MOESM7 of Delivery of oncolytic vaccinia virus by matched allogeneic stem cells overcomes critical innate and adaptive immune barriers

Additional file 7: Figure S7. Patients’ resistance to the Trojan horse is associated with critical HLA mismatches and the rapid induction of anti-stem cell cytotoxic and interferon responses. (A) The top table shows analysis of KIR Haplotypes as well as the presence of known KIR ligands including the Bw4 epitope (HLA-B) and the weak/strong C1/C2 epitopes (HLA-C). This table also includes analysis of the oligomorphic MICA/B molecules that serve as ligands for NKG2D activating receptors on NK cells. The bottom table shows the distribution and copy number of long(L)-inhibitory and short(S)-activating KIR receptors, with the total number of inhibitory and activating receptors present also summarized in the top table. Note the absence of clear correlation between permissiveness/resistance and KIR haplotype/KIR ligands, − 21 M/T dimorphism, and MICA/B oligomorphism. The RM58 stem cells manifest a potentially important KIR ligand C1/C2 mismatch with both the resistant SIBD01 and permissive SIBD02 blood donors, suggesting that such a mismatch alone is insufficient to confer resistance, which might also require additional and stronger HLA mismatching. (B–F) Flow cytometry analysis of gated live NK, NKT and T cells from the PBMC/ADSC/WT1 co-cultures, as in main Fig. 7, showing that all the 4 allogeneic stem cell lines tested induce much stronger CD107α and IFNγ responses in the NK and T cells from the resistant but not permissive blood donor even in the absence of the virus. The figure shows the average frequency and total numbers of IFNγ (B) or CD107α (C) single positive as well as the much lower-frequency IFNγ plus CD107α-double positive lymphocytes of each cell type. (E) Complete correlative analysis of gated live NK, NKT and T cells from the PBMC/ADSC/WT1 co-cultures as above (partly included in main Fig. 7c) showing the average percentages of CD107α or IFNγ single positive lymphocytes of each cell type based on triplicate wells and normalized to respective background (untreated controls). (F) Flow cytometry analysis as above showing that treatment with vaccinia virus or allogeneic stem cells doesn’t affect significantly the frequency of the gated lymphocyte populations, with the exception of the NKT-like cells from the resistant SIBD01 blood donor that expanded in response to the allogeneic stem cells alone and further in the presence of vaccinia virus infection. (G) NKT-like cells are the earliest produces of IFNγ. RM20 ADSCs (10,000 or 2000) were co-cultured with PBMC (250,000) from the resistant SIBD01 or permissive SIBD02 blood donors and WT1 VV (5000 pfu). Gated live NK, NKT and T cells were analyzed by flow cytometry for activation (CD69 surface stain) and effector functions (intracellular stain for IFNγ) at 6 h and 24 h timepoints. (H–J) Flow cytometry analysis of gated NK, NKT, and T cells from the PBMC of a resistant and several permissive blood donors co-cultured with allogeneic RM20 ADSCs untreated or pre-treated with 20 ng/ml IFNγ for 48 h. IFNγ pre-treatment enhances rather than suppresses NK and T cell responses in the permissive patients, in the presence (H) but also absence (I and J) of vaccinia virus. The data in (I) and (J) experiment also demonstrate that a later passage of the RM20 stem cells (p12) retains some T cell immunosuppression ability but loses ability to suppress NK cells. Stimulation of NK cells with K562 induces indirect T and NKT cell responses indicative of NK-NKT-T cell crosstalk. Data represent mean and SD of duplicate or triplicate wells showing % of gated population of cells or total number of cells. Asterisk was used to mark statistical significance (Student T-test, p