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Figure 3. Effect of tocilizumab administration on interleukin 6 and soluble interleukin 6 receptor levels based on conditioning regimen. (A). Concentration of IL-6 in the serum from patients that were treated with tocilizumab and received myeloablative or reduced intensity conditioning. (B). Concentration of soluble IL-6 receptor in the serum from patients that were treated with tocilizumab and received myeloablative or reduced intensity conditioning. ***P<0.001. IL: interleukin; sIL: soluble interleukin; RIC: reduced intensity conditioning.
were augmented in recipients of reduced intensity as opposed to MA regimens on day 14, but otherwise there were no differences at other time points (Online Supplementary Figure S2B). Of the other cytokines meas- ured in the blood, marginal increases were observed in IL- 2 at day seven and IL-10 at day 28 (Online Supplementary Figure S3). A direct comparison of cytokine levels between control and Toc-treated patients demonstrated a marked increase in IL-6 and sIL-6R levels in the latter group at all time points (Figure 4). There were also significant but very modest decreases in IL-2, IL-4 and IL-10 in these patients.
Immune reconstitution
Patients were tested by multi-parameter flow cytometry for reconstitution of lymphocytes and major lymphocyte subsets (CD3+, CD4+, and CD8+ T cells, B cells, and NK cells) at four intervals over the first year (Figure 5A,B). Patients recovered lymphocyte subsets to, or near, healthy control levels between six months and one year post- transplant, except for NK cells, which recovered early. The percentage of both regulatory T cells (Tregs) and T helper 17 (TH17) cells were within the expected range of healthy donors, and while Treg levels gradually decreased over the post-transplant period, the absolute number of TH17 cells remained stable (Online Supplementary Figure S4). The percentages and absolute number of B cells were particularly low at the one-month and three-month assessments (Online Supplementary Figure S5), and a subset analysis revealed other imbalances. Specifically, a func- tionally immature/transitional CD21− subset found to be
elevated in association with autoimmunity,24-26 infection,27 and a subset of patients with cGvHD27,28 was increased throughout the assessment period. However, this subset as well as most other abnormalities (including the percent- age of antigen inexperienced naïve B cells) recovered through the first year. There were no significant differ- ences seen when comparing patients experiencing aGvHD or cGvHD compared to those who did not experience GvHD for any assessment (data not shown).
Comparison to a matched control population
From the CIBMTR database, four controls were identi- fied for 30 patients, three controls for three patients, and two and one control for one patient each. The baseline characteristics for the patients in the phase 2 trial and the control cohort are detailed in Table 2. Median follow up was 15 months (range: 9-20 months) for patients receiving Toc/Tac/MTX and 13 months (range; 3-72 months) for the control cohort. The incidence of grades II-IV aGvHD at day 180 was significantly lower in the Toc/Tac/MTX cohort when compared to the Tac/MTX control popula- tion (17% versus 45% at day 180, HR=0.34 [0.17-0.69], P=0.003) (Figure 6A). Furthermore, corresponding proba- bilities of grade II-IV aGvHD-free survival were signifi- cantly higher in patients who received Toc/Tac/MTX than the matched cohort (69% versus 42% at day 180, HR=0.37, [0.21-0.67], P=0.001) (Figure 6B). There was no difference in the incidence of cGvHD between recipients in the Toc/Tac/MTX versus the Tac/MTX groups (38% ver- sus 45% at 12 months, HR=0.65, [0.37-1.13], P=0.13) (Figure 6C). There was also no difference in TRM,
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