Anti cd8a v450

Cells from blood, spleen, and lymph nodes were incubated with 1 µL per sample of mouse anti-Rat CD32 (clone D34-485, 20 µg/mL, BD biosciences, San Jose, CA, USA) diluted in staining buffer (0.5% bovine serum albumin (BSA) and 0.01% sodium azide in PBS), to prevent unspecific bindings. Then, cells were immunostained for 30 min on ice for the presence of T cells, B cells, NK cells, and myeloid cells by the following surface antibodies: anti-TCR-PerCP (clone R73, 4 µg/mL), anti-CD4-APC (clone OX-35, 4 µg/mL), anti-CD8a-V450 (clone OX-8, 8 µg/mL), anti-CD45R-PE (clone His24, 8 µg/mL), anti-CD161a-FITC (clone10/78, 2 µg/mL), anti-CD11b/c-PE-Cy7 (clone OX-42, 1.6 µg/mL), anti-CD40-FITC (clone HM40-3, 40 µg/mL), anti-CD86-PE (clone 24F, 16 µg/mL) all from BD biosciences. Cells stained with the corresponding isotype antibodies were used as control. Cells were washed 4 times in PBS and fixed with paraformaldehyde 1%. Samples were analyzed on a FACSCanto flow cytometer (Becton Dickinson), with acquisition of 10.000 events in gate for each sample, and data analyzed with FlowJo software.

For T cell phenotyping, single cell suspensions from pancreatic LNs or PA infiltrating cells were prepared and stained as described (26 (link)). For intracellular cytokine staining, T cells were restimulated ex vivo with hemagglutinin (HA)-specific peptides during 5 h before staining as previously described (26 (link)). The mAbs used were anti-CD61 (ITGβ3)-FITC, anti-CD51 (ITGαV)-PE, anti-CD49e (ITGα5)-APC, anti-CD183 (CXCR3)-Alexa Fluor 780, anti-CD29 (ITGβ1)-Pacific blue (BioLegend, San Diego, CA, USA); anti-CD4-V500, anti-CD4-FITC, anti-CD90.1 (Thy1.1)-PerCP, anti-CD90.1 (Thy1.1)-V450, anti-CD8a-V450, anti-CD62L-APC, anti-IL-2-APC, anti-IFNγ-PE (BD Pharmingen); anti-CD8a-APC-Alexa Fluor 780, anti-CD25-APC-Alexa Fluor 780, and anti-KLRG1-PE-Cy7 (eBioscience). Cells were analyzed on a FACSCanto II or a LSR Fortessa apparatus using Diva software (BDB).

The sialic acid mimetic Ac₅3F_axNeu5Ac was synthesized as described previously [21 (link), 23 (link)]. Carbo‐free blocking solution and biotinylated lectins MALII, SNA‐I, and PNA were purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Streptavidin‐PE was purchased from BD Pharmingen (Franklin Lakes, NJ, USA), eFluor 780 and 450 viability dyes from eBioscience, Inc. (San Diego, CA, USA), CellTrace™ Violet (PSBE) and CFSE Cell Proliferation Kits from Thermo Fisher Scientific. CpG ODN 1668 (‘5-TCCATGACGTTCCTGATGCT-3’) was purchased from Sigma Genosys (Haverhill, UK), lipopolysaccharide (LPS) (Escherichia coli O111:B4) from Sigma-Aldrich, and murine recombinant interleukin (IL)-2 from Immunotools. For flow cytometry experiments, the following antibodies were used: BioLegend: anti-I-A/I-E-BV510 (M5/114.15.2), anti-H-2 Kb/H-2D^b-PE (28–8-6), anti-CD86-APC/Cy7 (GL-1), Anti-CD90.1-APC/Cy7 (OX-7). Antibodychain: anti-CD80-A488 (16-10A1), anti-CD40-PE (23/3), anti-CD11c-APC (N418), anti-CD11b-PerCP (M1/70). eBioscience: anti-CD274-PE/Cy7 (MIH5). BD: anti-CD8a-V450 (53–6.7).