Anti cd3 clone 145 2c11

5 × 10⁴ CD4⁺ A1 Rag1^−/− × CBAc/a T cells or CD8⁺ OTI C57BL/6 T cells were incubated with pre-pulsed CHO-IE^K (with deadbox gene Dby peptide REEALHQFRSGRKPI) or CHO-SCD (Ovalbumin OVA_257-264 peptide SIINFEKL) for 48 hours. For trans-costimulation assays, 5 × 10⁴ IE^K expressing CHO cells were pulsed with 5μM of Dby peptide and incubated with A1 Rag1^−/− × CBAc/a T cells at a 1:1 ratio, together with the indicated number of unpulsed, CD80-expressing co-stimulatory CHO cells. For Figure 5, TCR stimulation was provided by immobilising 5μg/ml anti-CD3 (Clone 145-2C11; Biolegend catalogue number 100304) antibodies on streptavidin-coated plates (Sigma Aldrich catalogue number S6940). Supernatants were harvested after 48 hours and assayed for IL-2 by ELISA.
To analyse the data from CD28-WT of CD28-Y170F DO11.10 TCR transgenic mice, the dose response curves were fitted with a Gaussian function and the area under curve (AUC) calculated as a measure of IL-2 secretion, referred to as the integrated IL-2 response. In this analysis, data from both CD80-CD2 and CD80-CD4 were collated and the degree of IL-2 secretion impairment as a result of CD80 elongation was quantified by calculating the ratios of elongated CD80: CD80-WT. Paired T test analysis was used to determine if IL-2 secretion from elongated CD80 in CD28-WT T cells was significantly different from T cells with CD28-Y170F.

M-ATOs were fixed in 4% Formaldehyde (Sigma-Aldrich, Cat#
F8775) for 30 minutes at room temperature followed by 3×10
min washes in PBST (0.3% Triton X-100) and a 1-hour block in PBST/BSA (2%
BSA). M-ATOs were stained with anti-CD8a (clone 53–6.7;
Biolegend), anti-mDLL4 (clone HMD4–1;
Biolegend), anti-CD4 (clone RM4–5;
Biolegend), and anti-GFP (clone FM264G; Biolegend) at a
1:100 dilution, and anti-CD3 (clone 145–2C11; Biolegend)
at a 1:50 dilution overnight at 4°C. Secondary antibodies
AlexaFluor-594-conjugated anti-rat IgG (H+L) (Jackson ImmunoResearch,
Cat# 712–585-150) or Alexa-Fluor-488-conjugated anti-rat
IgG (H+L) (Jackson ImmunoResearch, Cat# 712–485-153)
were added at a 1:200 dilution for 2 hours at room temperature. For
anti-mDLL4, anti-hamster biotin (Jackson ImmunoResearch, Cat#
127–065-160) was added at a 1:500 dilution for 2 hours at
room temperature, and then AlexaFluor-594-conjugated Streptavidin
(Jackson ImmunoResearch, Cat# 016–580-084) was added
at a 1:800 dilution for 30 minutes at room temperature. Each M-ATO was
mounted individually in Vectashield Antifade Mounting Medium (Vector
Laboratories, Cat# H1000) on a concavity microscope slide
(Fisher Scientific). Immunofluo-rescence images were
acquired on a Zeiss LSM 880 confocal microscope equipped with Airyscan and
Zen software (Zeiss).

Naive OT-1 or P14 cells, respectively, from spleen were purified using naive CD8a⁺ T Cell Isolation Kit (Miltenyi Biotec) and subsequently separated by AutoMACS (Miltenyi Biotec). Purity of the cells (95%) was confirmed by flow cytometry. For in vitro stimulation, 2×10⁵ naive CD8⁺ T cells were resuspended in D-MEM supplemented with 10% FCS (fetal calf serum), 2 mM GlutaMAX (GIBCO), 10 mM HEPES (GIBCO), 100 U/mL Penicillin/Streptomycin (GIBCO), and 50 μM 2-Mercaptoethanol and stimulated with anti-CD3 (clone 145.2C11, Biolegend), anti-CD28 (clone 37.51, Biolegend) (1 mg/ml) and recombinant murine IL-12 (Peprotech) in 96-well plates pre-coated with 100 μg/mL goat anti-hamster IgG (H+L) secondary antibody (Jackson Immunoresearch).