Anti pe magnetic bead conjugated antibody

For human subjects HIP2, HIP3, HIP4 and HIP5 subsets of naive and memory CD4+ and CD8+ T cells were obtained by magnetic-activated cell sorting (MACS) prior to gDNA extraction and immunosequencing using the Adaptive Biotechnolgies Immunosequencing TCRβ kit. CD4+ were first bead isolated by negative selection (Miltenyi) then the CD45RA Naive CD4s were isolated by positive selection with the negative fraction including memory cells. CD8 subset include a large fraction of CD8+ effector memory cells that are CD45RA+ (TemRA) which are highly clonal and if included as “naïve” would skew the diversity in the naive fraction. Therefore, after negatively selecting CD8+ T cells, we needed two steps to isolate the naive from the memory cells. We first positively selected the CD45RO+ fraction. The negative fraction included CD8+ TemRA cells and CD8+naive cells. We then used CCR7-PE antibody (Miltenyi) and an anti-PE magnetic bead conjugated antibody (Miltenyi) to positively select the naive cells from the CCR7negative TemRA fraction. The TemRA cells were combined with the CD45RO fraction for CD8 memory TCR sequencing and the CD45RA+CCR7+ was naive. All cell sorted populations were assessed for purity and quantity using analytical flow cytometry. A summary of resulting enriched T cell fractions is provided in Table S1.