NR-V04 regulates B and T cells. (A) Schematic showing gating strategy for flow cytometry of B cell populations. (B) NR-V04 induces significant B cell proliferation in B16F10 tumors. B16F10 tumor-bearing mice were treated with vehicle and NR-V04 as in Fig. 8. N = 4, P = 0.0005 (B220+CD19+), P = 0.0002 (Plasmablast), P = 0.011 (IgD+IgM−), P = 0.0083 (IgD+IgM+); two experiments. (C and D) Splenic B cells from WT or NR4A1−/− mice were either untreated or treated with IgM to induce B cell proliferation that was detected using flow cytometry of dilution of Cell Trace Violet; two experiments for (C) showing representative images and (D) showing all the data points. N = 3, P < 0.0001. (E and F) Splenic B cells were treated with or without B16F10 lysis stimulation in vitro and B cell proliferation was assayed similarly using flow cytometry, with or without the presence of 250 or 500 nM of NR-V04 treatment for 24 h. N = 3. Representative images showing (E) NR4A1 protein or (F) Cell Trace Violet dilution, determined by flow cytometry. Con, control, NS, non-stimulated by B16F10 lysate; two experiments. (G and H) CD8+ T cell depletion diminishes therapeutic responses of NR-V04. Yummer1.7 tumors were established similarly as in Fig. 7 B, following i.p. injection of either Rat IgG2a or anti-CD8 antibody (Clone: 53-6.7; BioXCell) at 100 µg/mouse, twice a week starting day 0 of tumor cell injection. NR-V04 treatment started on day 7 similarly. (G) Histogram showing the effective depletion of CD8 but not CD4 T cells by anti-CD8 antibody. N = 3. (H) CD8+ T cell depletion diminishes therapeutic responses of NR-V04 in the Yummer1.7 model, similarly treated as in Fig. 7 B. N = 6, P = 0.3151, one experiment. (I) NR-V04 did not significantly reduce CD8+ Texh cells in (left panel) B16F10, N = 7, P = 0.1403, and (right panel) MC38 tumor models, N = 4, P = 0.1403, from experiments shown in Fig. 7 C, and Fig. 7 A, respectively, when tumors are different in size. (J–M) NR-V04 reduces CD8+ T cell exhaustion in vitro. (J and K) Human primary T cells isolated from blood and cultured with anti-CD3/CD28 Dynabeads (bead/cell = 1:1) for 7 days, and then treated with 500 nM of NR-V04 for 48 h prior to harvesting and analyzed using flow cytometry. (J) Flow gating of human CD8+ Texh cells. (K) 500 nM of NR-V04 significantly decreased human CD8+ Texh cells. N = 4, P = 0.0002, two experiments. (L and M) Mouse OT-1 CD8+ T cells supplemented with 10 ng/ml ovalbumin daily for 7 days to induce exhaustion, following with the treatment of 500 nM NR-V04 for 48 h prior to harvesting and analyzed using flow cytometry. (L) Flow gating of mouse CD8+ OT-1 Texh cells. (M). NR-V04 significantly reduced the percentage of OT-1 CD8+ Texh cells in CD8+ T cells. N = 4, P = 0.0479, two experiments. Two-way ANOVA was performed for all tumor growth curves with P values indicated. Others are shown as the mean ± SD. A two-sided unpaired t test was performed, with P values indicated. Supplementary to Fig. 8.
Figure S4.

NR-V04 regulates B and T cells. (A) Schematic showing gating strategy for flow cytometry of B cell populations. (B) NR-V04 induces significant B cell proliferation in B16F10 tumors. B16F10 tumor-bearing mice were treated with vehicle and NR-V04 as in Fig. 8. N = 4, P = 0.0005 (B220+CD19+), P = 0.0002 (Plasmablast), P = 0.011 (IgD+IgM−), P = 0.0083 (IgD+IgM+); two experiments. (C and D) Splenic B cells from WT or NR4A1−/− mice were either untreated or treated with IgM to induce B cell proliferation that was detected using flow cytometry of dilution of Cell Trace Violet; two experiments for (C) showing representative images and (D) showing all the data points. N = 3, P < 0.0001. (E and F) Splenic B cells were treated with or without B16F10 lysis stimulation in vitro and B cell proliferation was assayed similarly using flow cytometry, with or without the presence of 250 or 500 nM of NR-V04 treatment for 24 h. N = 3. Representative images showing (E) NR4A1 protein or (F) Cell Trace Violet dilution, determined by flow cytometry. Con, control, NS, non-stimulated by B16F10 lysate; two experiments. (G and H) CD8+ T cell depletion diminishes therapeutic responses of NR-V04. Yummer1.7 tumors were established similarly as in Fig. 7 B, following i.p. injection of either Rat IgG2a or anti-CD8 antibody (Clone: 53-6.7; BioXCell) at 100 µg/mouse, twice a week starting day 0 of tumor cell injection. NR-V04 treatment started on day 7 similarly. (G) Histogram showing the effective depletion of CD8 but not CD4 T cells by anti-CD8 antibody. N = 3. (H) CD8+ T cell depletion diminishes therapeutic responses of NR-V04 in the Yummer1.7 model, similarly treated as in Fig. 7 B. N = 6, P = 0.3151, one experiment. (I) NR-V04 did not significantly reduce CD8+ Texh cells in (left panel) B16F10, N = 7, P = 0.1403, and (right panel) MC38 tumor models, N = 4, P = 0.1403, from experiments shown in Fig. 7 C, and Fig. 7 A, respectively, when tumors are different in size. (J–M) NR-V04 reduces CD8+ T cell exhaustion in vitro. (J and K) Human primary T cells isolated from blood and cultured with anti-CD3/CD28 Dynabeads (bead/cell = 1:1) for 7 days, and then treated with 500 nM of NR-V04 for 48 h prior to harvesting and analyzed using flow cytometry. (J) Flow gating of human CD8+ Texh cells. (K) 500 nM of NR-V04 significantly decreased human CD8+ Texh cells. N = 4, P = 0.0002, two experiments. (L and M) Mouse OT-1 CD8+ T cells supplemented with 10 ng/ml ovalbumin daily for 7 days to induce exhaustion, following with the treatment of 500 nM NR-V04 for 48 h prior to harvesting and analyzed using flow cytometry. (L) Flow gating of mouse CD8+ OT-1 Texh cells. (M). NR-V04 significantly reduced the percentage of OT-1 CD8+ Texh cells in CD8+ T cells. N = 4, P = 0.0479, two experiments. Two-way ANOVA was performed for all tumor growth curves with P values indicated. Others are shown as the mean ± SD. A two-sided unpaired t test was performed, with P values indicated. Supplementary to Fig. 8.

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