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. 2024 Jul 4;12(7):e008677. doi: 10.1136/jitc-2023-008677

Figure 4.

Figure 4

anti-hOX40 hIgG1 monoclonal antibody activity requires both activating and inhibitory FcγR. hOX40het OT-I cells were transferred into hOX40KIhom, hOX40KIhomγ chain KO or hOX40KIhom FcγRIIB KO mice and then immunized as in figure 2A. (A) OT-I kinetic responses in hOX40KIhom recipients in response to challenge with anti-hOX40 hIgG1, hIgG2 and hIgG4 (solid lines) or isotype controls (dashed lines) n=3 except for Isotype hIgG4 where n=2 due to sick mouse being culled at start of experiment. Representative of two independent experiments. Splenic analysis of OT-I (B) and CD4+Foxp3+ (C) T cells on day 4 post OVA (5 mg) and antibody (100 µg) challenge. Isotype and anti-hOX40 hIgG1 n=6, isotype hIgG2, anti-hOX40 hIgG2 and anti-hOX40 hIgG4 n=5 and Isotype hIgG4 n=4. Representative of two (B) and three (C) independent experiments. (D) As in A except hOX40het OT-I purified CD8+T cells transferred in WT C57BL/6 recipients. OT-I kinetic responses in WT recipients in response to challenge with anti-hOX40 hIgG1, hIgG2 and hIgG4 (solid lines) or isotype controls (dashed lines) Isotype and anti-hOX40 hIgG1 n=5, isotype and anti-hOX40 hIgG2 n=4, isotype and anti-hOX40 hIgG4 n=3. Representative of two independent experiments. (E) OT-I kinetic responses in hOX40KIhom and hOX40KIhomγ chain KO mice in response to anti-hOX40 hIgG1, isotype hIgG1 n=4, anti-hOX40 hIgG1 n=3 due to one mouse excluded based on lack of OT-I response, hOX40KIhomγ chain KO Isotype hIgG1 n=5, hOX40KIhomγ chain KO anti-hOX40 hIgG1 n=4. Representative of two independent experiments. (F) Splenic analysis of OT-I T cells on day 4 post OVA (5 mg) and antibody (100 µg) challenge. hOX40KIhom Isotype hIgG1, anti-hOX40 hIgG1 and hOX40KIhomγ chain KO isotype hIgG1 n=4, hOX40KIhomγ chain KO anti-hOX40 hIgG1 n=5. (G) Splenic analysis of CD4+Foxp3+ T-cell fold change relative to Isotype control on day 4 post OVA (5 mg) and antibody (100 µg) challenge. hOX40KIhom anti-hOX40 hIgG1 n=14, hOX40KIhomγ chain KO anti-hOX40 hIgG1 n=14. (H) OT-I kinetic responses in hOX40KIhom and hOX40KIhom FcγRIIB KO mice in response to OVA and anti-OX40 hIgG1. hOX40KIhom—isotype hIgG1 n=16, OX40 hIgG1 n=12 due to four mice being excluded based on lack of response. hOX40KIhom FcγRIIB KO—isotype hIgG1 n=19, hOX40KIhom FcγRIIB KO anti-hOX40 hIgG1 n=15 due to four mice being excluded based on lack of response. Pooled from four independent experiments. (I) Blood analysis of OT-I T cells on day 40 post OVA (5 mg) and antibody (100 µg) challenge. hOX40KIhom isotype hIgG1 n=12, hOX40KIhom anti-hOX40 hIgG1 n=10 due to two mice being excluded due to lack of response, hOX40KIhom FcγRIIB KO isotype hIgG1 n=15 and hOX40KIhom FcγRIIB KO anti-hOX40 hIgG1 n=13 due to two mice being excluded due to a lack of response, data pooled from three independent experiments. (J) Splenic analysis of OT-I T cells on day 4 post OVA (5 mg) and antibody (100 µg) challenge. hOX40KIhom Isotype hIgG1 and anti-hOX40 hIgG1 n=4, hOX40KIhom FcγRIIB KO isotype hIgG1 and FcγRIIB KO anti-hOX40 hIgG1 n=5. (K) Splenic analysis of CD4+Foxp3+ T-cell numbers on day 4 post OVA (5 mg) and antibody (100 µg) challenge. hOX40KIhom Isotype hIgG1 n=4, anti-hOX40 hIgG1 n=6, hOX40KIhom FcγRIIB KO isotype hIgG1 n=4 and hOX40KIhom FcγRIIB KO anti-hOX40 hIgG1 n=6. (L) Splenic analysis of CD4+Foxp3+ T-cell fold change relative to isotype control on day 4 post OVA (5 mg) and antibody (100 µg) challenge. hOX40KIhom n=10, hOX40KIhom FcγRIIB KO n=11 data pooled from two independent experiments. ****p<0.0001, **p<0.01, *p<0.05 mean±SEM B–C, F and J—Sidak’s multiple comparison one-way ANOVA, G—unpaired t-test, I—Tukey’s multiple comparison one-way ANOVA. ANOVA, analysis of variance; FcγR, Fc gamma receptors; KI, knock-in; KO, knock-out; OVA, ovalbumin; WT, wildtype.