Supplemental Figure 1. Protein L

Transcription

1 Supplemental Figure 1 Protein L m19delta T m1928z T Suppl. Fig 1. Expression of CAR: B6-derived T cells were transduced with m19delta (left) and m1928z (right) to generate CAR T cells and transduction efficiency determined by flow cytometric analysis of Protein L expression. Mock transduced controls shown in the upper panel. Nature Medicine: doi:1.138/nm.4258

2 Supplemental Figure 2 BM + A2-TGL T cells +m1928z T cells Week 2 Week 3 Week 4 Suppl. Fig 2. Antilymphoma effects of CAR T cells: Lethally irradiated BALB/c recipients were reconstituted with B6 lin-depleted bone marrow cells and inoculated with A2-TGL lymphoma cells. Designated groups were treated with 1x16 B6 m19delta or m1928z T cells. Tumor growth was monitored by in vivo bioluminescence. Survival was monitored and data pooled this and other independent experiments are depicted in Figure 1D. Nature Medicine: doi:1.138/nm.4258

3 A Supplemental Figure 3 BM + A2 +.5M m19delta +.5M m1928z B C BM + A2 +.25M m19delta +.25M m1928z BM + A M m19delta +.125M m1928z Suppl. Fig 3. m1928z T cells eliminate CD19-expressing lymphoma at a range of doses. Lethally irradiated BALB/c recipients were reconstituted with 1x1 6 B6 lin - depleted bone marrow cells and inoculated with A2 lymphoma cells as described in Figure 1E. Designated groups were treated with.5x1 6 (A),.25x1 6 (B) or.125x1 6 (C) Thy1.1 + m19delta or m1928z T cells. Survival was monitored. Selected groups from this experiment have been depicted in Figure 1E. Nature Medicine: doi:1.138/nm.4258

4 Supplemental Figure x1 6 m19delta +1x1 6 m19delta +1x1 6 m19delta +.1x1 6 m1928z +1x1 6 m1928z +1x1 6 m1928z Suppl. Fig 4. At very high doses, m1928z T cells cause greater lethality: Lethally irradiated BALB/c recipients were reconstituted with 1x1 6 B6 lin - depleted bone marrow cells. Designated groups were treated with.1x1 6, 1x1 6 or 1x1 6 Thy1.1 + m19delta or m1928z T cells. Survival was monitored. Nature Medicine: doi:1.138/nm.4258

5 A Supplemental Figure Harvest BM only +m1928z C B B c Day 6 B cells s Day 6 Thy1.1 T cells % PBMCs BM only +m1928z C NS Suppl. Fig 5. B cell aplasia mediated by m1928z T cells: Data from the mice depicted in Figure 2B. (A) Survival monitored (B) Peripheral blood was drawn at day 6 and CD45- gated events analyzed for B22+ B cells and Thy1.1 T cells by flow cytometry. Nature Medicine: doi:1.138/nm.4258

6 Supplemental Figure 6 No. of cells (1^5) A B C CD4 6 CD8 4 5 mcd19-delta 4 2 CD4 CD8 No. of cells (1^5) Naive Act. EM CM B6 à BALB/c Naive Act. EM CM % Frequency GATA-3 T-bet Foxp3 mcd19-car Suppl. Fig 6. Lethally irradiated BALB/c recipients were reconstituted with B6 lin-depleted bone marrow cells. Designated groups were treated with B6 1x1 6 m19delta or m1928z T cells. Splenocytes harvested at day 7 post-hsct were counted and analyzed by flow cytometry. The absolute counts of CD4 + and CD8 + T cells (A), T cell subsets: Naive (CD62L + CD25 - CD44 - ), activated (Act., CD62L - CD25 + CD44 - ), effector memory, (EM, CD62L - CD25 - CD44 + ), central memory (CM, CD62L + CD25 - CD44 + ) (B), and the frequency of T-bet, GATA-3 and Foxp3 in splenic T cells (C) are shown. Nature Medicine: doi:1.138/nm.4258

7 A Supplemental Figure 7 GM-CSF IFN-gamma 15 BM+A2 4 BM+A2 1 +m1928z 3 +m1928z pg/ml pg/ml BM+A2 +m1928z BM+A2 +m1928z IL-6 TNF-a 4 BM+A2 6 BM+A2 3 +m1928z 4 +m1928z pg/ml 2 pg/ml 1 2 BM+A2 +m1928z BM+A2 +m1928z Suppl. Fig 7. Serum cytokine levels in allo-hsct recipients with established A2 tumor: Lethally irradiated BALB/c recipients were reconstituted with B6 lin-depleted bone marrow cells and inoculated with A2 lymphoma cells. Designated groups were treated with 1.x1 6 m19delta or m1928z T cells. Serum was obtained on day 5 post-transplant and cytokine levels assessed by Luminex. Data pooled from 2 experiments are depicted. Nature Medicine: doi:1.138/nm.4258

8 Supplemental Figure WT Host, BM Only CART +m1928z CART Rag2 KO host, mu deficient BM CART +m1928z CART Suppl. Fig 8. GVHD in mice B cell deficient mice when treated with donor m1928z vs m19delta T cells: Lethally irradiated BALB/c WT or Rag2 KO recipients were reconstituted with 1x1 6 B6 WT or mu deficient lin - depleted bone marrow cells Designated groups were treated with 1x1 6 m19delta or m1928z T cells. Survival was monitored. Nature Medicine: doi:1.138/nm.4258

9 Supplemental Figure 9 A LTR m1928z-ot-1 construct CD19-scFV CD8TM CD28 CD3ζ E2A OT-1 TCRα T2A OT-1 TCRβ LTR B TCR Vα2 42% 57% TCR Vβ5.1/5/2 CD19-His Suppl. Fig 9. Transduction of m1928-ot1 T cells (A) The m1928z-ot1 construct. (B) T cells transduced with m1928z-ot1. CD19-receptor expression was detected with CD19-His and OT-1 TCR expression detected with anti-vα2-and anti-vβ5.1/5/2 using flow cytometry. Cells were gated on OT-1 TCR + (42%, red histogram) and OT-1 TCR - (57%, orange histogram) populations of transduced T cells to demonstrate linked expression of the CAR and TCR constructs. Mock-transduced T cells are depicted for comparison (blue). Nature Medicine: doi:1.138/nm.4258