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. Author manuscript; available in PMC: 2026 Feb 7.
Published in final edited form as: Methods Cell Biol. 2025 Apr 5;199:37–49. doi: 10.1016/bs.mcb.2025.03.008

Methods for Analysis of Tertiary Lymphoid Structures and Immune Activity by Multiplex Immunofluorescence Histology

Leon Zheng 1, Priya Katyal 2, Ileana Soto Mauldin 1
PMCID: PMC12880810  NIHMSID: NIHMS2137600  PMID: 41106940

Abstract

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that are correlated with improved patient outcomes in several solid cancers, including melanoma. Multiplex immunofluorescent histology (mIFH) has been used in numerous studies to identify and characterize TLS. However, detailed studies evaluating immune cell subsets and markers of immune activity at TLS sites have been limited. Here, we introduce multiplex immunofluorescence histology methods to identify TLS, their associated immune cell components, and markers of immune activity. We outline two mIFH panels for evaluating and quantifying TLS, and markers of immune activity, offering methodologies that can be used to gain a more nuanced understanding of the role and immunological activity of TLS in cancer prognosis and therapy.

Keywords: melanoma, tertiary lymphoid structures, immunology, multiplex, immunofluorescence histology, IHC, immune infiltrates

Introduction:

Tertiary lymphoid structures (TLS) resemble lymph nodes in morphology and are found localized near solid tumors and in inflammatory diseases. TLS are composed of B-cells (CD20, CD19) and T-cells (CD3, CD4, CD8) organized with distinct areas for each cell type1. Localized within the T-cell area are high endothelial venules (HEV) that express peripheral node addressin (PNAd), a ligand for L-selectin which mediates leukocyte infiltration into lymphoid organs1,2. TLS are associated with increased survival across various solid tumor models, including head and neck squamous cell carcinoma3, ovarian cancer3, breast cancer4, and melanoma3,5. Furthermore, in melanoma, TLS are associated with response to anti-PD-1 checkpoint blockade therapy6–8.

TLS have primarily been identified in studies by evaluating for gene signatures in tumor biopsies or by multiplex immunofluorescent histology (mIFH) staining with antibodies to identify B and T cells and their spatial organization. The most common gene signature used to identify a TLS is a 12-chemokine signature of genes comprised of: CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11, and CXCL139. Interestingly, recent mIFH studies highlight that TLS are heterogeneous and can be classified into three maturation stages termed early, primary follicle-like, and secondary follicle-like, by their organization and B-cell follicle maturation10. In colorectal cancer, TLS maturation is prognostic, with a lower risk of reoccurrence observed in patients with secondary follicle-like TLS10. Advantageously, mIFH enables researchers to interrogate TLS individually to evaluate heterogeneity in maturation, cell densities, and the spatial relationship between immune cells which cannot be evaluated by gene profiling5,11. Disadvantages to mIFH are that panel development and optimization for mIFH staining is often time-consuming, labor-intensive, and costly. Additionally, data analysis, usually mediated by proprietary software, can be costly and time-intensive.

Here, we present methodologies for characterizing TLS and intratumoral immune infiltrates using mIFH, which facilitates the identification, quantification, and visualization of seven antigens in the same tissue specimen. Two mIFH panels are detailed, the first (Table 2) can be used to identify TLS structures in human tumors and tissues, using antibodies to: T-cells (CD3, CD8), B-cells (CD20), PNAd, DC-LAMP (activated dendritic cells) and Ki67 (proliferating cells). The second panel (Table 3) expands to include additional markers of T-cell activity: granzyme B, Thymocyte Selection Associated High Mobility Group Box (TOX), and Transcription Factor 7 (TCF7). Granzyme B, a serine protease utilized by CD8+ T-cells to induce apoptosis of target cells12, is associated with improved survival and anti-PD-1 therapy efficacy in breast cancer and non-small-cell lung cancer13,14. The transcription factor TOX is associated with T-cell exhaustion in cancer models, and in melanoma15, non-small-cell lung carcinoma15, and ovarian cancer16, TOX is associated with worse overall survival or impaired response to anti-PD-1 therapy. Transcription Factor 7 (TCF7) is the gene that encodes for the T-cell factor-1 (TCF-1) transcription factor, and identifies CD8+ T cells that have stem cell-like properties retaining the ability to proliferate, regenerate, and elicit long-term T-cell responses17,18. TCF7+ T-cells are associated with improved overall survival in primary small-cell esophageal carcinoma19 and oral squamous cell carcinoma20. In melanoma, TCF7+ T-cells are associated with improved responses to anti-PD-1 therapy21.

