Anti cd3 ucht1

Cells were analyzed using a BD LSR Fortessa™ flow cytometer following staining with fluorochrome conjugated antibodies. Prior to staining, cells were fixed using the fix buffer set (Biolegend), according to the manufacturer’s instructions. Antibodies used within this study were conjugated anti-CD3 (UCHT1, Biolegend), anti-CD56 (N901, Beckman Coulter), anti-NKp30 (P30-15, Biolegend), anti-NKp44 (P44-8.1, BD), anti-NKp40 (29A1.4, Biolegend), anti-TNF-α (Mab11, eBioscience), anti-IFN-γ (485.B3, Biolegend). Annexin V/PI staining was performed according to the manufacturer’s protocol (Biolegend). The data was analyzed using FlowJo 10.7.1 software.

The following antibodies (Ab) were used: anti-CD209 (DCN46; BD), anti-CD83 (HB1SE; Biolegend), anti-CD86 (IT2.2; BioLegend), anti-CD3 (UCHT1; BioLegend), anti-CD19 (HIB19; BioLegend), anti-CD8 (RPA-T8; BioLegend), anti-Vα24 Jα18 TCR (6B11; Biolegend), anti-CD137 (4B4-1; BioLegend), anti-Ki67 (Ki67; BD Pharmingen), PE-HLA-A*0201 NLVPMVATV and APC-HLA-A*0201 GILGFVFTL (both Immudex). Cells were stained for 15 min at room temperature, washed 2 × in flow buffer (PBS + 5% FBS) and fixed in 1:1 4% formalin:PBS before analysis. In some experiments (Fig. 3A–F), MHC class I dextramers PE-HLA-A*0201 NLVPMVATV or PE-HLA-A*0201 GILGFVFTL (Immudex) were added after live/dead staining, for 10 min prior to surface Ab staining. All flow cytometry was performed on a BD Biosciences LSRFortessa or LSRII SORP flow cytometer. Cells were stained with Fixable Live/Dead NIR (Invitrogen). Data analysis was performed using FlowJo software (Tree Star, Inc.). For all analyses, dead cells and doublets were first excluded by gating.

Polarized CD4⁺ effector T cells were generated by activating purified HD naïve CD4 T cells with plate-bound anti-CD3 (UCHT1) and anti-CD28 (CD28.2) (both 5 μg/ml, Biolegend) in the presence of IL-2 (50 U/ml), IL-12 (1 ng/ml) and anti-IL4 (10 μg/ml) (Th1 conditions) or IL-2 (50 U/ml), IL-4 (20 ng/ml), anti-IL12 (10 μg/ml) and anti-IFNγ (10 μg/ml) (Th2 conditions). Cells were transferred into fresh media on day 3 and IL-2 was added, as needed. Cells were re-activated every 7 days using the same cultures conditions for 3 rounds of polarization. All cytokines and Abs except IL-2 (Peprotech) were purchased from R and D and T cell polarizing media contained Iscove’s DMEM supplemented with penicillin (200 μg/ml), streptomycin (200 μg/ml), gentamicin (40 μg/ml), 10% FBS and 5% human serum blood type AB.

For polyclonal Treg induction, human naive CD4⁺T cells were defined as CD3⁺, CD4⁺, CD45RA⁺, CD45RO⁻, CD127⁺, CD25⁻, HLA-DR⁻ and sorted with the BD FACSAria III for purity (see Supplementary Fig. 6). CD4⁺T cells were cultured for 12 h in a 96-well plate pre-coated with 5 μg ml⁻¹ anti-CD3 (UCHT1, BioLegend) and 5 μg ml⁻¹ anti-CD28 (CD28.2, BioLegend) and 100 U ml⁻¹ IL-2 (Peprotech). Limited TCR stimulation was achieved by pipetting the cells into new, uncoated wells, after 12 h, where they were cultured for additional 36 h without further TCR stimulation. To assess Treg induction using continuous TCR stimulation naive CD4⁺T cells were stimulated in pre-coated wells as described above for a time period of 54 h and analysed accordingly. For antigen-specific Treg induction, human naive CD4⁺T cells were defined as CD3⁺, CD4⁺, CD45RA⁺, CD45RO⁻, CD127⁺, CD25^-, HLA-DR⁻ and sorted with the BD FACSAria III for purity. Naive CD4⁺T cells were co-cultured with autologous CFSE-labelled DCs isolated as described above in the presence of insulin mimetopes, natural insulin B:9-23 epitope (0.001 and 0.01 ng ml⁻¹). After 12 h APCs were removed by sorting CD4⁺T cells as CFSE⁻ followed by a culture for additional 36 h in new wells without further peptide stimulation.

For flow cytometry cell sorting, cells were stained with anti-CD4 (RPA-T4) and anti-CD3 (SK7) antibodies (BioLegend); anti-CD4 (RPA-T4), anti-CD3 (SK7) and anti-HLA-DR (L243) or with anti-CD4 (SK3), anti-CD3 (UCHT1), and anti-CD14 (63D3) according to the sorting strategy. Gating strategies are depicted in Supplementary Figs 1–3. Sorted populations cell purity was routinely >98%. For intracellular cytokines assays sorted CD3^highCD4^high, rested for at least 3 h, were stimulated with 5 μg mL⁻¹ of anti-CD3 (UCHT1, BioLegend) and 2 μg mL⁻¹ of anti-ICOS (C398.4 A, BioLegend), crosslinked with 5 μg/mL anti-mouse IgG1 (BioLegend) plus 10 μg mL⁻¹ anti-hamster IgG (Thermo Fisher Scientific) at 37°C in the presence of Brefeldin-A (Life Technologies) for 14 h. Cells were fixed in paraformaldehyde 1% (Sigma-Aldrich) and permeabilized with saponin (Carl Roth). Antibodies used are listed in Supplementary Data 2. When indicated 1.7 μg mL⁻¹ LPS (Sigma-Aldrich), TNC (Merck Millipore), or cell-depleted synovial fluid (SF) was added. For TLR4 blocking, CLI-095 (InvivoGen) was added at 10 μg mL⁻¹ 1 h before stimulation. Cell sorting was performed in a BD FACSAria III instrument (BD Biosciences).

Upon Treg induction using limited or continuous TCR-stimulation in vitro sort-purification of CD127^lowCD25^highCD4⁺ Tregs was performed. The Tregs were then stimulated for 36 h in the presence 5 μg ml⁻¹ anti-CD3 (UCHT1, BioLegend) and 5 μg ml⁻¹ anti-CD28 (CD28.2, BioLegend) antibodies without addition of TGFβ followed by the analysis of CD25, CD127 and Foxp3 by intracellular staining and FACS as described above.

Tregs were sort-purified as DAPI⁻CD4⁺CD3⁺CD25^high T cells, and DAPI⁻CD4⁺CD25⁻CD44⁻CD62L⁺ T cells are sort-purified as activated responder T cells (Tresp). Splenocytes from Rag1^-/- mice were irradiated with 30 Gy of X-ray. Tresp cells (5 × 10⁴) were then labeled with Cell Trace Violet (CTV) according to the manufacturer’s protocol (Invitrogen). Labeled Tresp were cultured with Treg cells at ratios of 1:1, 1:2, 1:4, 1:8, and 1:16 for 72 h in the presence of anti-CD3 (UCHT1, BioLegend). Dleu2-17aa or scrPEP were added in the concentration of 10 μM. The suppression function of Treg cells was analyzed by determining the dilution of the CTV label in responder cell proliferation.