1 / 5

Supplemental Figure 1

chaeli
Download Presentation

Supplemental Figure 1

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Supplemental Figure 1: IL13 mutein-CAR T cells show no difference in T-cell phenotype. The phenotype of IL13 mutein-CAR T cells and NT T cells was evaluated using a panel of antibodies. CAR and NT T-cell products consisted of CD4-positive and CD8-positive subpopulations (mean 34.8±3.73%; mean 65.9±2.4%), and contained naïve (CD3+/45RA+) and memory (CD3+/45RO+) T cells. Central (CD3+/CD62L+: 47.1±8.6%) as well aseffector memory T cells (CD3+/CD62L-/CCR7-: 33.0±8.5%) were present. Supplemental Figure 1

  2. (A) IL13Rα1 (mg/mL) (B) IL13Rα2 (mg/mL) • Supplemental Figure 2: IL13 mutein-CAR T cells secret IFNγ in an antigen-dependent manner. IL13K-CAR, IL13Y-CAR or non-transduced (NT) T cells were stimulated with increasing amounts of recombinant IL13Rα1 (A) or IL13Rα2 (B) proteins. After 24h IFNγ was measured by ELISA (n=2, assay performed in duplicate for each donor; mean and SD is shown); ≥250mg of IL13Rα1 and ≥ 125mg of IL13Rα2 protein induced significant IFNγproduction (p<0.05) of IL13K-CAR or IL13Y-CAR T cells in comparison to unstimulated T cells. Supplemental Figure 2

  3. K NT Y YR KR Donor 3 Donor 2 Donor 1 U373 U373 U373 IL13Ra1+/a2+ A431 Raji A431 Raji T cells 293T Lysis (%) IL13Ra1+/a2- IL13Ra1-/a2- Supplemental Figure 3: IL13 mutein-CAR T cells kill IL13Ra1+ and IL13Ra2+ cells. All four IL13mutein-CAR constructs (K, KR, Y, YR) killed IL13Ra1+/a2+ cells (U373), IL13Ra1+/a2- cells (A431 or 293T). In contrast IL13Ra1-/a2- cells (Raji or T cells) were not killed. Non-transduced (NT) T cells had no cytolytic activity. E:T ratio Supplemental Figure 3

  4. A B GL261 B16F10 Murine T cells Lysis (%) E:T ratio • Supplemental Figure 4:IL13 mutein-CAR T cells recognize and kill murine IL13Ra1- and IL13Ra2-positive cells. (A) qRT-PCR analysis of murineglioma (GL261),melanoma (B16F10), and murine T cells. (B) Human IL13-mutein CAR T cells (IL13K, IL13KR) killed IL13Ra1-positive (GL261) andIL13Ra2-positive (B16F10) cells. In contrast murine IL13Ra1- and IL13Ra2-negative cells (T cells) were not killed. NT T cells had no cytolytic activity. Supplemental Figure 4

  5. A 50.6% GFP CAR B C • Supplemental Figure 5: Limited persistence of 2nd generation IL13 mutein-CAR T cells in vivo.IL13KR-CAR T cells were transduced to express eGFP.ffLuc. (A) FACS analysis confirmed the expression of CAR and eGFP.ffLuctransgenes.(B) IL13KR-CAR and IL13KR.eGFP.ffLuc T cells killed IL13Ra1+/IL13Ra2+ cells (U373) in contrast to IL13Ra1-/IL13Ra2- cells (Raji cells) in a standard 4hchromium release assay. (C) 1 x 105 unmodified U373 cells were injected intracranially into mice. On day 7 mice received 2 x 106 IL13KR.eGFP.ffLucCAR T cells intracraniallyusing the same tumor coordinates. Bioluminescence imaging was used to monitor T-cell persistence. Supplemental Figure 5

More Related