Figure 5: RNAseq analysis shows no substantive difference in CD22 expression; Bryostatin1-mediated increased CD22 augments CART potency; and CART exposure to Bryostatin1 decreases IFN-gamma production, but increases Granzyme B production, and does not adversely affect tumor clearance in vitro or in vivo.

(A-B) KOPN8 or SEM cell lines were exposed to 1nM Bryostatin1 for 16, 24, 48, or 72 hours. RNA was extracted and analyzed by RNAseq. (C-E) Cell lines were co-incubated with 1nM of Bryostatin1 for 24 hours. Then 1×10⁵ target tumor cells were co-cultured with 1×10⁵ CD22 CAR for 16hrs. IL-2 (C), IFN-γ (D), and granzyme B (E) were measured by ELISA from cell culture supernatants. Statistics were calculated using unpaired t-test (**** p<0.0001, *** p<0.0002, ** p<0.0021, * p<0.0332). (F-G) CD22 CART cells were co-incubated with 1nM of Bryostatin1 for 24 hours and washed. Then 1×10⁵ target tumor cells were co-cultured with 1×10⁵ CD22 CAR for 18 hrs. IFN-γ (F), and granzyme B (G) were measured by ELISA from cell culture supernatants. Statistics were calculated using unpaired t-test (**** p<0.0001, *** p<0.0002, ** p<0.0021). (H) Mock or CD22 CAR T cells were co-incubated at 1:1 effector-to-target ratio with either CD22^neg, CD22^lo or Nalm6 cells. Cell death was monitored by loss of GFP-positive cells using IncuCyte ZOOM. (I) NSG mice were injected with CD22^neg, CD22^lo, or Nalm6 on Day 0, CD22 CAR T cells on Day 3, and then were given either DMSO control or Bryostatin1 at 40ug/kg once weekly for 2 weeks. Leukemia progression was monitored using IVIS technology and luciferin-D IP injections.