Clone fjk 16s

Single-cell suspensions of spleen were generated as has been described previously (Taylor et al., 2018 (link)). Blood was obtained from SS and SR rats, which had taken 20% fructose or tap water for 4 weeks. PBMCs were separated by Ficoll-Paque PLUS gradient centrifugation (GE Healthcare, Chicago, IL, USA). Briefly, phenotypic and intracellular analyses were performed by incubating cells with antibodies for T cell surface markers – fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat CD3 (1:100; clone G4.18, #554832; BD Biosciences, Franklin Lakes, NJ, USA) and PE-conjugated mouse anti-rat CD4 (1:100; clone OX-35, #554838; BD Biosciences) – for 30 min on ice in the dark. After washing, the cells were fixed and permeabilized using fix/perm concentrate (BD Biosciences) before incubation with antibodies for intracellular staining of APC rat anti-rat IL-17A (1:100; clone eBio17B7, #17-7177-81; Thermo Fisher Scientific) to identify Th17 cells and PerCP-cyanine5.5 rat anti-rat FOXP3 (1:100; clone FJK-16s, #14-5773-82; Thermo Fisher Scientific) to identify Treg cells. Cells were then washed and run through a four-color flow cytometer (FACSCalibur; BD Biosciences) and data were collected using CellQuest or FlowJo v10.0 software. An example of the flow cytometry gating strategy used is shown in Fig. S3.

The single-cell suspension method of PBMCs was described in our previous study [13 (link)]. The single-cell suspension method of the spleen and kidney was described in previous studies [23 (link), 24 ]. Rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) for sacrifice, and then, the serum, spleen, and kidney were taken and stored at -80°C in liquid nitrogen until further studies. Monoclonal antibodies FITC mouse anti-rat CD3 (1 : 100, clone G4.18, #554832, BD Bioscience, Franklin Lakes, NJ, USA) and PE mouse anti-rat CD4 (1 : 100, clone OX-35, #554838, BD Bioscience) as T cell surface markers stained the PBMCs, spleen, and kidney from WKY and SHR. CD3 and CD4 were used to identify T helper (Th) cells. To identify Th17 cells or Treg cells, the stained CD3 and CD4 cells were washed and fixed and then were permeabilized using fix/perm concentrate (BD Bioscience). The cells were incubated with the monoclonal antibody APC rat anti-rat IL-17A (1 : 100, clone eBio17B7, #17-7177-81, Thermo Fisher Scientific) for Th17 cells or PerCP-Cyanine5.5 rat anti-rat FoxP3 (1 : 100, clone FJK-16s, #14-5773-82, Thermo Fisher Scientific) for Treg cells. After incubation, the cells were washed and run through a four-color flow cytometer (FACSCalibur; BD Biosciences). Data were collected using CellQuest.

For the evaluation of Treg cells, single-cell suspensions of splenocytes were stained using the Anti-Mouse/Rat Foxp3 Staining Set PE of eBioscience. Briefly, cells were fixed, permeabilized and stained according to the manufacturer’s protocol (eBiosciences) using anti-forkhead-box-protein 3 (FOXP3) PE (eBioscience, clone FJK-16s), anti-CD4 FITC (clone RM4-5) and anti-CD25 APC (clone PC61.5) antibodies. Cells were acquired on a flow cytometer and gated on CD4+ in the living population, before gating on CD25+Foxp3+ cells. Treg numbers per spleen were calculated by multiplying % of CD4+CD25+Foxp3+ cells with the total numbers of isolated cells per spleen.

Tricolor flow cytometry was used to identify the influence of MPA on the population of ConA and PHA-stimulated regulatory T (Treg) cells and the expression of CD25 –a late activation marker. Surface antigens were stained using anti-canine mAbs (Table 1): rat anti-CD4:FITC (clone YKIX302.9, MCA1038F, AbD Serotec), mouse anti-CD25:eFluor 660 (clone P4A10, 50–0250, eBioscience, San Diego, USA) according to protocol of staining extracellular antigens described above [48 (link), 49 (link)]. Next, forkhead box P3 (FoxP3) expression was evaluated using a cross-reactive anti-FoxP3:PE antibody as described previously by Biller et al. using a FoxP3/Transcription Factor Staining Buffer Set (00–5523, eBioscience) [48 (link), 50 (link)]. Briefly, the samples were incubated overnight in the 1X Fixation/Permeabilization solution at 4°C in the dark. The samples were then washed in a 1X Permeabilization Buffer and incubated with the rat anti-FoxP3:PE mAb (clone FJK-16s, 12–5773, eBioscience) cross-reacting with dog cells at room temp. for 30 min (Table 1). After staining, the samples were washed again, suspended in the cytometric buffer and analyzed. Samples incubated with corresponding isotype were used as a negative control: rat IgG2a:FITC (MCA1212F, AbD Serotec), mouse IgG1:eFluor 660 (50–4714, eBioscience) and rat IgG2a:PE (12–4321, eBioscience).

Excised tumor and spleen tissues were fixed in 4% formaldehyde overnight and then cryopreserved in 30% sucrose for 24 h. Tissue samples were embedded in Tissue-Tek OCT (Sakura Finetek, Alphen aan den Rijn, NL) and sectioned at 8 μm using a cryostat (Leica Microsystems, Diegem, BE). Sections were stained with antibodies directed against murine CD4, CD8, and FoxP3. Briefly, sections were first blocked with 0.2% (v/v) Triton X-100 (Sigma), 10% (w/v) goat serum, 5% rat serum, and 2% BSA in PBS for 1 h at room temperature. Subsequently, the primary antibodies (rat CD8a-FITC 1:500 [clone 53–6.7, BioLegend] and CD4-FITC 1:500 [clone GK1.5, BioLegend] and mFoxP3-APC 1:500 [clone FJK-16s, eBioscience] were applied to the slides for 1 h at room temperature protected from the light. After washing with PBS, the sections were mounted using Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI to visualize the cell nuclei. The slides were imaged using a structured illumination AxioImager microscope (Zeiss, Oberkochen, GE). The results are presented in Supplemental Information.