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1 Complete but curtailed T-cell response to very-low-affinity antigen Dietmar Zehn, Sarah Y. Lee & Michael J. Bevan Supp. Fig. 1: TCR chain usage among endogenous K b /Ova reactive T cells. C57BL/6 mice were infected with recombinant Listeria monocytogenes expressing Ova protein. On days 4.5 (black symbols) and 7.5 p.i. (red symbols) splenocytes from 10 mice were harvested and briefly in vitro re-stimulated with N4ova peptide. Cells were than co-stained with anti-cd8, anti-ifn and the depicted anti-v specific antibodies. K b /Ova specific cells were identified by their ability to make IFN The data show the fraction of K b /Ova specific cells expressing the indicated V chains. The asterix highlights changes with p-values <

2 Supp. Fig. 2: Defining H-2K b binding and stimulatory potency of the altered peptide ligands. A, Dose response curves of the capacity of individual APL and native Ova peptide (N4) to stabilize surface H-2K b on TAP deficient RMA-S cells are shown. B, The functional avidities of native Ova peptide and the APL in stimulating IFN production in an OT-1 T cell line are depicted. C, The ratios of the concentration of an APL divided by the concentration of native Ova peptide required for inducing a half-maximum IFN response (EC 50 ) in OT-1 T cells are shown. 2

3 Supp. Fig. 3: In vivo OT-1 response to Listeria encoding native Ova peptide or different APL. C57BL/6 mice grafted with 10 4 naïve congenic OT-1 cells were infected with non-recombinant Listeria monocytogenes (wt) or with Listeria expressing Ova protein containing the indicated epitopes. Shown are CD8 + gated white blood cells collected on day 6 p.i. The annotated numbers represent % OT-1 of total CD8 + T cells. 3

4 Supp. Fig. 4: Early termination of OT-1 expansion correlates with reduced BrdU incorporation. A and B, C57BL/6 mice grafted with 10 4 naïve congenic OT-1 cells were infected with recombinant Listeria monocytogenes expressing Ova protein containing the indicated epitopes. 5.5 days post infection BrdU was injected i.p. and 6 hours later splenocytes were harvested and stained for BrdU. Representative OT-1 T cell gated flow cytometry plots (A) and data for N=3 mice (B) of the frequency of BrdU positive cells within the OT-1 population are shown. 4

5 Supp. Fig. 5: Phenotypes and effector function of OT-1 T cells stimulated by native Ova peptide or different APL. A, B, C57BL/6 mice grafted with 10 4 naïve CD45.1 congenic OT-1 T cells were infected with Listeria monocytogenes expressing the indicated epitopes. A, Granzyme B production by CD8 + gated total splenocytes harvested on day 4 p.i. are shown. B, The cytokine production by splenic OT-1 T cells collected on day 7 p.i., re-stimulated for 5 hours with various peptides in vitro, is presented. In C the ability of OT-1 T cells primed by Lm-N4ova or Lm-V4ova to lyse V4 presenting target cells in vivo was tested. To exclude target lysis by high affinity V4 reactive endogenous T cells, P14xTCR -/- TCR transgenic mice were used as hosts OT-1 cells and Lm-N4ova, 5x10 4 and Lm-V4ova, or no OT-1 and Lm-V4ova were transferred into these mice. On day 5 post infection a 50:50 mix of V4 peptide pulsed CFSE high and control CFSE low cells were transferred into the mice. 24 hours later, cells recovered from the spleen were analysed for CFSE. Representative flow plots, the magnitude of target cell lysis and the frequency of OT-1 cells among total splenocytes are shown. Despite more than 30-fold lower OT-1 numbers after Lm-V4ova compared to Lm-N4ova stimulation, about 40-50% of target cells were lysed within 24 hours, indicating that V4 primed OT-1 possess cytotoxic capacity. 5

6 Supp. Fig. 6: PALS retention of the clonal progeny after high affinity ligand stimulation. A very low input mixture of 80 CD45.1 OT-1 and 80 Thy1.1 OT-1 cells was transferred into C57BL/6 mice, which were subsequently immunised with Lm-N4ova. Spleen sections were examined at day 4 p.i. and costained with anti-cd45.1 (green) and anti-thy1.1 (red) as well as with anti-b220 (blue). At this limiting dilution dose of OT-1 cells and at this stage, more than 80% of the OT-1 containing PALS carried either CD45.1 or Thy1.1 OT-1 cells and not both. The lack of a mixture of CD45.1 and Thy1.1 OT-1 cells implies a single clone origin of OT-1 cells in one PALS and that N4 stimulated OT-1 cells are, at least until day 4, retained in the same PALS where they had met antigen and expanded. 6

