Anti tcrαβ

The following conjugated antibodies were used for flow cytometric phenotyping and analysis: anti-CD34, anti-CD43, anti-KDR, anti-CD45, anti-CD7, anti-CD5, anti-CD4, anti-CD8, anti-CD3, antiTCRαβ, anti-CD56, anti-CD15, anti-CD14, and anti-CD235a purchased from BD Biosciences. All antibodies were used in a 1:30 dilution. Dead cells were excluded from analysis in all experiments by staining with DAPI. Flow cytometry analysis was conducted on an LSRII cytometer (BD Biosciences, Paris, France), a FACS CANTO (BD Biosciences, Paris, France), or a FACS CELESTA (BD Biosciences, Paris, France) and analyzed using FlowJo software (BD Biosciences, Ashland, OR, USA).

For flow cytometric analysis, splenocytes (2 × 10⁵) were labelled with mouse anti-rat monoclonal antibodies (mAb) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridininchlorophylla protein (PercP), allophycocyanin (APC) or APC-Cyanine (Cy)7, as in previous studies^{60 (link)}. In this case, the antibodies used were anti-CD4, anti-CD8α, anti-CD8β, anti-TCRαβ, anti-TCRγδ, anti-NKR-P1A, anti-CD25, anti-CD45RA (BD Biosciences, San Diego, USA), anti-CD62L, anti-CD103 (Biolegend, San Diego, CA, USA), anti-TLR-4 (Novus Biologicals, Littlon, CO, USA) and anti-Foxp3 (eBioscience). After staining with standard procedures^{20 (link)}, analyses were performed using a Gallios^TM Cytometer (Beckman Coulter Inc., Miami, FL, USA) at the CCiT-UB. All results were assessed by the Flowjo v.10 software (TreeStar, Inc., Ashland, OR, USA).

IEL (2 × 10⁵) were stained with anti-rat monoclonal antibodies (mAb) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridininchlorophylla protein (PercP), allophycocyanin (APC) or APC-cyanine (Cy)7, as in previous studies (24 (link)). In this case, the mAb used were anti-CD4, anti-CD8α, anti-CD8β, anti-TCRαβ, anti-TCRγδ, anti-NKR-P1A, anti-CD25, anti-CD45RA (BD Biosciences, San Diego, USA), anti-CD62L, and anti-CD103 (Biolegend, San Diego, CA, USA). After standard procedures (20 (link)), samples were analyzed using a Gallios^TM Cytometer (Beckman Coulter Inc., Miami, FL, USA) in the Scientific and Technological Centers of the University of Barcelona (CCiT-UB). The results were assessed by FlowJo version 10 software (TreeStar, Inc., Ashland, OR, USA). Lymphocyte subsets were defined as follows: Th (TCRαβ⁺ NKR-P1A⁻ CD4⁺) cells; Tc TCRαβ⁺ (TCRαβ⁺ NKR-P1A⁻ CD8⁺) cells; Tc TCRγδ⁺ (TCRγδ⁺ CD8⁺) cells; natural killer (NK) (NKR-P1A⁺ TCRαβ⁻) cells; NKT (NKR-P1A⁺ TCRαβ⁺) cells; CD4⁺ (CD4⁺ CD8⁻) cells; CD8⁺ (CD4⁻ CD8⁺) cells; CD8αα⁺ (CD8α⁺ CD8β⁻); CD8αβ⁺ (CD8α⁺ CD8β⁺). Results are expressed as percentages of positive cells in the lymphocyte population selected according to their forward-scatter cells (FSC) and side-scatter characteristics (SSC) or in a particular selected population.