Eight-Color Multiplex Immunohistochemistry for Simultaneous Detection of Multiple Immune Checkpoint Molecules within the Tumor Microenvironment

Mark A. J. Gorris, Altuna Halilovic, Katrin Rabold, Anne van Duffelen, Iresha N. Wickramasinghe, Dagmar Verweij, Inge M. N. Wortel, Johannes C. Textor, I. Jolanda M. de Vries and Carl G. Figdor
J Immunol January 1, 2018, 200 (1) 347-354; DOI: https://doi.org/10.4049/jimmunol.1701262

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    DAB single staining on tonsil tissue and CHO cells transfected with constructs cDNA encoding immune checkpoint molecules. DAB IHC screening was performed at dilutions recommended by the manufacturer on tonsil control tissue (A). CHO cells were transfected with constructs cDNA encoding immune checkpoint molecules. CHO AgarCyto sections were subjected to DAB IHC (B). Wild type cells do not show positive staining (B, top panel), whereas the transfected cells do show a positive signal (B, bottom panel). Original magnification ×20. Immune checkpoint molecules expression of the wild type and transfected CHO cells was confirmed by flow cytometry, stained with Ab clones suitable for this technique (C). Gray line represents CHO wild type, and black lines represents transfected CHO.

  • FIGURE 2.
    FIGURE 2.

    IHC staining of immune checkpoint molecules on tonsil control tissue. DAB IHC was performed at dilutions recommended by the manufacturer (A). Equal concentrations used in the TSA method resulted in a more intense staining and an increase in the number of positive cells (B). Primary Ab titrations were performed with the TSA method (C), and final dilutions (marked light gray) were chosen that yielded similar results to DAB IHC (D). Stainings were performed on sequential slides. Images are split up into composites (upper right halves) and results of the cell scoring (lower left halves). Cells were scored with inForm Cell Analysis by counting the number of positive cells of three ×20 fields (red = positive, blue = negative). Original magnification ×20.

  • FIGURE 3.
    FIGURE 3.

    Primary Ab order optimization of multiplex IHC. Optimal multiplex IHC determined by comparing the TSA single staining signal (A) with the multiplex signal (B) on sequential slides of tonsil control tissue. Optimal combination of PD-1 and PD-L1 in duplex was determined first, followed by implementation of OX40 in triplex, CD27 in 4plex, TIM3 in 5plex, and finally CD3 in 6plex (B). Optimal Ab order was determined by visual inspection in combination with cell positivity score determined by inForm Software analysis of three ×20 fields. Mean positive cell score is shown per incorporated position (C). Only the cell positivity score of the single, first, last, and chosen position is shown to reduce complexity of the graphs. Final position in the multiplex panel is marked with light gray. Original magnification ×20.

  • FIGURE 4.
    FIGURE 4.

    Eight-color multiplex IHC in different tumor types. Sections containing invasive margin tumor cores were stained with multiplex IHC with the optimal Ab order and tumor marker. Representative multicolor composite pictures of each tumor type are shown. Original magnification ×20. TM, tumor marker.

  • FIGURE 5.
    FIGURE 5.

    Assessment of immune checkpoint expression molecules in different tumor types. Representative overviews of each tumor type with stroma (gray), tumor (black), and immune marker–positive cell clusters (red circles) (A). DLBCL was tumor marker/cytokeratin negative. Expression of immune checkpoint molecules was determined on tumor cells and on immune marker–positive cells, subdivided into CD3+ and CD3− cells (B). Immune checkpoint molecule expression is shown as percentage of total tumor cells or total immune marker–positive cells. n = 3 per tumor type.

Additional Files

Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section