Table 2:

Tertiary Lymphoid Structures Identification Staining Panel

Staining Order Antigen Retrieval Primary Antibody and working dilution Secondary Antibody Opal
1. AR9 CD3 (1:100) Opal Anti-Ms+Rb HRP 520
2. AR9 CD8 (1:500) Opal Anti-Ms+Rb HRP 540
3. AR6 CD20 (1:2000) Opal Anti-Ms+Rb HRP 620
4. AR6 PNAd (1:100) Rat Secondary Super Picture HRP 570
5. AR9 DC-LAMP (1:500) Opal Anti-Ms+Rb HRP 650
6. AR6 Ki67 (1:20) Opal Anti-Ms+Rb HRP 690
7. AR6 DAPI Opal Anti-Ms+Rb HRP -
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Table 3:

T-cell Activity Panel

Staining Order Antigen Retrieval Primary Antibody and working dilution Secondary Antibody Opal
1. AR9 CD3 (1:100) Opal Anti-Ms+Rb HRP 520
2. AR9 CD8 (1:500) Opal Anti-Ms+Rb HRP 540
3. AR6 CD20 (1:2000) Opal Anti-Ms+Rb HRP 620
4. AR9 TCF7 (1:1000) Opal Anti-Ms+Rb HRP 570
5. AR9 TOX (1:100) Sigma Opal Anti-Ms+Rb HRP 650
6. AR9 Granzyme-b (1:100) Opal Anti-Ms+Rb HRP 690
7. AR6 DAPI Opal Anti-Ms+Rb HRP -
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In summary, this method provides a comprehensive approach to investigate immune activity markers at TLS and tumor sites. The described panels can be used to identify and quantify immune biomarkers such as: TLS presence; T-cell, B-cell, and dendritic cell density and location within the tumor microenvironment; and quantity, proportion, and location of activated T- and B-cells and their relationship with patient survival or clinical outcomes. Collectively, the use of these panels may lead to a better understanding of the tumor microenvironment and may lead to the identification of new prognostic indicators for cancer immunotherapies.

1. MATERIALS

1.1. Specimen Preparation

  1. Paraffin embed tissue specimen

  2. Freshly Cut 5-μm sections mounted on SuperFrost Plus Microscope Slides

  3. Xylene

  4. Ethanol (EtOH)

  5. 10% Buffered Formalin Phosphate

1.2. Immunofluorescent staining

  1. Slide humidity chamber

  2. Antigen Retrieval Buffer 9 (Akoya Biosciences)

  3. DIVA Decloaker, 10X Antigen Retrieval Buffer (Biocare Medical)

  4. Opal 7-Color Manual IHC kit (Akoya Biosciences), which includes Opals, blocking/antibody diluent, secondary antibody recognizing mouse or rabbit species, spectral DAPI, AR6 Antigen Retrieval Buffer, and amplification diluent.

  5. Antibodies for staining (Table 1)

  6. Super Picture HRP Polymer Conjugate Broad Spectrum (Life Technologies)

  7. ProLong Diamond Antifade Mountant (ThermoFisher Scientific).

  8. 24×50mm cover glass

  9. Opal Staining jar (Akoya Biosciences)

  10. Wash Buffer: Tris-buffered saline containing 0.05% Tween 20, PH 7.5 (TBST)

  11. Tissue-Tek slide staining dishes and slide rack for immersing slides into solutions

  12. Panasonic Microwave Oven with Inverter Technology and Genius Sensor, 1200W

Table 1:

Antibodies for Immunofluorescence Staining

Antibody Clone Supplier
CD3 MRQ-39 Cell Marque
CD8 C8/144B Agilent
CD20 L26 Agilent
Ki67 SP6 Abcam
PNAd MECA-79 BioLegend
DC-LAMP EPR24265-8 Abcam
TCF7 1D2 Sigma
TOX Cat # HPA018322 Sigma
Granzyme-b GB7 Bio-Rad
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1.3. Image Acquisition

