Fig. 1

Workflow for immunohistochemistry and image analysis. a Diagram of the immunohistochemical workflow. b Quantification of the efficiency of elution for three antibodies: anti-PD1, anti-FOXP3 and anti-CD4. Black bars represent antibody staining intensity (mean ± SEM) and gray bars represent staining intensity following elution. Each experiment was carried out three times. The dotted line is the background intensity produced by an unstained tissue section. c Flow diagram to show Visiopharm methodology (www.visiopharm.com) used to analyse tissue sections stained sequentially with anti-CD4, anti-PD1 and anti-FOXP3 antibodies. The algorithm first identified cell nuclei by haematoxylin counterstain following which CD4 expressing cells were marked green. Next CD4 expressing cells were interrogated for co-expression of FOXP3. There was then a branch point in the algorithm. If FOXP3 expression was detected but there was no co-expression of PD1 at a high level (PD1^hi), as previously defined by comparison with tonsillar Tfh cells, then the cell was determined to be Treg and the nucleus was labelled yellow. However, if the cell co-expressed CD4, FOXP3 and was PD1^hi then it was determined to be a Tfr cell and the nucleus was labelled turquoise. Cells that were CD4 expressing and PD1^hi, but not FOXP3 expressing were determined to be Tfh cells and the nuclei were labelled orange. Cells of each subset were then counted digitally. d Identification of specific T-cell subsets by multiplex immunohistochemistry. Tissue sections were stained with anti-CD4, anti-PD1 (EuroMabNet, clone NAT105) and anti-FOXP3 (EuroMabNet, clone 236A). Example Tfh cell (CD4⁺PD1⁺FOXP3⁻) painted orange, Treg cell (CD4⁺PD1⁻FOXP3⁺) painted yellow and Tfr cell (CD4⁺PD1⁺FOXP3⁺) painted turquoise are presented