Fig. S1. Accumulation Analysis of Endogenous and MCC-Derived Peptides

Supplementary information

Supplementary Figure legends

Fig. S1. Accumulation analysis of endogenous and MCC-derived peptides. a, Accumulation of pMHC complexes at the T-cell-APC-interface. 5C.C7 TCR transgenic T cells were loaded with fura-2 and mixed with CH27 APCs that had been previously loaded with the biotin-tagged peptide and labeled with Cy3-streptavidin. The upper panel show differential interference contrast (DIC) images overlaid with calcium ratio images obtained with fura-2 (340/380 nm excitations) in a false color scale as indicated below. The middle panel shows epifluorescent midplane acquisitions of Cy3.

The lower panel show 3D reconstruction of Cy3-labeled pMHC fluorescence in the T- cell-APC interface, viewed from the T-cell looking at the APC illustrated in a false color scale as indicated in the color bar below. b, Images show three representative 3D reconstructions of Cy3-labeled pMHC fluorescence in the T-cell-APC interface after T- cell contact and induction of elevated Ca2+ in response to the indicated peptides presented by CH27 APC. c, Quantitative analysis of the accumulation frequency measured as the percentage of cells (accumulation ratio ± SD, n = 20) with an accumulation ratio

(synapse/membrane) >1.2 in response to the indicated peptides. d, The average of synapse associated fluorescence intensity to membrane fluorescence intensity (synapse/ membrane) (average accumulation ± SD, n = 20). e, Percentage of cells that showed increase (i) in fluorescence at the synapse was classified as high (more than 2-fold), moderate (between 1.6-fold and 2-fold), elevated (between 1.2-fold and 1.6-fold) and low

(less than 1.2) (n = 20). 2

Fig S2. Binding and stability analysis of endogenous/null or MCC-derived peptides. a, Comparison of the stability or b, affinity of peptide binding of various IEk binding peptides at 37 C (% of maximal binding  SD, n = 3). Fitting of the data (symbols) accordingly to a one-site competition model (solid lines) (GraphPad Prism 4) generated the indicated EC50-values. c, T-cell activation data for various peptides presented by

CH27 cells (IEk) measured as IL-2 production in 5C.C7 T cells which was detected by

ELISA (Eu3+ counts  SD, n = 3). d, Comparison of K5-biotin-peptide exchange of IEk-

K5, IEk-99A and IEk-99E complexes at different temperatures and peptide concentrations after 7 days incubation (Eu3+ counts  SD, n = 3) . Red arrows indicate the molar ratio between peptide and IEk molecules as present in the individual pMHC dimer mixtures.

Please note that no peptide exchange was observed at this molar ratio at 4C. Because no exchange of peptide could be observed after incubating IEk-null complexes for a prolonged time (>1 week) with K5 peptide, we concluded that the stimulating effect observed for heterodimers was not the result of exchange of null/endogenous ligand with

Fig S3: Functional characterization of generated pMHC dimers. a, Analysis of cross-linking efficiency. Cross-linker and cysteine-labeled monomeric K5-

IEk molecules were mixed in various molar concentrations to generate K5-K5 dimers

Cross-linking was verified by SDS PAGE analysis followed by Comassie Blue staining.

For comparison a K5- IEk sample is included without addition of cross-linker. Please note that IEk-molecules are not SDS-stable when produced in E. coli and therefore appears as bands at approximately 25 and 28 kDa, which correspond to the IEk - and IEk -chains 3 respectively. b, K5-derived dimers. MCC-MCC, K5-K5, K5-ER60, K5-99A and K5-99E pMHC dimers, K5 monomer and 99E monomer were generated, boiled in the presence of

SDS, analyzed by SDS-PAGE followed by silver-staining. The analysis revealed the expected -subunit crosslinks for all five types of dimer with no tendency to aggregate.

Because of the lacking SDS-stability of IEk molecules, the band at approximately 60 kDa represents two cross-linked IEk- chains. c, Activation by MCC-derived dimers. Primed

5C.C7 TCR transgenic T cells were loaded with fura-2 and mixed with the soluble pMHC dimers MCC-MCC, MCC-ER60, MCC-99A, MCC-B2M, MCC-cCyt, MCC-

MSA, MCC-99E at 60 g/ml or the 99A-99A dimer or MCC-monomer at 250 g/ml.

