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. Author manuscript; available in PMC: 2026 Feb 7.
Published in final edited form as: Methods Mol Biol. 2021;2265:573–587. doi: 10.1007/978-1-0716-1205-7_40

Multiplex Immunofluorescence Histology for Immune Cell Infiltrates in Melanoma-associated Tertiary Lymphoid Structures

Ileana S Mauldin 1, Adela Mahmutovic 1, Samuel J Young 1, Craig L Slingluff Jr 1
PMCID: PMC12880811  NIHMSID: NIHMS2135379  PMID: 33704741

Abstract

The presence of tertiary lymphoid structures (TLS) is correlated with prolonged patient survival in a variety of solid cancers, including melanoma. However, few methods have been described that could enable a more comprehensive understanding of the organization and functionality of TLS in solid cancers. In this chapter, we describe multiplex immunohistochemistry and microscopy approaches for identifying, characterizing, and quantifying TLS and intra-tumoral immune infiltrates in melanoma. The described methods are not limited to melanoma alone and could be used to evaluate tertiary lymphoid structures in a wide variety of human cancers.

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

1. INTRODUCTION

Tertiary lymphoid structures (TLS) have been observed in a variety of solid tumors in humans, and their presence is a favorable prognostic indicator for melanoma survival [1–4]. TLS have morphological similarities to conventional secondary lymphoid organs, typically containing organized T- and B-cell compartments, lymphatic vessels, and high endothelial-like vessels that express peripheral node addressin (PNAd). Additionally, dendritic cells (DC) are often localized in TLS, presumably to aid in antigen presentation [5–7]. The three multiplex IHC panels described here are designed to enable researchers to evaluate tertiary lymphoid structures (TLS), which are generally localized peritumorally, but could be used generally to analyze intratumoral immune infiltrates as well.

Patient immune responses are often analyzed by flow cytometric analysis. However, since the tissue is dissociated for this technique, information about the organization or location of immune infiltrates is lost, and thus identifying and analyzing TLS would not be feasible. Traditional bright-field immunohistochemistry (IHC) usually only allows for the visualization of 3 antigens in a tissue specimen at best, thus limiting studies of immune response. Furthermore, traditional bright-field immunohistochemistry is also challenging to perform with multiple antibodies simultaneously, especially when two or more antigens are co-expressed in the same cell or subcellular region, as they can block each other. The availability of techniques that could enable a more comprehensive understanding of the organization of immune infiltrate or TLS in cancers has been limited, until now. In this chapter, we describe methods for the characterization of TLS and intratumoral immune infiltrates in melanoma tumor specimens, using multiplex immunohistochemistry which enables the identification, quantification, and stable visualization of six antigens in one tissue specimen. An additional advantage of multiplex IHC is that it can be used to visualize multiple antigens co-expressed in the same subcellular region and enables users to perform spatial proximity analyses of the distance between two different cell types, data that is useful in informing patient outcome [8,9].

The three multiplex IHC panels presented here can enable studies that evaluate: the location of immune infiltrate within the tumor microenvironment, the organization of immune infiltrate relative to other neighboring immune cells, and the context of immune activity at these sites, which could further the understanding of the context of anti-tumor immune responses generated against cancer and could help to inform patient outcomes. They could easily be modified to incorporate new immunological targets of interest as well. The panel detailed in Table 2 is designed to aid researchers in identifying TLS from patient tumor specimens; for these experiments, normal lymph node or tonsil specimens can be utilized as positive staining control samples to evaluate staining success and reproducibility, since TLS have morphological similarities to conventional secondary lymphoid organs (Fig. 2 A–C). The markers encompassed in these panels include CD4 and CD8 to identify T cells and CD20 to identify the B cell compartment, which is usually organized and distinct in lymph nodes and TLS. PNAd is used to identify high endothelial venules usually localized in the T cell area of a lymph node and TLS, and CD83 is used to identify mature dendritic cells [5–7]. Once TLS have been identified in patient samples by utilizing the multiplex panel detailed in Table 2, the additional two multiplex panels can be used to further interrogate immune activity in tumor samples, in addition to the markers Foxp3 to identify Tregs [10] and Ki67 to identify proliferating T cells [11], which are also included in panel 2.

Table 2:

Tertiary Lymphoid Structures Identification Staining Panel

Staining Order Antigen Retrieval Primary Antibody and working dilution Secondary Antibody Opal
1. AR9 CD8 (1:500) Opal Polymer HRP 540
2. AR6 CD20 (1:1000) Opal Polymer HRP 520
3. DIVA FoxP3 (1:500) Opal Polymer HRP 570
4. AR6 PNAd (1:1000) Super Picture HRP 620
5. AR6 CD83 (1:200) Opal Polymer HRP 650
6. AR6 Ki67 (1:20) Opal Polymer HRP 690
7. AR6 - Opal Polymer HRP Spectral DAPI
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Fig. 2:

Fig. 2:

Fig. 2:

Fig. 2:

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Paraffin embedded tissue specimens were sectioned and stained for immunofluorescent microscopy as outlined in Sections 3.1 and 3.2. Tumor sections were stained with the antibodies and Opals detailed in Table 2, TLS Identification Panel. Images were acquired at 20x magnification and spectrally compensated using single stain positive controls. (A-B) Spectrally compensated images of peritumoral TLS found in melanoma specimens. (C) Image of tonsil used as a staining control. Scale bars are indicated and are 500μm or 100 μm.

