Figure 5.

Either SHP-1 or SHP-2 Suffices for PD-1 Coinhibition in Jurkat T Cells

(A) Immunoblot analysis of equal amounts of proteins from total lysates of representative clones of WT Jurkat, Jurkat-PD-1, Jurkat-PD-1^OST, Jurkat-PD-1^OST SHP-1^–, Jurkat-PD-1^OST SHP-2^–, and Jurkat-PD-1^OST SHP-1^–SHP-2^– cells probed with antibodies to SHP-1, SHP-2, or VAV1 (loading control). Left margin, molecular size in kilodaltons. Data are representative of two independent experiments.

(B) Jurkat-PD-1^OST, Jurkat-PD-1^OST SHP-1^–, Jurkat-PD-1^OST SHP-2^–, and Jurkat-PD-1^OST SHP-1^– SHP-2^– cells were analyzed using flow cytometry for expression of CD3, CD28, PD-1, PD-L1, PD-L2, BTLA, and HVEM. Gray shaded curves correspond to isotype-matched control antibody (negative control), and data are representative of two independent experiments.

(C) IL-2 production by Jurkat-PD-1^OST, Jurkat-PD-1^OST SHP-1^–, Jurkat-PD-1^OST SHP-2^–, and Jurkat-PD-1^OST SHP-1^–SHP-2^– clones stimulated with Raji cells or Raji-PD-L1 cells in the presence of 1 ng/mL SEE. Analysis of IL-2 production was performed 24 h after stimulation. Expression of IL-2 (ng/mL) using boxplot with the median, boxed interquartile range, and whiskers extending to the most extreme point up to 1.5 times the interquartile range. Each dot corresponds to a clone of the specified Jurkat cells. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, and ^∗∗∗∗p ≤ 0.0001; ns, non-significant (unpaired Student’s t test). Data are representative of two independent experiments.