Journal: Cancer Discovery

Article Title: IL2 Targeted to CD8 + T Cells Promotes Robust Effector T-cell Responses and Potent Antitumor Immunity

doi: 10.1158/2159-8290.CD-23-1266

Figure Lengend Snippet: Molecular design and characterization of AB248, a CD8 cis -targeted IL2. A, Overview of the objectives for a CD8 + T-cell cis -targeted IL2 molecule. B and C, Human PBMCs were assessed for expression of CD8α and CD8β by flow cytometry ( n = 10). Shown are the constituent cell types for CD8α + or CD8β + lymphocytes by percentage ( B ), and the percentage staining positive for CD8α or CD8β within the indicated cellular subsets ( C ). D, Overview of the molecular characteristics of the development candidate AB248. E, pSTAT5 was assessed by flow cytometry following a 25-minute stimulation of human blood with rhIL2, CTRL-not-α-hIL2, or AB248 ( n = 10). F and G, Human CD8 + T-cell subsets were flow sorted from peripheral blood and cultured for 24 hours with AB248, rhIL2, or media, and transcriptional changes were analyzed via RNA-seq. Shown is the sorting strategy for a representative donor ( F ) and gene expression (by average log 2 fold changes of cpm) over media control. APC; antigen presenting cell. ( G ) with each differentially expressed gene represented by a point. P value cutoffs of 0.2 are applied to fold change data. A reference dashed line with a slope of 1 is shown; R is the correlation coefficient from the linear regression model ( n = 3 donors). H and I, human PBMCs were cultured with the indicated concentrations of rhIL2, CTRL-not-α-hIL2, or AB248 for 5 days and Ki-67 expression was assessed by flow cytometry. Shown is Ki-67 staining in one representative donor at 10 nmol/L ( H ) and Ki-67 expression (%) within the indicated cell subsets as a function of concentration (I; n = 2). J, Isolated peripheral blood CD8 + T cells were incubated with AB248 for 48 hours in the presence or absence of suboptimal TransAct CD3/CD28 stimulation (1:10,000) and supernatant IFNγ and TNFα were quantified using MSD ( n = 5). Studies are representative of 2–3 independent experiments.

Article Snippet: Samples were submitted to SeqMatic to generate 10x libraries using 10 × 5′v2 Single-Cell RNA-seq, V(D)J enrichment and feature barcoding.

Techniques: Expressing, Flow Cytometry, Staining, Cell Culture, RNA Sequencing Assay, Control, Concentration Assay, Isolation, Incubation