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Tertiary Lymphoid Structures – A Promising Biomarker for Immunotherapy

What we know, and what we don’t

Tertiary lymphoid structures (TLS) have been a hot topic in the field of immuno-oncology, where it is believed they play a critical important role in influencing the tumor microenvironment.1-6 Commonly seen in invasive carcinoma, their presence is associated with improvement in clinical outcomes. While the mechanisms that underlie the role of TLSs have yet to be fully elucidated, the prevailing hypothesis is that TLSs are a major player in antitumor immune responses.

TLSs are unique in that they fail to adhere to classical definitions of organs. Their structures do not have consistent organizational schemes, they lack capsules, and do not develop in pre-determined locations. TLS arise in tissues where the main function is not related to the generation of immune cells such as the thymus or lymph nodes, but rather they present as a lymphoid neogenesis in response to a chronic stimulus such as infection, autoimmunity, and in chronic allograft rejection. TLSs are a site of immune privilege for antigen presentation and clonal differentiation to support immunological memory.1-4

Since the presence of TLSs are associated with improvement in clinical outcomes, their presence or induction following cancer therapies could be a valuable predictor of therapeutic responses.

How we study a TLS

Hematoxylin and eosin (H&E) staining is the simplest technique enabling the detection of TLSs in formalin-fixed paraffin-embedded (FFPE) tumor tissue sections.1-2 Mature TLSs correspond to lymphoid follicles including a dense cellular aggregate resembling germinal centers found in secondary lymphoid structures. Less differentiated structures such as lymphoid aggregates and lymphoid follicles without germinal centers can also be detected.1-2

However, the established methods such as H&E and chromogenic IHC staining are limited in the amount of information they allow to be extracted from FFPE tissue sections. Typically, chromogenic IHC is constrained to one or two protein markers, meaning the full depth and breadth of cellular interactions in the tumor microenvironment cannot be wholly examined.4

Multispectral imaging of multiplex immunofluorescence (mIF) has emerged as a tool with significant predictive capabilities for immunotherapy response. Multispectral mIF enables deeper interrogation into the spatial biology of the tumor microenvironment, including the capture of spatially resolved, cell-to-cell interactions.4 Thus, the development of clinically useful biomarkers like TLSs to select responders to immunotherapies will be critical for the advancement of such treatments and mIF and spatial context will be critical for the reliable detection of TLS. In a recent talk given by Dr. Karina Silina, Swiss National Science Foundation (SNSF) PRIMA group leader at the Institute of Pharmaceutical Sciences (IPW) at the ETH Zurich, outlines her ongoing research efforts aimed at defining the physiological role and mechanisms of TLS development in cancer. To study TLS development in cancer, she utilized a 6-plex Opal mIF panel (CD20, PNAD, CD3, DC-LAMP, CD23 and CD21). She found that TLS development happens in a stepwise fashion and begins when lymphocytes start to cluster around high endothelial venules, marked by peripheral-node-addressin (PNAd) expression. In this early stage, there are no follicular dendritic cells (FDCs) present yet in the TLS. Then a network or clusters of these FDC (CD21+) can be visualized. In the final stage, active germinal centers are visible (CD21+/CD23+). Interestingly, in all of these identified stages the presence of mature antigen presenting dendritic cells (DC-LAMP+) can be detected. Her finding show that the development of TLS appear similar in various tissues including bladder, lung, colon, skin, kidney, and many more. The way she does this is by utilizing mIF, particularly the PhenoImager HT to understand TLS’s potential to be used for deliberate TLS induction as a novel immunotherapeutic approach.5 There is growing evidence that spatial biology is key to understanding immunotherapy response and clinical outcomes, and thus it is critical to develop mIF assays that can be translated to the clinic and biomarkers such as TLSs represent a valuable piece of information for patient stratification. PhenoImager platforms make use of Opals, automated mIF staining, high-throughput multispectral slide image acquisition, and advanced machine learning–based image analysis algorithms for segmenting and characterizing the cell-level immuno-biology occurring in the TME. In particular, Opal enables the development of multiplexed assays with balanced, quantitative signal for rare and abundant targets, imaged in a single final scan with the Akoya portfolio of multispectral imagers. The PhenoImager solution enables quick and accurate spatial phenotyping of tissue across whole slides. This would allow researchers the ability to develop spatial phenotypic signatures across whole slides. The development of a spatial phenotypic signature requires unbiased measurement of the interactions and cell densities of tumor and immune cells within the tumor microenvironment. Again, focusing this technological approach on TLSs we can see the potential for a valuable tool for patient stratification into specific prognostic and therapeutic populations.

The technological approaches discussed above have been associated with higher accuracy in the detection of TLSs compared to H&E staining.1 It is these capabilities that will offer researcher the ability to comprehensively evaluate TLS density, size, and cellular content. The ability to characterized TLSs and their composition is key for understanding ongoing adaptive immune response and it starts with procuring the clearest possible picture.5

Interested in learning more? Talk to us or check out some other PhenoImager HT applications.


  1. Sautès-Fridman, C., Petitprez, F., Calderaro, J. et al. Tertiary lymphoid structures in the era of cancer immunotherapy. Nat Rev Cancer 19, 307–325 (2019).
  2. Munoz-Erazo, L., Rhodes, J.L., Marion, V.C. et al. Tertiary lymphoid structures in cancer – considerations for patient prognosis. Cell Mol Immunol 17, 570–575 (2020).
  3. Werner, F., Wagner, C., Simon, M., et al. Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration. Front. Immunol., (2021).
  4. Hoyt, C., Multiplex Immunofluorescence and Multispectral Imaging: Forming the Basis of a Clinical Test Platform for Immuno-Oncology. Front. Immunol., (2021).
  5. Pagliarulo F, Cheng PF, Brugger L, et al. Molecular, Immunological, and Clinical Features Associated With Lymphoid Neogenesis in Muscle Invasive Bladder Cancer. Front Immunol. (2022)
  6. Pimenta EM, Barnes BJ. Role of Tertiary Lymphoid Structures (TLS) in Anti-Tumor Immunity: Potential Tumor-Induced Cytokines/Chemokines that Regulate TLS Formation in Epithelial-Derived Cancers. Cancers (Basel). (2014).

Author: James DeRosa, MPH

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