Fig. 2: In vitro-generated Th9 cells mediate the GVT effect selectively toward B-cell malignancies.

a Th9 cells were generated in vitro from B6 (H-2^b) mice. Lethally irradiated B6D2F1 (H-2^bxd, CD45.2) recipient mice were reconstituted with TCD-BM from B6 (H-2^b, CD45.2) mice in the presence or absence of B6-derived (H-2^b) SCs or Th9 cells. Th9 cells were given only once at the day of BMT (1×) or twice at the day of BMT and 6 days later (2×). Tumor cells were injected either on the day of BMT (A20 (H-2^d), Bcl-1 (H-2^d), T8-28 (H-2^d), P815 (H-2^d) or seven days later (Bcr-Abl⁺ B-ALL (H-2^d, GFP⁺)) as indicated by arrows. Surviving animals/total animals treated are indicated in brackets. b Th9 cells were generated in vitro from B6.SJL (H-2^b, CD45.1) mice and co-injected into lethally irradiated B6D2F1 (H-2^bxd, CD45.2) recipient mice reconstituted with B6-derived TCD-BM (H-2^b, CD45.2). At the day of BMT, mice were injected with B-cell lymphoma A20 (H-2^d) or mastocytoma P815 (H-2^d). 14 days after BMT, splenocytes and liver lymphocyte were stained for CD45.1⁺ Th9 cells, which were intracellulary stained for IFN-γ, TNF-α, IL-13 and IL-9 expression. c B6.SJL-derived in vitro-generated Th9 cells were co-cultured in direct cell-to-cell contact or in transwells with A20 (H-2^d, CD45.2) or P815 (H-2^d, CD45.2). After 48 h, Annexin V expression was determined on CD45.1⁻ tumor cells. d Downregulation of MHC class II by CRISP/Cas9-mediated targeting of CIITA in A20 cells was confirmed by flow cytometry. Two MHC class II downregulated A20 cell lines (A20-II^-1 and A20-II^-2, GFP⁺) and the control A20 cells transfected with nontargeting sgRNA (A20PC, H-2^d, GFP⁺) were stained for I-E^d and I-A^d or isotype control. Lethally irradiated B6D2F1 mice were reconstituted with B6-derived TCD-BM in the absence of presence of in vitro-generated B6.SJL-derived Th9 cells. Mice were injected at the day of BMT with a 1:1 mixture A20-II^-1 and A20-II^-2 or the MHC Cl II expressing control A20PC. Surviving animals/total animals treated are indicated in brackets. a *P ≤ 0.0, **P ≤ 0.01, ***P ≤ 0.001; ns not significant. Data represent 1 (Bcr-Abl), 2 (Bcl-1, T8-28) or three independent experiments (A20, P815) for each tumor cell line. b Data represent the mean value ± SD of three mice/group. c Data represent the mean value ± SD of triplicates of two independent experiments with ***P ≤ 0.001; ns not significant, paired Student’s t test. d Representative FACS staining from more than five stainings performed. Data represent a single experiment with indicated mice numbers. ***P ≤ 0.001, ****P ≤ 0.0001; ns not significant.