Figure 2.
T-cell immunotherapy-induced MHC-II expression is mediated by IFN-γ. (A-C) Human THP-1, AML-250167, or AML-329614 cells were treated with vehicle (PBS), 10 ng/mL FLZ, human HLA-mismatched CD3+ T cells (E:T of 1:1), or FLZ and human T cells (FLZ + T) for 48 hours. Supernatant IFN-γ concentrations were measured by enzyme-linked immunosorbent assay (ELISA). (D-F) THP-1, AML-250167, or AML-329614 cells were treated with human UTD T cells (E:T of 0.1:1), or human T cells expressing CART-123 (E:T of 0.1:1) for 48 hours. Supernatant IFN-γ concentrations were measured by ELISA. (G-H) THP-1 (G) or AML-250167 (H) cells were treated with vehicle (PBS), 50 ng/mL IFN-γ, human HLA-mismatched CD3+ T cells (E:T of 1:1), or FLZ and human T cells (FLZ + T) for 48 hours in the presence or absence of antibodies to human IFN-γ (100 μg/mL) and the IFN-γR1 or CD119 (10 μg/mL). Isotype control antibodies were used as a negative control. MHC-II rMFI on the THP-1 (G) and AML-250167 (H) cells was measured by flow cytometry. (I-J) THP-1 IFN-γR1 (I) and β-actin (J) KO cell lines were generated using CRISPR-Cas9. Specifically, a lentivirus expressing CAS9, red-fluorescent protein (RFP), and IFN-γR1 or β-actin single guide RNA was used to infect THP-1 cells. IFN-γR1 KO (RFP+; IFNγR1−) and wild-type (RFP−; IFN-γR1+) cells or β-actin KO (RFP+; β-actin−) and wild-type (RFP−; β-actin+) cells were treated with vehicle (PBS), 50 ng/mL IFN-γ, human HLA-mismatched CD3+ T cells (E:T of 1:1), or FLZ and human T cells (FLZ + T) for 48 hours, and THP-1 MHC-II rMFI was determined by flow cytometry. (K-L) THP-1 (K) or AML-250167 (L) cells were treated with vehicle (PBS), 50 ng/mL IFN-γ, human HLA-mismatched CD3+ T cells (E:T of 1:1), or FLZ and human T cells (FLZ + T) for 48 hours in the presence or absence of ruxolitinib (1000 nM) or baricitinib (1000 nM). MHC-II rMFI on the THP-1 (K) and AML-250167 (L) cells was determined by flow cytometry. (M-P) THP-1 (M-N) or AML-250167 (O-P) cells were added to the upper and bottom chambers of a transwell plate with a 0.4-μm pore size to prevent cell migration. Cells in the upper chamber were treated with vehicle (PBS), 50 ng/mL IFN-γ, human HLA-mismatched CD3+ T cells (E:T of 1:1), or FLZ and human T cells (FLZ + T) for 24 hours and MHC-II rMFI on an aliquot of the THP-1 (M) and AML-250167 (O) cells in both the upper and lower chambers was determined by flow cytometry. Remaining THP-1 (N) and AML-250167 (P) cells in the lower chamber were cultured for an additional 3 days in the absence of the upper chambers and MHC-II rMFI was determined longitudinally by flow cytometry. Bars represent means and error bars represent standard errors above and below (when applicable) the mean. P values were calculated using an unpaired, 2-sided Student t test. ∗P < .05; ∗∗P < .001; ∗∗∗P < .0001; ∗∗∗∗P < .00001.

T-cell immunotherapy-induced MHC-II expression is mediated by IFN-γ. (A-C) Human THP-1, AML-250167, or AML-329614 cells were treated with vehicle (PBS), 10 ng/mL FLZ, human HLA-mismatched CD3+ T cells (E:T of 1:1), or FLZ and human T cells (FLZ + T) for 48 hours. Supernatant IFN-γ concentrations were measured by enzyme-linked immunosorbent assay (ELISA). (D-F) THP-1, AML-250167, or AML-329614 cells were treated with human UTD T cells (E:T of 0.1:1), or human T cells expressing CART-123 (E:T of 0.1:1) for 48 hours. Supernatant IFN-γ concentrations were measured by ELISA. (G-H) THP-1 (G) or AML-250167 (H) cells were treated with vehicle (PBS), 50 ng/mL IFN-γ, human HLA-mismatched CD3+ T cells (E:T of 1:1), or FLZ and human T cells (FLZ + T) for 48 hours in the presence or absence of antibodies to human IFN-γ (100 μg/mL) and the IFN-γR1 or CD119 (10 μg/mL). Isotype control antibodies were used as a negative control. MHC-II rMFI on the THP-1 (G) and AML-250167 (H) cells was measured by flow cytometry. (I-J) THP-1 IFN-γR1 (I) and β-actin (J) KO cell lines were generated using CRISPR-Cas9. Specifically, a lentivirus expressing CAS9, red-fluorescent protein (RFP), and IFN-γR1 or β-actin single guide RNA was used to infect THP-1 cells. IFN-γR1 KO (RFP+; IFNγR1−) and wild-type (RFP−; IFN-γR1+) cells or β-actin KO (RFP+; β-actin−) and wild-type (RFP−; β-actin+) cells were treated with vehicle (PBS), 50 ng/mL IFN-γ, human HLA-mismatched CD3+ T cells (E:T of 1:1), or FLZ and human T cells (FLZ + T) for 48 hours, and THP-1 MHC-II rMFI was determined by flow cytometry. (K-L) THP-1 (K) or AML-250167 (L) cells were treated with vehicle (PBS), 50 ng/mL IFN-γ, human HLA-mismatched CD3+ T cells (E:T of 1:1), or FLZ and human T cells (FLZ + T) for 48 hours in the presence or absence of ruxolitinib (1000 nM) or baricitinib (1000 nM). MHC-II rMFI on the THP-1 (K) and AML-250167 (L) cells was determined by flow cytometry. (M-P) THP-1 (M-N) or AML-250167 (O-P) cells were added to the upper and bottom chambers of a transwell plate with a 0.4-μm pore size to prevent cell migration. Cells in the upper chamber were treated with vehicle (PBS), 50 ng/mL IFN-γ, human HLA-mismatched CD3+ T cells (E:T of 1:1), or FLZ and human T cells (FLZ + T) for 24 hours and MHC-II rMFI on an aliquot of the THP-1 (M) and AML-250167 (O) cells in both the upper and lower chambers was determined by flow cytometry. Remaining THP-1 (N) and AML-250167 (P) cells in the lower chamber were cultured for an additional 3 days in the absence of the upper chambers and MHC-II rMFI was determined longitudinally by flow cytometry. Bars represent means and error bars represent standard errors above and below (when applicable) the mean. P values were calculated using an unpaired, 2-sided Student t test. ∗P < .05; ∗∗P < .001; ∗∗∗P < .0001; ∗∗∗∗P < .00001.

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