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. 2022 Feb 7;10(2):e003382. doi: 10.1136/jitc-2021-003382

Figure 4.

Figure 4

HMBD-002 neutralizes VISTA activity by inhibiting VISTA-VSIG3 binding, attenuating VSIG3-mediated suppression of IFN-γ release from activated T cells, reducing MDSC-mediated T cell suppression and inhibiting neutrophil chemotaxis. (A) Inhibition of VISTA:VSIG3 binding, analyzed by competition ELISA. Data shown are mean of n=3 measurements and error bars are SEM. (B and C) VSIG3 suppression of IFN-γ levels, measured by ELISA at 24 hours, from PBMC cultured in plates coated with αCD3 monoclonal antibody (OKT3) and VSIG3-Fc at ratios of 1:2, in presence and absence of test articles as indicated. Data shown are mean of n=3 measurements; error bars are SEM and p value was obtained by (B) paired t-test, *p<0.05 and (C) one-way analysis of variance (Tukey’s multiple comparison test), **p<0.01. (D) Anti-CD3-induced IFN-γ secretion from MDSC co-cultured with autologous PBMCs after 96 hours, in presence or absence of indicated test articles as measured by ELISA. Data were normalized to isotype control. Data shown are mean of n=6 measurements and error bars are SEM and p value was obtained by unpaired t-test. *p<0.05, **p<0.01 (E) Inhibition of neutrophil migration to the bottom chambers of transwell coated with C5a and measured using CellTiter–Glo. Data shown are mean of n=3 measurements and error bars are SEM. IFN, interferon; MDSC, myeloid-derived suppressor cells; VISTA, V-domain Ig suppressor of T cell activation; VSIG3, V-Set and Immunoglobulin domain containing 3.