Based on the ratiometric analysis of fura-2, the percentage of cells (n>100) with an elevated calcium signal (>1.5) was determined. It is somewhat surprising that an effect could be observed for MCC-99A dimer when previous tetramer experiments from our laboratory have demonstrated that there was no effect of 99A-IEk combined with MCC-

IEk 1. Recently Stern and colleagues2 demonstrated that pMHC molecules coupled through shorter spacers are more efficient inducing T-cell activation, and it is likely that the scaffold provided by streptavidin tetramers does not provide the optimal spacing leading to less efficient T-cell activation. The small response observed with MCC-99E dimer and MCC-2M is caused by contamination with trace amounts of MCC-MCC dimer. To avoid this problem, the K5 heterodimers (Fig. 2) were purified twice on the anti-T7-column. d, Comparison of the dose-response of T-cell activation by pMHC dimers (K5-K5 and MCC-MCC) was analyzed as described in (c). e, Comparison of activation of naïve 5C.C7 T cell versus primed 5C.C7 T cells. Also shown in the figure is the effect of adding cross-linked biotinylated CD28 antibody (PharMingen) at 20 g/ml 4 to naïve 5C.C7 before adding the indicated homo- and heterodimers. Biotin-labeled

CD28 antibody was crosslinked via streptavidin (Sigma) (> 100 cells, fura ratio  SD, n =

Figure S4. T-cell activation potency and binding affinity of pMHC dimers. a, Quantitative analysis of Ca2+ response. Fura-2 ratio data (340/380 nm) were measured every 20 s for 20 T cells in a randomly chosen microscope field (fura ratio  SD, n = 20) and integrated for 10 min after imaging began as a measure of calcium elevation

(integrated ratio  SD, n = 20). The integrated ratio was calculated by taking the sum of the ratio measured for each 20 s intervals for n number of cells and then subtracting the number of cells considered. This number for each 20 s interval was then averaged over a total of 10 min. b, The percentage of cells (>150 cells, percentage of cells ± SD, n = 4) with membrane-localized pH(Akt)-YFP in response to the indicated concentrations of pMHC dimer. c, Quantitative analysis of pH(Akt) accumulation. The ratio between diffuse and accumulated pH(Akt)-YFP (fluorescence at the membrane relative to total fluorescence) was determined (integrated ratio  SD, n = 20) and integrated for 10 min after the start of imaging as a measure of PI3 kinase activity. The integrated ratio was determined as described for the Ca2+ analysis. d, Effect of mixing K5-ER60 pMHC dimers with K5-99E or 99E-99E dimers. 5C.C7 T cells were mixed with K5-ER60 pMHC dimer at 10 g/ml and K5-99E or 99E-99E dimers, respectively at 100 g/ml.

Based on the ratiometric analysis of fura-2, the percentage of cells (fura ratio ± SD, n =

4) with an elevated calcium signal (>1.5) was determined. No inhibiting effect could be observed for neither K5-99E nor 99E-99E. 5

Fig. S5. Effect of endogenous peptides when presented by membrane-associated

5C.C7 TCR transgenic T-cells loaded with fura-2 were added to APCs or bilayers. The

percentage of cells (>150 cells, percentage of cells ± SD, n = 5) with an elevated

calcium signal (>1.5) was determined using ratiometric analysis (340/380 nm). a,

CHO-gpi-IEk APC pulsed with a 100 M mixture containing a 1:1000 ratio of

agonist peptide (MCC) to endogenous peptide. b, lipid bilayers containing B7-1

and ICAM-1 (1g/ml) pulsed with a 0.08 M mixture containing a 1:100 ratio

MCC-IEk to endogenous-IEk. (c-d) CHO-gpi-IEk pulsed with a 100 M mixture

containing the indicated ratio of endogenous peptide to agonist peptide (MCC).

(e-h), Fura-2 ratio data (340/380 nm) were measured every 15 s for 6 T cells in a

randomly chosen microscope field (fura ratio  SD, n = 6) when added to (e-f)

CHO or (g-h) lipid bilayers pulsed with mixtures of endogenous/agonist peptide

as described above and the average calcium ratio was plotted as function of time.

Figure S6. The effect of CD4 binding mutations on pMHC dimer-induced T-cell activation by endogenous-agonist pMHC dimers. 6 a, Activation by pMHC dimers (60 g/ml) measured as the percentage of cells with an

increased calcium signal with and without blocking CD4 binding with an anti-

CD4 mAb (GK1.5). b, Schematic of the K5-ER60 dimers, indicating the location

of the CD4 binding site mutations, denoted by the prefix “m” and a star at the site.

c, 5C.C7 T cells expressing PH(Akt)-YFP were loaded with fura-2, mixed with the

indicated pMHC dimers and imaged. The fura ratio (340/380 nm) was determined

(integrated ratio  SD, n = 20) for the amount of calcium over background was

determined for 10 minutes after imaging began. d, Dose response assay of the

percentage of cells with membrane-localized pH-Akt-YFP (n≥ 15) in response to

various concentrations of pMHC dimers. e, The integrated ratio between diffuse

and accumulated pH(Akt)-YFP over 10 min is shown as a measure of PI3K

activity (integrated ratio  SD, n≥ 15).

