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Blood, Vol. 113, Issue 1, 193-203, January 1, 2009
Anti-CD3 prevents factor VIII inhibitor development in hemophilia A mice by a regulatory CD4+CD25+-dependent mechanism and by shifting cytokine production to favor a Th1 response
Blood Waters et al.
113: 193
Supplemental materials for: Waters et al
Files in this Data Supplement:
- Figure S1. Optimization of FVIII concentration for restimulation of splenocytes from hemophilia A Balb/c mice (JPG, 44.8 KB)
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Splenocytes were pooled from anti-CD3–treated, HBSS-treated or untreated animals (n = 2–3/treatment group) 1 week after the final of 4 FVIII immunizations. Splenocytes (5 × 106/mL) were restimulated in vitro with FVIII for 72 hours and culture supernatant was analyzed by sandwich ELISA for IL-2. There was no apparent dose response in the untreated mice, most likely due to a very low frequency of FVIII-specific lymphocytes in the culture. In contrast, there was a clear dose response in the HBSS-treated and anti-CD3–treated mice. We chose 1.0 µg FVIII/mL as the optimal dose for all subsequent experiments because this resulted in a clearly measurable response in HBSS-treated controls and anti-CD3 mice, but not in the untreated animals.
- Figure S2. Anti-CD3 treatment in vivo increases regulatory CD4+CD25+ T cells (JPG, 36.4 KB)
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The spleens from hemophilia A Balb/c mice (n = 5/time point) were analyzed 1, 8 and 15 days after treatment with anti-CD3 (50 µg/d for 5 d) for expression of CD4+, CD8+ and CD4+CD25+ (gated on CD4+) cells. Untreated and age-matched hemophilia A Balb/c mice (n = 6) were also studied to determine basal T cells levels. The transient nature of this response may be due to the very short half-life of anti-CD3 F(ab)′2 fragments (<1 day).22 Data are shown as the mean ± S.E.M.
- Figure S3. CD4+CD25+ cells from anti-CD3–treated mice and controls express high levels of GITR and FoxP3 (JPG, 64.3 KB)
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Splenocytes were pooled and analyzed from anti-CD3–treated, HBSS-treated or untreated animals (n = 2–3/treatment group) 1 week after the final of 4 FVIII immunizations (1 of 3 and 1 of 2 representative experiments for the Balb/c and C57Bl/6 mice, respectively, is shown). All groups of Balb/c mice expressed high levels of the Treg markers GITR (A) and FoxP3 (B) in the CD4+CD25+ population. A similar result was observed for C57Bl/6 mice (C) and (D). There was a slight decrease in expression of GITR in tolerant C57Bl/6 mice (C) relative to the HBSS-treated, but the non-tolerant anti-CD3–treated mice also produced high levels of GITR. Importantly, there was significant decrease in expression of FoxP3 in the anti-CD3 (nt) relative to the anti-CD3 (tol.) mice (D). Anti-CD3 (tol.) and anti-CD3 (nt) refer to anti-CD3–treated C57Bl/6 mice inhibitor negative or positive, respectively, 1 week after FVIII immunizations. ns, not significant.
- Figure S4. Anti-CD3 treatment in vivo increases the absolute number of regulatory CD4+CD25+ T cells (JPG, 32.3 KB)
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The spleens from hemophilia A Balb/c mice (n = 5/time point) were analyzed 1, 8 and 15 days after treatment with anti-CD3 (50 µg/d for 5 d) for expression of CD4+CD25+ cells (not gated on CD4+). Untreated and age-matched hemophilia A Balb/c mice (n = 6) were also studied to determine basal T cells levels. The transient nature of this response may be due to the very short half-life of anti-CD3 F(ab)′2 fragments (<1 day).22 Data are shown as the mean ± S.E.M.
- Figure S5. FVIII rechallenge does not break anti-CD3–meditated tolerance (JPG, 24.2 KB)
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Mice received the optimal dose of anti-CD3 (10ug/d for 5 d) or HBSS followed 3 days later by 4 weekly FVIII immunizations. Six weeks after the final FVIII immunization, plasma was sampled and directly afterward mice were rechallenged with 0.2 µg FVIII intravenously. Seventy-two hours after rechallenge plasma was sampled and inhibitor formation was evaluated by the Bethesda assay.
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