  1. Vectra 3 microscope (Akoya Biosciences) or comparable microscope

  2. Inform Image analysis software (Akoya Biosciences)

2. METHODS – Immunofluorescent Staining on Formalin-fixed Tumor Sections

2.1. Deparaffinization, Rehydration, Fixation, and Antigen Retrieval

  1. Immerse slides in Tissue-Tek slide staining dishes that are filled with the following solutions: xylene x3 dishes, 100% EtOH, 95% EtOH, 70% EtOH, DH20 ×3 dishes, TBST x1 dish, Neutral Buffered formalin x1 dish. The slide rack containing slides is moved from dish to dish in sequential steps with all steps done at room temperature.
    1. Xylene dish 1 (10 min)
    2. Xylene dish 2 (10 min)
    3. Xylene dish 3 (10 min)
    4. 100% EtOH (5 min)
    5. 95% EtOH (5 min)
    6. 70% EtOH (2 min)
    7. DH20 (2 min)
    8. TBST (2 min)
    9. DH20 (2 min)
    10. Neutral Buffered Formalin (20 min)
    11. DH20 (2 min)

  2. Place the slides in an Opal slide processing jar and add Antigen Retrieval Buffer. Rinse the slides for 2 min then discard Buffer.

  3. Completely fill the processing jar with AR Buffer that has been diluted to 1X. Use the appropriate antigen retrieval buffer associated with the antibody that will be used for staining. For example, for Table 2 which starts with a CD3 stain post AR9 antigen retrieval, AR9 Buffer would be used.

  4. Microwave slides for approximately 2 min at 100% power until the solution is at a boil. Once the solution is boiling stop the microwave and place at 20% power, allow slides to heat at 20% power for 18 min. During the initial high-power microwave step, it is critical to watch the slides so that too much solution does not boil out; if this happens add more buffer. It is critical that the tissue on your slides is completely submerged in buffer during the whole microwaving process and subsequent cooling process.

  5. Allow slides to cool at room temperature for 30 min, then proceed to do further staining steps or store slides in fridge at 4 degrees C overnight.

2.2. Multiplex Stain

Specific antibodies, antigen retrieval buffers, and Opal dye details for each panel are found in Tables 1–3 for the TLS Identification and T-cell Activity Panels, respectively. All subsequent steps are done at room temperature.

  1. Rinse Slides in TBST in Tissue-Tek staining dish.

  2. Remove slides from TBST and place into humidified chambers.

  3. Pipette 300μl of Antibody Diluent/Block solution on each slide allow to block for 10 min. Discard blocking solution by tipping slides over a beaker, place slides back in staining chamber.

  4. Primary Antibody diluted in Antibody Diluent/Block should be pipetted on slides, 300μl per slide, and allowed to incubate for 30 min in a humidified chamber. For example, for Table 2, the first antigen stained will be CD3 diluted at 1:100 in Antibody diluent.

  5. Set up 4 Tissue-Tek dishes filled with TBST washing buffer. The first dish should contain a clean slide rack that will be moved from dish to dish.

  6. After Primary antibody incubation discard the primary antibody over a beaker.

  7. Wash slides in staining rack 4× 2min each in TBST.

  8. Remove slides from TBST and place into humidified staining chambers.

  9. Pipette 300μl of ready to use Secondary Antibody (Opal Polymer HRP or Super Picture HRP, detailed in the staining protocols below Tables 2–3) to each slide, and allow to stain for 10 min.

  10. Discard Secondary antibody and wash slides 4x (2min each) in TBST as previously described.

  11. Remove slides from TBST and place into humidified staining chambers.

  12. Pipette 200μl of Opal staining solution that has been diluted in Amplification Diluent and allow to stain for 10 min (Opal staining solution: Opal stock should be made in 150μl DMSO; dilute Opal 1:50 in Amplification Diluent). Use the Opal that you assigned to each primary antibody.

  13. Discard Opal solution and Wash Slides 4x (2min each) in TBST

  14. Rinse Slides in the AR Buffer (1X) that will correspond to the next staining antigen of interest, in an Opal slide processing jar then discard buffer. For example, for Table 2, that would be AR9 which corresponds to CD3.

