For the in vitro suppression assay, CD4+ T cells from untreated Tg4 mice were stimulated either alone or in the presence of a titrated number of CD4+

T cells from i.n. Ac1–9[4K]-, [4A]- or [4Y]-treated Tg4 mice that had been re-stimulated in vitro in order to maximize IL-10 secretion 12. As shown in Fig. 5A, T cells from untreated mice proliferated optimally in response to Ac1–9[4K] stimulation, whereas CD4+ T cells from i.n. Ac1–9[4K]-, [4A]- or [4Y]-treated Tg4 mice responded poorly. When co-cultured with Hydroxychloroquine cost CD4+ T cells from untreated mice at a 1:1 ratio, CD4+ T cells from Tg4 mice treated with i.n. Ac1–9[4A] or [4Y] appeared suppressive, inhibiting naïve CD4+ T-cell proliferation by 55 and 64% at a ratio of 1:1, titrating out to 1:2 and 1:4, respectively (Fig. 5A). Supernatants from the in vitro suppression assays were collected and analyzed for IL-2 levels by sandwich ELISA. As shown in Fig. 5B, CD4+ T cells from all three peptide-treated groups produced

very small amounts of IL-2 when compared with untreated CD4+ T cells. The amount of IL-2 detected in the co-cultures reflected the amount of suppression observed in Fig. 5A. Taken together, these results demonstrate a hierarchy in the ability of the tolerizing Copanlisib in vitro peptides to induce Treg as significant suppression of T-cell proliferation and IL-2 secretion was only detected in co-cultures containing CD4+ T cells from i.n. Ac1–9 [4A]- and [4Y]-treated Tg4 mice. An in vivo model of T-cell-mediated suppression has been described previously 6 whereby CFSE-labeled Tg4 cells were transferred into either untreated or peptide-treated recipient mice and their proliferation to subsequent peptide challenge assessed by CFSE dilution. This assay was used here to address the capacity of the different affinity

peptides to mediate suppression in vivo. Figure 6 shows the proliferation of naïve Tg4 CD4+ T cells adoptively transferred to untreated or peptide-treated recipient mice. The baseline CFSE level was determined by administering a single dose of i.n. PBS to untreated recipient mice. Upon challenge with Ac1–9[4A], CFSE+CD4+ T cells divided in the untreated recipient mice with a division only index of 0.32. The division index of CFSE+CD4+ T cells transferred to i.n. Ac1–9[4K]-treated recipients was lower (0.28) but not significantly different from the above. However, when transferred to i.n. Ac1–9[4A]- or [4Y]-treated recipient mice, the division index of the same cells was only 0.13 and 0.06, respectively. Thus, the proliferation of transferred T cells was significantly suppressed upon transfer to i.n. Ac1–9[4A]- and [4Y]-treated recipient mice. These results are consistent with those depicted in Fig. 5 and demonstrate that the observed hierarchy in the ability of the tolerizing peptides to induce Treg and thus mediate suppression extends to in vivo suppression of T-cell proliferation.