Figure S7. The effect of CD4 binding mutations on pMHC dimer-induced T-cell activation by null-agonist pMHC dimers. 7 a, Schematic of the K5-ER60 dimers indicating the location of the CD4 binding site

mutations, denoted by the prefix “m” and a star at the site. b, 5C.C7 T cells

expressing PH(Akt)-YFP were loaded with fura-2, mixed with the indicated

pMHC dimers and imaged.A dose-response curve with respect to the percentage

of cells with elevated Ca2+ signaling (340/380 nm) was determined (>100 cells). c,

The fura ratio (340/380 nm) was determined (integrated ratio  SD, n = 20) and

integrated for 10 min after imaging began as a measure of calcium elevation. d,

Dose response assay of the percentage of cells with membrane-localized pH-Akt-

YFP (n≥ 15) in response to various concentrations of pMHC dimers. e, The

integrated ratio between diffuse and accumulated pH(Akt)-YFP over 10 min is

shown as a measure of PI3K activity (integrated ratio  SD, n≥ 15).

Supplementary Videos

Videos S1-S5

Fura-loaded in-vitro primed 5C.C7 T cells expressing PH(Akt)-YFP were mixed with

K5-K5 (Video S1), K5-ER60 (Video S2), K5-99A (Video S3), K5-99E (Video S4) or

K5-2M (Video S5) dimers at 50 g/ml as described in Fig. 2. The left panels show differential interference contrast (DIC) images overlaid with calcium ratio images obtained with fura-2 (340/380 nm excitations) in a false color scale and the right panel show the localization of PH(Akt)-YFP in 5C.C7 T cells.

Supplementary Methods

Functional analysis of IEk binding peptides. 8

Peptide dissociation and peptide binding was determined as previously described32.

Peptide exchange experiments were performed by incubating peptide-IEk complexes with biotin-labeled K5-peptide for 7 days at the indicated temperatures and concentrations.

After incubation, pMHC complexes were immobilized for 30 min at 4C onto microtiter- plates that had been coated with 14.4.4 antibody (anti-IEk) and blocked with 10% FCS in

PBS. Biotin-labeled K5 peptide was detected with Europium-labeled streptavidin

(Wallac).

T-cell activation assay.

5 x 104 of irradiated APCs (CH27) were pulsed in round-bottomed wells with dilutions of various peptides and mixed with 2 x 104 primed and Ficoll-purified (Histopaque-1119,

Sigma) primed 5C.C7 T cells for 24 h at 37C, 5% CO2. After incubation, the supernatant was tested for IL-2 in an IL-2-specific sandwich ELISA using Europium-labeled streptavidin (Wallac)32.

IL-2 staining

Primed 5C.C7 TCR transgenic T cells were mixed with various soluble pMHC monomer

or dimers at 50 g/ml and incubated at 37 C, 5 % CO2 for 4 hours. After incubation cells were diluted in PBS and allowed to adhere onto coverslips previously coated with poly-

L-lysine (Sigma-Aldrich) at 10 mg/ml in H20 for 5 min at RT. Cells were fixed with 3.7

% paraformaldehyde for 10 min at RT and washed one time with PBS followed by neutralization with 15 mM NH4Cl2 in PBS for 10 min at RT. Cells were permeabilized with 0.2 % Triton X-100 in PBS for 2 min at RT followed by four washes with PBS. 9

Cells were blocked with PBS containing 5% donkey serum (blocking solution) for 2 hours at 4C, washed and incubated with rat-anti-mouse IL-2 antibody (Pharmingen) in blocking solution. Cells were washed 4 times with PBS and stained with Cy3-labeled donkey-anti-rat IgG antibody (Jackson ImmunoResearch Laboratories). After four washes in PBS, coverslips were mounted in Vectashield (Vector laboratories) mounting medium.

Supplementary references

1. Boniface, J. J. et al. Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [corrected]. Immunity 9,

459-66 (1998).

2. Cochran, J. R., Cameron, T. O., Stone, J. D., Lubetsky, J. B. & Stern, L. J. Receptor proximity, not intermolecular orientation, is critical for triggering T-cell activation. J Biol

Chem 276, 28068-74 (2001).