  15. Completely fill jar with AR Buffer and Microwave for ~2 min at 100% power until solution is boiling and then, 18 min at 20% power, making sure that the slides are always completely submerged in buffer.

  16. Allow Slides to cool at room temperature for 30 min, then proceed to do further staining steps or store slides in fridge at 4 degrees C overnight.

These steps are repeated 5 more times until all primary antibodies have been successfully stained with the corresponding assigned Opal and Antigen Retrieval Buffer. The order of staining can impact staining results, thus following the staining order 1–7 is important and is detailed in Tables 2–3.

2.3. Spectral DAPI Stain, Mount, and Coverslip

  1. Set up 2 Tissue-Tek dishes filled with TBST washing buffer, and 2 Tissue Tek dishes filled with DH20. The first dish should contain a clean slide rack that will be moved from dish to dish.

  2. Wash slides in DH20 (2 min).

  3. Wash slides in TBST (2 min).

  4. Pipette 300μl DAPI staining solution to each slide, allow to incubate for 5 min (DAPI solution: 2 drops of spectral DAPI per ml TBST).

  5. Wash slides in TBST (2 min).

  6. Wash slides in DH20 (2 min).

  7. Move slides to chamber apply ProLong Diamond Antifade Mountant and coverslip.

  8. Allow slides to dry for a few hours to overnight.

  9. Clean slides with EtOH and image with fluorescent microscope.

2.4. Identification and characterization of immune infiltrate and TLS in melanoma by immunofluorescence

Acquisition and analysis of immunofluorescent images can be performed with a fluorescent microscope such as the Vectra 3 (Akoya Biosciences). Spectral unmixing or compensating is performed using the inForm image analysis software (Akoya Biosciences) and single stain controls. See Note 1 for suggested staining controls. Representative staining images are shown in Fig. 1–2 and staining protocols detailed in Tables 2–3. See Note 2 for image acquisitions suggestions and Note 3 for cell enumeration.

Figure 1.

Figure 1.

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Images of tissues stained with the TLS Identification Panel (Table 2). Images in (A-H) are of TLS found peritumorally in metastatic melanoma and images in (I-P) are of a tumor-involved lymph node specimen. The T-cell area of the TLS are seen in (B, C, J, K). Evidence of B-cell follicles are evident in (D, L). Evidence of PNAd+ vasculature found in the T-cell area are evident in (F, N), as well as mature DC (E, M). Evidence of proliferating cells occurring for B and T-cells are seen in (G, O). Scale bars and markers are indicated.

Figure 2.

Figure 2.

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Images of tissues stained with the T-cell Activity Panel (Table 3). Images in (A-H) are of TLS from metastatic melanoma; this TLS was identified using the TLS identification panel. Images in (I-P) are of a tonsil specimen used as a staining control. Evidence of T-cells (B, C, J, K), B-cells (D, L), Granzyme-B (E, O), TCF7 (F, M), and TOX (G, N) expression is shown. Scale bars and markers are indicated.

NOTES

  1. We include human lymph node, spleen, and tonsil specimens as positive staining and imaging controls. Positive staining controls are done side by side with specimens of interest as well as single stain controls that will be used for unmixing; no Opal staining controls (specimen is stained with all reagents/steps except the Opal dye staining steps are omitted) and no primary antibody controls (specimen is stained with all reagents except primary antibodies). Fluorescence Minus One controls (FMO) may also be useful during panel development.

  2. We acquire images of the whole tumor at 10x and select regions of interest to enable infiltrate analyses. For cell enumeration all regions of interest are scanned at 20x. For our analyses, we scan and analyze the entire tumor area. Potential TLS regions are selected based on CD20 staining. TLS are usually localized on the tumor border or peritumorally.

  3. We use Halo (Indica Labs) to enumerate immune cells on 20x scanned regions of interest. Annotations can be drawn around TLS using Halo software to enable enumeration of cell subsets at the TLS site. However, enumeration of immune cells can be done manually or by using other commercially available software packages such as inForm (Akoya Biosciences).

Funding:

This work was supported by the University of Virginia Harrison Research Award Grant and the University of Virginia Department of Surgery Research Grant

Footnotes

Conflicts of Interest: The authors have no conflicts of interest to declare.

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