TLS containing germinal centers (GC) have been correlated with improved survival for patients with hepatocellular carcinoma [12]. A recent study in patients with lung squamous cell carcinoma found that corticosteroid treatment during chemotherapy impaired the development of tertiary lymphoid structures with mature GC and abrogated their prognostic value [13]. Thus, the state of maturation of GC has been correlated to patient outcome [13,14]. TLS maturation stages have been described as early, primary follicle-like, and secondary follicle-like, and they are defined by expression of CD21 and CD23 markers [13,14]. CD21 stains follicular DCs, and CD23 is expressed by mature B cells and follicular DCs localized in GC. Early TLS contain dense B and T cell lymphocytic aggregates but lack CD21 and CD23 expression. Primary follicle-like TLS contain dense B and T cell aggregates and CD21+ cells but lack CD23 expression. Secondary follicle-like TLS contain dense B and T cell lymphocytic aggregates with CD21 and CD23 expression [13]. The panel detailed in Table 3 is designed to aid researchers in identifying the maturation of TLS and lymph node follicles from primary non-GC-containing follicles to mature GC-containing follicles (Fig. 3 A-C). Furthermore, activation-induced cytidine deaminase (AID) expression can also be evaluated in GC B cells using the panel described in Table 3. AID generates mutations which induce antibody diversity in lymph node-associated B cells [15,16] and thus may lend new insight into antibody production by B-lineage cells in TLS.

Table 3:

Tertiary Lymphoid Structures Maturation Panel

Staining Order Antigen Retrieval Primary Antibody and working dilution Secondary Antibody Opal
1. AR9 ILT4 (1:100) Opal Polymer HRP 620
2. AR9 AID (1:100) Opal Polymer HRP 570
3. DIVA FoxP3 (1:500) Opal Polymer HRP 650
4. AR6 CD20 (1:1000) Opal Polymer HRP 520
5. AR6 CD23 (1:100) Opal Polymer HRP 540
6. AR9 CD21 (1:500) Opal Polymer HRP 690
7. AR6 - Opal Polymer HRP Spectral DAPI
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Fig. 3:

Fig. 3:

Fig. 3:

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(A) Spectrally compensated images of peritumoral TLS from melanoma specimens, visualized post-staining with antibodies and Opals detailed in Table 3, TLS Maturation Panel. (B) Image of a lymph node specimen, used as a staining control. Scale bars are indicated and are 500μm or 100 μm.

The T helper cells (Th cells), also known as CD4+ cells, can adopt different functional states depending on the transcription factors they express. Th1, Th2, Th17, and Treg lineages are defined by expression of T‐bet, Gata‐3, RORγt, and FoxP3 expression respectively [17,18]. Recently, the transcription factor Eomesodermin (Eomes) has also been described as a master transcription factor of the Th1 phenotype, in addition to T-bet [19]. Eomes is highly expressed in CD8+ T cells, and may impact the effector function of CD8+ T cells [20]. Higher expression of T-bet is associated with a favorable outcome of cancer patients [21]. Both T-bet and Eomes have been implicated in anticancer responses [19,22]. Conversely, Tregs have been shown to mediate suppression of immune responses [23], and high Treg densities have been correlated with poor prognosis in some cancer models [24,25]. Another recently identified immunosuppressive molecule, ILT4, is predominantly expressed by innate immune cells: monocytes, macrophages, dendritic cells, and granulocytes. However, the role of ILT4 in cancer development and progression is not well defined (Fig. 3A-C) [26]. Markers to aid in identifying and quantifying immune suppressive FoxP3+ Tregs or ILT4+ innate immune cells (Figs. 2A–C, 3A-C), and immune activating T-bet or Eomes expressing T cells (Fig. 4A-C), have been included in multiplex staining panels 1–3, with a focused selection of antibodies for assessing Th lineage presented in Table 4. In addition, proliferating lymphocytes and tumor cells can be identified by expression of Ki67 [11,27] (Figs. 2A–C, 4A-C). Thus, in this chapter we describe three multiplex IHC panels that can be used to further understand immune activity and tumor control mediated by TLS. Without multiplex IHC, these types of analyses would not be possible.

