Kaluza version 1

Seminiferous tubules from decapsulated testes or epididymes were minced in PBS, warmed to 37°C and incubated for 15 min at room temperature with agitation (200 rpm on an orbitary shaker). Diffused germ cell suspension were collected in PBS, filtered through a 70μM cell strainer (Ref 352350, BD Falcon), fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences). DNA was stained by incubation with DAPI for 30 min at room temperature. Flow cytometry analysis was performed using a BD-FORTESSA (BD Biosciences) cytometer, and the results were analyzed using BD-DIVA version 8 (BD Bioscience), FlowJo version 10 (TreeStar) and Kaluza version 1.3 (Beckman Coulter Inc.) softwares.

For phenotypic T cell analysis and comparison of the microenvironment of PT and TDLN, multi-color flow cytometry was carried out using the LSR Fortessa X-20 (BD Biosciences). 150,000 T-cells per sample were stained using the following surface antibodies: CD3, CD4, CD8, CD25, CD45RA, CD27, CD127, HLA-DR, PD-1, TIM-3 and LAG-3. See Additional file 2: Table S2 for antibody specifications. After surface staining, cells were permeabilized and stained for FoxP3, Ki67, and CTLA-4 using a FoxP3 staining kit (00–8332-56, 00–5223-56, 00–5123-43; eBioscience) according to the manufacturer’s instructions. Data were analyzed using Kaluza version 1.3 (Beckman Coulter). CD8⁺ T cell data were visualized in t-Distributed Stochastic Neighbor Embedding (t-SNE) density plots generated in FCS express 6 (De Novo software).

For each sample, the fluorescence from 5000 platelets, gated by their characteristic forward and side scatter (Supplementary Figure S1A (online)),^{24 –26} was measured on a FACSVia flow cytometer (BD Biosciences, Oxford UK). For the analysis of fibrinogen binding, percentage positive platelet values were determined by gating 2% of the negative population using EDTA (10 µM) (Sigma, Gillingham UK) (Supplementary Figure S1B (online)). For P-selectin exposure, the mean fluorescence intensity (MFI) was calculated from the platelet population as defined by the forward and side light scatter (Supplementary Figure S1C (online)). Data were acquired using BD FACSVia research loader software (BD Biosciences, Oxford UK) and analysed using Kaluza version 1.5 (Beckman Coulter, High Wycombe, UK). The stability of performance of the flow cytometer was tested daily using FACSVia Cytometer Setup and Tracking beads (BD Biosciences, Oxford UK).

Multiparameter flow cytometry was performed in accordance to BD Pharmingen™ Protocol. In brief, splenocytes were harvested at day 14 post second boost; 5 × 10⁶ cells were stimulated in a Nunc™ 96-well conical-bottom plate (Thermo Scientific™, USA) for 6 h at 37 °C with 10 μg of M2eh peptide and 1 μg of influenza virus A/Aichi/2/68 (H3N2) in the presence of 1 μg/ml of Brefeldin A (BD Bioscience, USA) and purified hamster anti-mouse CD28. The cells were then washed, and Fc receptors were blocked using CD16/CD32 antibodies (Mouse BD Fc Block, BD Pharmingen, USA) for 30 min. Next, cells were incubated with Zombie Aqua (Zombie Aqua™ Fixable Viability Kit, Biolegend, USA) to enable gating of live cells during analysis and subsequently stained with antibodies (CD3e-FITC, CD8a-APC-Cy7, CD4-PerCP, CD62L-PE-Cy7, CD44-APC) at 4 °C for 30 min (antibodies from BD Pharmingen, USA). Cells were permeabilized using the BD Cytofix/Cytoperm Plus (BD Bioscience, USA) protocol and stained with anti-TNF-α-BV421 or anti-IFN-γ-PE (BD Pharmingen, USA). Sample acquisition (50,000 live CD3+ were collected) was performed with a BD FACS Canto II flow cytometer (Becton Dickinson, USA) and analyzed using Kaluza, version 1.5 (Beckman Coulter, USA).