Uchl1

Immunophenotype of T cells was performed using standard staining and flow cytometry techniques as described before^{41 (link),42 (link)}. Briefly, combinations of fluorophore-conjugated anti-human monoclonal antibodies specific for CD3 (BioLegend, OKT3, Cat#317344, 1:500), CD4 (BioLegend, OKT4, Cat#317438, 1:500), CD8 (BioLegend, SK1, Cat#344724, 1:500), CCR7 (BioLegend, G043H7, Cat#353214, 1:500), CD45RO (BioLegend, UCHL1, Cat#304244, 1:500), PD1 (eBioscience, EBIOJ105, Cat#12-2799-42, 1:500), TIM3 (BioLegend, F38-2E2, Cat#345006, 1:500), LAG3 (eBioscience, 3DS223H, Cat#15-2239-42, 1:500) were used to label T cells in flow cytometry staining buffer for 30 min on ice after Fc blocking, followed by intracellular cytokine staining with the BD Fixation/Permeabilization Solution Kit. Data were acquired on LSRFortessa (BD Biosciences) and analyzed with FlowJo software (Treestar). Cytokine antibodies included IFN-γ (eBioscience, B27, Cat#MHCIFG04, 1:500), IL9 (BioLegend, MH9A4, Cat#507614, 1:500), IL2 (BioLegend, MQ1-17H12, Cat#500342, 1:500), GrzB (BD Biosciences, GB11, Cat#561142, 1:500), or annexin V (BD Bioscience, Cat#550475, 1:50).

Conventional hematoxylin/eosin and Masson’s Trichrome staining were used to assess cardiac inflammation and fibrosis, respectively (see Supplemental methods). Immunopositive cells (for CD3, CD45, CD45.1, CD90.1) and fibrotic markers (Masson’s Trichrome; αSMA; periostin, vimentin, both Abcam) were quantified using Olympus BX51 microscope and cellSens (Olympus) or Fiji software. Formalin fixed, paraffin embedded human heart tissue from 10 lymphocytic myocarditis patients were obtained from the Biobank of the Institute for Pathology and Neuropathology, University Hospital Tubingen in accordance with local ethical regulations and stained using rabbit anti-human CD4 (EPR6855, Abcam), mouse anti-human CD45RO (UCHL-1, eBioscience) and mouse anti-human CD45RA (HI100, eBioscience); see Supplemental Methods. Image acquisition was performed with a Zeiss AxioObserver Z1 widefield microscope and processed by ImageJ.
Human and mouse cardiac fibroblasts were stained with anti-mouse/human αSMA antibody and phalloidin (Sigma); see Supplemental methods. Image acquisition was performed with a Leica DM IRE2 microscope equipped with the Nomarski Interference Contrast and the Operetta high-content screening platform (Perkin–Elmer).

Cell purity was assessed by flow cytometry staining with antibodies specific for CD4 (SK3, eBioscience), CD45RA (HI100, eBioscience), CD45RO (UCHL1, eBioscience). Assessment of activation markers was conducted with antibodies to CD25 (BC96, eBioscience), CD69 (FN50, Biolegend), CD62L (DREG-56, Biolegend), CD40L (24–31, eBioscience), CD71 (OKT-9, eBioscience). Data was acquired on an LSRII (BD Biosciences) and analyzed in FlowJo (Treestar).