Fig. 4:

Fig. 4:

Fig. 4:

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(A) Spectrally compensated images of peritumoral TLS in melanoma specimens, visualized post-staining with the antibodies and Opals detailed in Table 4, TLS T Helper Cell Lineage Identification Panel. (B) Image of a lymph node, used as a staining control. Scale bars are indicated and are 500μm or 100 μm.

Table 4:

Tertiary Lymphoid Structures T Helper Cell Lineage Identification Staining Panel

Staining Order Antigen Retrieval Primary Antibody and working dilution Secondary Antibody Opal
1. AR9 CD4 (1:100) Opal Polymer HRP 520
2. AR9 CD8 (1:500) Opal Polymer HRP 540
3. AR9 Eomes (1:200) Opal Polymer HRP 620
4. AR6 CD20 (1:4000) Opal Polymer HRP 570
5. DIVA T-bet (1:200) Opal Polymer HRP 650
6. AR6 Ki67 (1:20) Opal Polymer HRP 690
7. AR6 - Opal Polymer HRP Spectral DAPI
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2. MATERIALS

2.1. 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

2.2. Immunofluorescent staining

  1. Slide humidity chamber

  2. Antigen Retrieval (AR) 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. Monoclonal antibodies for staining (Tables 1–4)

  6. Super Picture HRP Polymer Conjugate Broad Spectrum

  7. ProLong Diamond Antifade Mountant

  8. 24×50mm cover glass

  9. Opal Staining jar

  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
CD4 SP35 Cell Marque
CD8 C8/144B Agilent
CD20 L26 Agilent
FoxP3 D2W8E Cell Signaling Technology
Ki67 SP6 Abcam
PNAd MECA-79 BioLegend
CD83 Abcam, catalog# ab205343
LILRB2/ILT4 LS Bio, catalog# LS-B9762
AID zA001 Thermo
CD23 1B12 Leica Biosystems
CD21 EP3093 Abcam
Eomes Abcam, catalog#ab23345
T-bet 4B10 Santa Cruz Biotechnology
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2.3. Image acquisition and analysis

  1. Vectra 3 fluorescent microscope or comparable microscope

  2. inForm® image analysis software

  3. Halo® image analysis platform (Indica Labs)

3. METHODS

3.1. Immunofluorescent staining of cell surface markers on formalin-fixed tumor sections

3.1.1. Deparaffinization, Rehydration, Fixation, and Antigen Retrieval.

  1. Immerse slides in Tissue-Tek slide staining dishes that are filled with the following solutions: xylene x 3 dishes, 100% EtOH, 95% EtOH, 70% EtOH, DH20 × 3 dishes, TBST x 1 dish, Neutral Buffered formalin x 1 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 AR Buffer. Rinse the slides for 2 min then discard Buffer.

  3. Completely fill chamber with AR Buffer that has been diluted to 1X. For example, for the staining panel described in Table 2, which starts with a CD8 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. If the buffer boils out and the slides are exposed, refill the chamber and start by getting the solution to boiling at 100% power and then 20% power for the remaining time.

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

3.1.2. Multiplex Stain.

Specific antibody staining protocols/details are found in Tables 2 – 4 for TLS Identification, TLS Maturation, and Th Lineage Identification, 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 CD8 diluted at 1:500 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 and add slides to staining rack in TBST.

  7. Wash slides in staining rack 4x (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 in Tables 2–4) 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 AR6 which corresponds to CD20.

  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; therefore, the staining order 1–7 is important and is detailed in Tables 2–4.

3.1.3. 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.

3.2. 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. Spectral unmixing or compensating is performed using the inForm image analysis software and single stain controls. See Note 1 for suggested staining controls. Representative staining images are shown in Fig. 1–3, and staining protocols are detailed in Tables 2–4. See Note 2 for image acquisitions suggestions and Note 3 for cell enumeration.

Fig. 1:

Fig. 1:

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Human melanoma specimens were stained for immunofluorescent microscopy as outlined in Sections 3.1 and 3.3 and Table 2. Specimens were scanned at 10x and regions of interest (shown in green boxes) were selected on spectrally uncompensated images. The regions of interest are of TLS located peritumorally around one melanoma specimen. Scale bar = 800 μm. Regions of interest are subsequently scanned at 20x for digital analyses which include spectral unmixing and analysis of immune markers.

4. Notes

  1. We include human lymph node and colon specimens as positive staining 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 that the Opal dye staining steps are omitted) and no primary antibody controls (specimen is stained with all reagents except primary antibodies) are also included. 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 and identification of all TLS. Regions of interest are then rescanned at 20x for cell enumeration. TLS are usually localized on the tumor border or peritumorally.

  3. We use the Halo® image analysis platform to enumerate immune cells on 20x scanned regions of interest. However, enumeration of immune cells can be done manually or by using other commercially available software packages such as inForm®.

Funding:

This work was supported by the University of Virginia Cancer Center (NIH P30 CA044579, Biorepository and Tissue Research Facility, Molecular and Immunologic Translational Science Core)

Footnotes

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

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