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. 2023 May 18;142(3):290–305. doi: 10.1182/blood.2022018937

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© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.

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Figure 4. — CD4⁺ T-cell proliferation in response to FVIII requires a select set of APCs. (A) Inactivation of MFs. C57BL/6 HA mice (n = 4-5) received GdCl₃ or PBS (control) IP, 1 day before, and on, the day of IV FOVA (5 μg) administration. (B) Depletion of MZ B cells. C57BL/6 HA mice (n = 3-4) received anti-CD11a and anti-CD49d or isotype control antibodies IP, 4 and 2 days before IV FOVA (5 μg) administration. (C) Depletion of DCs, MZMs, and MMMs. C57BL/6 CD11c-DTR/GFP mice (n = 3) received DT or PBS (control) IP, 1 day before, and on, the day of IV FOVA (5 μg) administration. (D) Depletion of pDCs. C57BL/6 BDCA2-DTR/GFP mice (n = 5) received DT or PBS (control) IP 1 day before, 1 day after, and 3 days after IV FOVA (5 μg) administration. (E) Depletion of cDC1s. C57BL/6 XCR1-DTRvenus mice (n = 4) received DT or PBS (control) IP, 1 day before and 2 days after IV FOVA (5 μg) administration. (F) S129/C57BL/6 HA mice (n = 5-12 per group) received a single FVIII +/− ODN1826 injection after MZ B-cell depletion. One week later, pDC frequencies were analyzed by flow cytometry in naïve (control), FVIII control (FVIII + isotype), ODN + FVIII control (ODN + FVIII + isotype), and MZ B cell–depleted (FVIII + αMZB and ODN + FVIII + αMZB) mice. Shown are means ± SD and P values by analysis of variance. (A-E) In each experiment, 1 day after FOVA administration, CD4⁺ T cells were magnetically sorted from OT-II mice, labeled with CTV and adoptively transferred to the experimental animals. Four days later the spleens were collected for flow cytometry analysis of proliferation. The plots show a summary of percentage proliferation (mean ± SD), representative proliferation histograms, and respective dot plots. P values come from unpaired Student t test. GdCl_3, gadolinium chloride; MMMS, marginal metallophilic macrophages; PBS, phosphate buffered saline.

Figure 4. — CD4⁺ T-cell proliferation in response to FVIII requires a select set of APCs. (A) Inactivation of MFs. C57BL/6 HA mice (n = 4-5) received GdCl₃ or PBS (control) IP, 1 day before, and on, the day of IV FOVA (5 μg) administration. (B) Depletion of MZ B cells. C57BL/6 HA mice (n = 3-4) received anti-CD11a and anti-CD49d or isotype control antibodies IP, 4 and 2 days before IV FOVA (5 μg) administration. (C) Depletion of DCs, MZMs, and MMMs. C57BL/6 CD11c-DTR/GFP mice (n = 3) received DT or PBS (control) IP, 1 day before, and on, the day of IV FOVA (5 μg) administration. (D) Depletion of pDCs. C57BL/6 BDCA2-DTR/GFP mice (n = 5) received DT or PBS (control) IP 1 day before, 1 day after, and 3 days after IV FOVA (5 μg) administration. (E) Depletion of cDC1s. C57BL/6 XCR1-DTRvenus mice (n = 4) received DT or PBS (control) IP, 1 day before and 2 days after IV FOVA (5 μg) administration. (F) S129/C57BL/6 HA mice (n = 5-12 per group) received a single FVIII +/− ODN1826 injection after MZ B-cell depletion. One week later, pDC frequencies were analyzed by flow cytometry in naïve (control), FVIII control (FVIII + isotype), ODN + FVIII control (ODN + FVIII + isotype), and MZ B cell–depleted (FVIII + αMZB and ODN + FVIII + αMZB) mice. Shown are means ± SD and P values by analysis of variance. (A-E) In each experiment, 1 day after FOVA administration, CD4⁺ T cells were magnetically sorted from OT-II mice, labeled with CTV and adoptively transferred to the experimental animals. Four days later the spleens were collected for flow cytometry analysis of proliferation. The plots show a summary of percentage proliferation (mean ± SD), representative proliferation histograms, and respective dot plots. P values come from unpaired Student t test. GdCl_3, gadolinium chloride; MMMS, marginal metallophilic macrophages; PBS, phosphate buffered saline.