T cells from CD4-cre NKAP cKO mice show reduced sialylation via α2,8-linkage on cell surface that leads to decreased binding of inhibitory receptor Siglec-E. A, Splenocytes were freshly harvested from Rag1-GFP WT (grey filled histogram) or Rag1-GFP CD4-cre NKAP cKO mice (black open histogram) and were stained with plant lectins PNA, SNBL, or MAL II. PNA recognizes unsialylated O-glycans. SNBL specifically recognizes sialic acids via α2,6-linkage while MAL II specifically recognizes sialic acids via α2,3-linkage. Data shown are representitive of at least 3 mice per group from 4 independent experiments. B, Quantitative PCR analysis comparing mRNA levels of NKAP, α2,8-sialyltransferases (ST8sia1, ST8sia4 and ST8sia6) in sorted splenic CD4+Rag1-GFP+ RTEs from Rag1-GFP WT or Rag1-GFP CD4-cre NKAP cKO mice is shown. The relative expression of these genes was normalized to 18S rRNA. Data shown is the mean ± SEM of 5 mice in each group, with all data normalized to one of the WT mice in each group. Data were analyzed for significance by an unpaired Student’s t test.