Isolation of Central Memory T Cells

Leukapheresis products were obtained from healthy donors under protocols approved by the City of Hope Institutional Review Board. Central memory T cells defined as CD45RO+CD62L+ were isolated on Miltenyi autoMACS (Miltenyi Biotec Inc). Briefly, PBMC were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare) and incubated with anti-CD14 and anti-CD45RA microbeads (Miltenyi Biotec Inc) for 30 minutes at room temperature (RT). CD14+ and CD45RA+ cells were then immediately depleted using the autoMACS. The unlabeled negative fraction of cells was labeled with biotinylated-DREG56 mAb (COHNMC CBG) followed by incubation with anti-biotin microbeads (Miltenyi Biotec Inc). The CD62L+ T_CM were purified with positive selection on auotMACS. Immediately after selection, the T_CM were activated, transduced, and maintained as described (13 (link)). Cultures were supplemented with lenalidomide every 48 hours for 21 days before in vitro analysis.

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Wang X., Walter M., Urak R., Weng L., Huynh C., Lim L., Wong C.W., Chang W.C., Thomas S., Sanchez J.F., Yang L., Brown C.E., Pichiorri F., Htut M., Krishnan A, & Forman S.J. (2017). Lenalidomide enhances the function of CS1 chimeric antigen receptor redirected T cells against multiple myeloma. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(1), 106-119.

Publication 2017

Miltenyi autoMACS Ficoll-Paque anti-biotin microbeads

Biotin Cd45ro Cd62l Central memory t cells Density gradient centrifugation Donors Ficoll Institutional review board Lenalidomide Leukapheresis Microbeads

Corresponding Organization : City of Hope

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Variable analysis

independent variables

Lenalidomide treatment

dependent variables

T cell activation and transduction
T cell maintenance

control variables

Central memory T cells (CD45RO+CD62L+) isolated from leukapheresis products of healthy donors
PBMC isolation by density gradient centrifugation over Ficoll-Paque
Depletion of CD14+ and CD45RA+ cells using autoMACS
Positive selection of CD62L+ T cells (T_CM) using autoMACS

controls

Positive control: None specified
Negative control: None specified

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