Extended Data Fig. 5: cpLECs can present OVA_323-339 to OT-II T cells.

(a – f): Healthy or EAE score 3.0 cpLECs were FACS sorted as CD31⁺ Podoplanin⁺ after excluding CD45^intermediate microglia, CD45⁺ leukocytes, Ghost⁺ dead cells, and doublets. Sorted cpLECs were then co-cultured with magnetically purified CD4 OT-II T cells from splenocytes using negative selection after Cell Trace Violet labeling in the presence of 100 µg/mL of OVA_323-339 for either 24 hours (a – c) or 72 hours (d – f). (a – b): Gating strategy for measuring the early T cell activation marker CD69 by OT-II T cells after 24 hours of co-culture. (c): Quantitation of the average percentage of 2D2 T cells expressing CD69 after 24 hours of co-culture. n = 3 replicates per group, pooled from 4 healthy and 4 EAE mice; data are represented as mean ± standard error of the mean. For percent of CD69 OT-II cells, p < 0.0001. (d – e): Gating strategy for measuring T cell proliferation by the dilution of Cell Trace Violet by OT-II T cell after 72 hours of co-culture. (f): Quantitation of the average percent of proliferated OT-II T cells after 72 hours of co-culture. n = 3 replicates per group, pooled from 4 healthy and 4 EAE mice; data are represented as mean ± standard error of the mean. For percent of proliferated OT-II cells, p = 0.0012.

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