Fig. 5: MPXV M2 affects the interactions of B7.1/2 with CD28 and CTLA4.

a, b A flow cytometry analysis of the role of M2 on the binding of CD28 to hB7.1/2. c, d A flow cytometry analysis of the role of M2 on the binding of CTLA4 to hB7.1/2. hB7.1-transduced (293T-hB7.1) and hB7.2-transduced (293T-hB7.2) cells were first stained with human CD28-hFc or CTLA4-hFc proteins in the presence or absence of the MPXV M2, then the binding capacity of CD28-hFc and CTLA4-hFc to the 293T-hB7.1/2 was visualized by secondary antibodies (anti-human IgG/488) via flow cytometry. Representative flow cytometric plots of two independent experiments performed in duplicate (a, c). The data averaged from two independent experiments are shown as mean (b, d). 293T-hB7.1/2 cells stained with secondary antibody alone were used as a negative control (black). Gating strategy is shown in Supplementary Fig. 11b. Data were analyzed by two-tailed unpaired Student’s t test. *P < 0.05; **P < 0.01. e–h CD28/CTLA4 and M2 competition for the binding to hB7.1 (e, f) and hB7.2 (g, h) were tested by BLI. hB7.1/2-hFc were first loaded on the protein A biosensors, then the biosensors were dipped into buffer with or without M2 (the first arrow), followed by immersion into the buffer containing either CD28 or CTLA4 (the second arrow). hB7.1/2-buffer-hB7.1/2 were included as controls to indicate the unoccupied Fc-binding sites on the biosensor. The BLI traces are representative of two independent experiments. Source data are provided as a Source data file.