7 Supp. Fig. 7: The earliest wave of endogenous K b /Ova specific T cells released into the blood stream is dominated by low avidity T cells. C57BL/6 mice were infected with 10 3 CFU Lm-N4ova and bled on days 4.5 and 7.5 post infection. PBMC were isolated and in vitro expanded via stimulation with anti-cd3 and anti-cd28 antibody coated beads plus IL-2. 6 days later the T cell lines were briefly re-stimulated using titrated doses of N4ova peptide. Shown are dose response curves of the number of IFN producing T cells as the fraction of maximum response seen at 10µM peptide, error bars show standard error for N=5. 7

8 Supp. Fig. 8: OT-1 T cells responding to Lm-Q4ova or Lm-V4ova stimulation in the absence of competition from endogenous CD8 + T cell responders naïve congenic OT-1 T cells were transferred into C57BL/6 or TCRa -/- xp14 TCR transgenic mice which lack endogenous CD8 + T cells that can respond to Q4 or V4. These mice were infected with the indicated Listeria strains and bled on day 7 p.i. The frequencies of OT-1 cells among CD8 + white blood cells are shown. 8

9 Supp. Fig. 9: Recall responses of OT-1 memory T cells primed by N4 or V4 stimulation congenic OT-1 memory T cells isolated from C57BL/6 host mice on >day 35 p.i. with Lm-N4ova or LmV4ova were transferred into naïve C57BL/6 mice which were subsequently infected with Lm-N4ova or Lm-V4ova. On days 5 and 7 the secondary hosts were bled and the frequency of OT-1 among total CD8 + T cells was determined. Error bars show standard error for N=5. 9

10 Supp. Fig. 10: Response of OT-1 T cells to additional SIINFEKL derived altered peptide ligands. C57BL/6 mice grafted with 10 4 naïve congenic OT-1 cells were infected with nonrecombinant Listeria monocytogenes (wt) or with Listeria expressing Ova protein containing the indicated epitopes. Shown are the frequency of OT-1 among total CD8 + T cells on day 6 post infection. 10

11 Supplemental methods: Mice: C57BL/6.SJL and TCR -/- P14 TCR transgenic mice were obtained from Taconic farms. H-2K b stabilization: TAP deficient RMA-S cells were first incubated overnight at 26 C to increase the level of empty surface H-2K b molecules and then loaded with titrated doses of soluble peptide for 2 hours at 26 C and for 1 hour at 37 C. Cells were washed twice and stained with biotinylated anti-h-2k b specific mab Y3 and streptavidin PE (Molecular probes). Potency in stimulating OT-1: RMA-S cells loaded with titrated doses of peptide were used to stimulate an OT-1 T cell line for 5 hours in the presence of Brefeldin A. Subsequently, the OT-1 cells were stained for intracellular IFN. Data analysis. The data obtained were analysed and fit to sigmoidal doserespose curves and the EC 50 values for half-maximum response concentrations were calculated using GraphPad Prizm software. BrdU incorporation: At 5.5 days post infection mice were injected i.p. with 1mg BrdU (Sigma) dissolved in 0.5 ml sterile PBS. 6 hours later splenocytes were harvested and stained using the BrdU flow cytometry kit from BD. In vivo cytotoxicity assay: CD45.1 congenic total splenocytes were labelled with 2µM or 10µM CFSE. Then the high dose CFSE cells were pulsed with 10µM V4ova peptide for 1h at 37 C. Cells were washed three times and than a 50:50 mix of CFSE high and CFSE low (total of 2x10 6 per mouse) were injected i.v. 24 hours later cells were harvested. 11

12 Expansion of PBMC: Similarly as described (Zehn et al. Immunity Aug;25(2):191-3) PBMC were purified from total blood using lympholyte-mammal (Cedarlane Laboratories). Cells were expanded using anti-cd3/cd28 coated beads (Invitrogen) and T cell growth factor ( -methylmannopyranoside-blocked supernatant from concavalin A-stimulated rat splenocytes). 6 days later cells were analysed in an ICS assay as described in the main methods section. Transfer of memory OT-1 cells. Splenocytes isolated from C57BL/6 mice containing OT-1xLy5.1 memory cells (>day 35 post infection) were incubated with Fc block (24G2 mab) and then stained with biotinylated anti-cd45.1 mab (Becton Dickinson) and anti-biotin MACS beads (Miltenyi). Cells were positively selected using LS selection columns (Miltenyi) and transferred into naïve mice which were subsequently infected with Lm-N4ova or Lm-V4ova. 12

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