prognostic information during the decision-making process.

Tertiary lymphoid structures (TLSs), which consist of B cells, T cells, follicular dendritic cells and high endothelial venules, have recently been found to be associated with effective antitumor immune responses in patients with cancer. Tumor-infiltrating T cells and B cells have each been demonstrated to be associated with survival in patients with cancer. We hypothesized that TLSs, an assembly of immune cells, may be important for the initiation and/or maintenance of T cell and B cell responses against tumors. The aim of the present study was to examine the cellular mechanism of B cells in TLSs within gastric cancer and to understand the antitumor immune response of TLSs. Each B cell subset in a tumor was examined using flow cytometry to evaluate B cell differentiation and the functional status of B cells. In addition, B cell clonality was investigated by analyzing the B cell antigen receptor gene using PCR, and the function and formation/maintenance of TLSs were evaluated using reverse transcription-quantitative PCR. Tumor-infiltrating B cells were more differentiated compared with that in distant non-tumor tissues and tumor-draining lymph nodes. The PCR results revealed specific BCR gene expression in tumor-infiltrating B cells. The expression of co-stimulatory factors, CD80 and CD86, was observed, in addition to the constantly expressed major histocompatibility complex molecules (HLA-ABC and HLA-DR). CD70 was expressed in addition to CD27 in both CD20+ B cells and CD8+ T cells, indicating that these factors are activated together through their interaction. The mRNA expression levels of CCL21, CXCL13, PD-L1, perforin and granzyme B in TLSs was significantly higher compared with that in non-TLSs. The majority of tumor-infiltrating B cells in gastric cancer exist in the form of TLSs around the tumor and have been antigen-sensitized and differentiated, and proliferated in TLSs but not in the lymph nodes. In addition, B cells in TLSs might primarily function as antigen-presenting cells and be associated with the induction of cytotoxic T cells.

Lymphoid organ neogenesis takes place in chronically inflamed tissues including cancer and yields the development of tertiary lymphoid structures (TLS). TLS are ectopic lymphoid organs that activate antigen specific T-cells and B cells in infection and autoimmunity and correlate with prolonged survival in various cancer types. This suggests that TLS contribute to protective anti-tumor immunity. Therefore, promoting the development of tumor-associated TLS could be a novel immunotherapeutic approach. However, the molecular and cellular mechanisms of TLS development in human cancer or how TLS contribute to survival are largely not understood. Here we used multiparameter immunofluorescence and digital pathology to quantify TLS and to characterize their cellular composition and tissue context in cohorts of lung squamous cell carcinoma (LSCC, n=138), colorectal cancer (CRC, n=111), clear cell renal cell carcinoma (ccRCC, n=50) and bladder cancer (BC, n=33) patients. Furthermore, we established an experimental model to characterize TLS development and its impact on tumor-specific immunity. We discovered that TLS development and maturation followed the same steps in all analyzed tumor types as well as in the lungs of mice in our experimental model. First, B and T lymphocytes accumulated around blood vessels. Second, a network of follicular dendritic cells developed within the lymphocytic aggregate, and third, a germinal center (GC) reaction was activated. Additionally, we identified a niche of CXCL13+ perivascular stroma and CXCL12+LTB+ and PD-L1+ epithelial cells that were associated with TLS in LSCC. We found that the number of tumor-associated TLS was an independent prognostic factor for prolonged survival in untreated LSCC, CRC and BC, but not in ccRCC patients or in LSCC and BC patients who were treated with neoadjuvant chemotherapy. By comparing the chemotherapy-treated and untreated cohorts we observed that the number of TLS was not changed but TLS maturation (i.e. GC formation) was significantly impaired after chemotherapy. This difference was at least partially dictated by corticosteroids, which are commonly used to treat the side effects of chemotherapy of LSCC patients. We further studied the mechanisms underlying TLS development using the experimental model. We identified a combination of stimuli that induces the development of mature TLS in the lungs of mice. Besides inflammatory stimuli, a foreign antigen was necessary to achieve a significant increase in TLS numbers and maturation stage, suggesting that cognate interactions are crucial for lymphoid organ neogenesis. This is further supported by our observation that CRC patients with microsatellite instability, which presumably results in more neoantigens, had an increased proportion of mature TLS. The negative impact of corticosteroids on TLS development was confirmed in this model. In summary, we propose that GC+ TLS represent the relevant TLS phenotype contributing to survival in different tumor types. Lymphoid organ neogenesis is negatively affected by corticosteroids, which might impair the spontaneous as well as therapy-induced anti-tumor immunity. The established experimental model will allow investigation of the mechanisms of TLS development and function in cancer and assessment of their therapeutic potential. Citation Format: Karina Silina, Alex Soltermann, Chiara Burkhardt, Farkhondeh Movahedian Attar, Ruben Casanova, Alessandra Curioni-Fontecedro, Holger Moch, Florian Posch, Thomas Winder, Nick van Dijk, Charlotte Voskuilen, Michiel van der Heijden, Maries van den Broek. Harnessing lymphoid organ neogenesis as a novel prognostic biomarker and therapeutic target [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A113.