Morphological analysis
Muscle biopsy specimens from participating partner sites were cryopreserved immediately after removal at − 80 °C before analysis.
8 µm cryostat sections were stained according to standard protocols, including H&E, Gomori trichrome, Elastica van Gieson, non-specific esterase, acid phosphatase, Kongo red, SDH and COX-SDH.
A Benchmark XT Immunostainer (Ventana Medical Systems, Illkirch, France) was used for immunohistochemical staining in a standardized manner. The primary antibodies are listed as follows (clone, dilution, company): mouse anti-human: CD8 (C8/114B, 1:100, DAKO), CD20 (L26, 1:400, DAKO), CD45 (UCHL1, 1:400, DAKO), CD56 (ERIC-1, 1:200, Serotec), CD68 (EBM11, 1:100, DAKO), C5b-9 (aE11, 1:100, DAKO), HLA-ABC/MHC cl. I (W6/32, 1:1000, DAKO), HLA-DR/MHC cl. II (CR3/43, 1:200, DAKO), MHCneo (NB-MHCn, 1:20, Novocastra), CD57 (SPM 129, 1:50, Zymed), PD1 (NAT105, 1:100, Abcam); rabbit anti-human: CD4 (SP35, 1:100, Zymed), CD3 (polyclonal, 1:100, DAKO), p62 (polyclonal, 1:100, Abcam), KLRG1 (polyclonal, 1:50, Proteintech), CD27 (EPR8569, 1:500, Abcam), PD-L1 (E1L3N, 1:100, Cell Signalling); rat anti-human: PD-L2 (TY25, 1:100, Abcam).
Fluorescence staining with luminescent conjugated oligothiophenes (LCO) and fluorescence double staining was performed in staining chambers after fixation in acetone for 10 min. For LCO staining, the sections were incubated with pFTAA for 30 min and afterwards washed with phosphate-buffered saline (PBS) and distilled water.
For double immunostaining, serum of the secondary antibody species was added first for 30 min to avoid non-specific binding. The incubation with the first primary antibody was at 4 °C overnight and was followed by the first fluorescent secondary antibody with an incubation time of 1 h at room temperature.
The protocol was repeated in the dark with a second primary antibody and a second fluorochrome-coupled secondary antibody on the same sections, each with an incubation time of 1 h. Each incubation was followed by a washing step with PBS for 2 × 5 min. Finally, the sections were aqueously mounted with Vectashield mounting medium with DAPI and stored at 4 °C.
Fluorochromes and other fluorescence staining solutions are listed as follows (dilution and company): goat anti-rabbit: AF488 (1:100, Invitrogen), Cy3 (1:100, Dianova); goat anti-mouse: AF488 (1:100, Invitrogen), Cy3 (1:100, Dianova); LCO pFTAA (1:500, Institute of Chemistry Linköping University, Sweden).
We used irrelevant antibody stains (either mouse/rabbit monoclonal isotype controls) as negative control, as well as omission of the primary antibodies. For positive controls, we used the respective tissues as mentioned by the companies.
Semi-quantitative scores and overall severity score
To evaluate the expression of calibre variance and atrophy, connective/fat tissue proliferation, necrosis, regeneration, MHC cl.-I/MHC cl.-II expression, rimmed vacuoles, mitochondrial alterations, autophagy and cellular infiltration, a semi-quantitative score was used to differentiate between low, medium, and severe levels.
Mitochondrial accumulation was categorized into cap-like subsarcolemmal accumulations, subsarcolemmal accumulations surrounding the entire fibre, and “ragged blue” fibres. Autophagy was separated into singular fine-granular sarcoplasmic labelling by p62, presence within vacuoles in a few fibres and presence in vacuoles in many fibres. The stages of cellular infiltration were divided into 1–4 cells per high power field (HPF = 0.096 mm2), 4–20 cells per HPF and > 20 cells per HPF. Capillary pathology, complement activation and amyloid deposits were divided into present or absent. A severity level of 0–3 or 0–1 was assigned for each category, and the sum of these results in an overall severity score of 0–10. The results are illustrated in a heat map. For the quantitative counting of the cells, 10 HPF were averaged. The values are presented as boxplots with 1.5*IQR (Interquartile range) whiskers.
RNA extraction and quantitative real-time PCR
According to the manufacturer's instructions, RNA was isolated using the triazole/chloroform method (Invitrogen, Carlsbad, CA, USA). The concentration of RNA and the degree of purity were measured with the Infinite M200 Microplate Reader (Tecan, Grödig, Austria, RRID: SCR_020543). Afterwards the complementary DNA was produced by reverse transcription using the High-Capacity cDNA Archive Kit (Applied Biosystems, Forster City, USA). To measure the expression level of the gene transcripts and an endogenous control gene (PGK1), quantitative PCR was performed using the 5' nuclease method on an Applied Biosystems™ QuantStudio™ 6 Flex Real‐Time PCR System (ThermoFischer, Waltham, MA; USA) with the following conditions: 95 °C 0:20, 95 °C 0:01, 60 °C 0:20, 45 cycles (values above 40 cycles were defined as not expressed). All genes were run as triplicates. The Ct values of the target genes were related to the endogenous control gene and ΔCt (= Ct—Ct of the endogenous control) was calculated.
The following Taq-Man Gene Expression Assays from Life Technologies/ThermoFisher were used: B3GAT1 (CD57), Hs01024500_m1; CD244, Hs00175569_m1; CD27, Hs00609654_g1; KLRG1, Hs00195153_m1; PD1, Hs01550088_m1; PDL1, Hs00204257_m1; PDL2, Hs00228839_m1; PGK1, Hs99999906_m1; TBX21, Hs00894392_m1.