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Single cell suspensions from the spleen, secondary lymph nodes, or thymii were acquired by mechanical disruption, stained with phycoerythrin (PE) and/or allophycocyanin (APC)-conjugated tetramers (1 hour, room temperature), and then enriched using magnetic beads and columns as described (Moon et al., 2007 (link)). Samples were acquired on a BD FACSCanto or LSR II Fortessa, and analyzed with FlowJo software (Treestar). MOG_35–55:I-A^b and influenza A virus NP_311–325:I-A^b specific CD4 T cells were gated on lymphocytes, single cells, B cell and myeloid (B220, CD8α, CD11b, CD11c, F4/80) negative, CD3⁺CD4⁺CD8⁻ tetramer positive cells (Figure S2). p31:I-A^g7 (YVRPLWVRME) and InsB_10-23:I-A^g7 (HLVERLYLVCGEEG) specific CD4 T cells were gated on lymphocytes, single cells, B cell and myeloid (B220, CD8α, CD11b, CD11c) negative, CD3⁺CD4⁺CD8⁻ tetramer double positive (PE and APC) cells, as previously described (Judkowski et al., 2001 (link); Pauken et al., 2013 (link)). Tetramer geometric MFI was calculated in FlowJo by gating on tetramer⁺ CD4 T cells. TCR affinity was calculated as [(tetramer geometric MFI)/(CD3ε geometric MFI)].