Figure 1. Peptide tolerisation leads to functional impairment of virus-specific CD4⁺ T cells.

A–G: 5×10⁴ splenocytes from mice transgenic for a T cell receptor recognizing the LCMV helper epitope GP61 (LCMV-glycoprotein_61-80/I-A^b-specific TCR, SMARTA mice) and expressing the T cell marker Thy1.1 were adoptively transferred into C57BL/6 mice on day -10. One group of mice was treated with 100 µg GP61 dissolved in IFA (squares), while control mice were treated with IFA alone (circles) at days -9, -6, -3. At day 0 mice were infected with 200pfu LCMV-WE or left untreated. Seven days after infection mice were analyzed for CD4⁺ T cell function. (A) Frequencies of GP61-specific CD4⁺ T cells (Thy1.1⁺ T cells) were analysed in spleen and blood. (B) For further phenotyping of thy1.1⁺ CD4⁺ cells, cells were gated as shown in the gating tree. (C) Cells were re-stimulated with GP61 in vitro and after six hours Thy1.1⁺ T cells were analysed for intracellular expression of IL-4, IL-10, IFN-γ and TNF-α by FACS analysis (one of four representative dot blots is shown. (D) Bar charts show data analyzed in C (n = 4,*p<0.001). (E) Cells were re-stimulated with PMA/Ionomycin in vitro and after six hours Thy1.1⁺ T cells were analyzed for intracellular expression of IL-4, IL-10, IFN-γ and TNF-α by FACS analysis (n = 4, *p<0.001). (F) GP61-specific Thy1.1⁺ CD4⁺ T cells and CD4⁺ T cells from untreated C57BL/6 mice were analysed for the activation markers CD69, IL-7Rα, CD62L, CD44 and CXCR3 (marker is set on the activated phenotype). (G) Statistically analysis from the data derived in F (n = 4, *p<0.05). H: 5×10⁶ splenocytes from mice transgenic for a T cell receptor recognizing the LCMV helper epitope GP61 (LCMV-glycoprotein_61-80/I-A^b-specific TCR, SMARTA mice) and expressing the T cell marker Thy1.1 were transferred into a total of six C57BL/6 mice on day -11. Three of those mice were treated with 100 µg GP61 dissolved in IFA (GP61-IFA-pretreated), while the other three mice were treated with IFA alone (IFA-pretreated) at days -10, -7, -4. In addition six C57BL/6 mice were treated with 100 µg GP61 dissolved in IFA (GP61-IFA-treated) and six control mice were treated with IFA alone (IFA-treated) at days -10, -7, -4. At day -1 splenocytes from each of the three GP61-IFA-pretreated mice were transferred into one GP61-IFA-treated, one IFA-treated and one untreated C57BL/6 mouse (one untreated C57BL/6 mouse died during transfer). In parallel splenocytes from each IFA-pretreated mouse was transferred into one GP61-IFA-treated, one IFA-treated and one untreated C57BL/6 mouse (transfer scheme Figure S2). Number of transferred splenocytes was adapted to the frequencies so that all mice received the same numbers of Thy1.1⁺ CD4⁺ T cells at day -1 (data not shown). On day 0 mice were infected with 200pfu LCMV-WE. Six days after infection mice were analyzed for Thy1.1⁺ CD4⁺ T cells by FACS analysis.