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In vivo IL-12/IL-23p40 neutralization
inumab above 1 μg/mL were observed throughout the treatment period, and were associated with neutralization of circulating IL-12/IL-23p40 (Figure 1B). From day 0 to peak level post HCT, placebo-treated patients had a 14.1- fold increase in IL-12/IL-23p40, while ustekinumab-treat- ed patients had only a 2.7-fold increase.
Donor alloreactive T-cell polarization
When stimulated with third-party alloantigen, donor T cells collected on day 30 from blood of ustekinumab-treat- ed patients produced less IFN-γ (P=0.001) and IL-17 (P=0.03), and similar amounts of IL-4 (P=0.38) compared to donor T cells from blood of placebo-treated patients (Figure 2). Other Th2 cytokines (IL-5, IL-13) were not test- ed. The sensitivity to detect host-specific alloresponses was low because the transplants were from HLA compat- ible donors. However, lower production of IL-17 by host- specific donor T cells was also observed in ustekinumab- treated patients compared to placebo at day 30 (P=0.02). Findings on day 30 and day 90 samples were similar (Figure 2). IFN-γ production in response to PMA/iono- mycin was higher among the ustekinumab group at day 30, yet similar at day 90. Among other tested supernatant cytokines, there were no significant differences in TNF-α, TGF-β, IL-10, IL-2, IL-6, IL-12p40, or IL-23. We did not observe significant differences in Th1/Th2/Th17 pheno- type by flow cytometry at days 30 and 90 post HCT, and did not detect significant differences in serum cytokines at days 7 and 28 post HCT, except a significant reduction in IL-12/IL-23p40 in the ustekinumab-treated group at day 28 (Online Supplementary Figures S2-S4). No significant dif- ferences in gene expression were observed among periph- eral blood mononuclear cells assayed with the NanoString nCounter GX Human Immunology V2 Kit (data not shown).
Regulatory T-cell frequency and suppressive function
There was no significant difference in Treg/total CD4 at day 30 (ustekinumab: median 13, range 2-22; placebo:
median 11, range 3-20), both without (P=0.4) and with (P=0.4) adjustment for donor type (Figure 3). There was also no significant difference in absolute number of Treg or Treg suppressive function between groups (Figure 3).
Engraftment and early toxicity
There were no significant differences in neutrophil (P=0.2) or platelet (P=0.5) engraftment between the ustek- inumab- and placebo-treated patients, and all cases achieved these milestones promptly. No differences in thrombotic microangiopathy (TMA) (n=1 for each arm), TMA grade, or median time to TMA onset (48 vs. 62 days) were observed. No difference was observed in hepatic VOD (n=3 vs. n=2), VOD severity (moderate in all cases), or median time to VOD onset (28 vs. 31 days). In all cases, the syndrome resolved without intervention beyond diuresis, and no deaths occurred from VOD.
Acute and chronic GvHD
Acute GvHD organ staging and overall grade are pre- sented in Table 2. The day 100 cumulative incidence of grade II-IV acute GvHD was 33% for the ustekinumab arm versus 40% for the placebo arm (Figure 4A). Median time to acute GvHD onset was significantly longer among ustekinumab-treated patients compared to placebo (56 days vs. 28 days; P=0.02). Examining validated serum bio- markers (REG3α, ST2) of lethal GvHD and non-relapse mortality (NRM) at day 7 post HCT (analyzed by ELISA using methods previously described),21,22 we found ustek- inumab-treated subjects had significantly lower REG3α compared to the placebo group [median: 55 (range 8-234) vs. 135 (21-387) ng/mL; P=0.014]; ST2 was not significant- ly different [median: 11,817 (range 3493-40,898) vs. 19,923 (range 3791-97,662) pg/mL; P=0.68] (Figure 5). There was no significant difference in cumulative incidence of any grade chronic GvHD or NIH moderate/severe chronic GvHD (Figure 4B) between groups. In addition, no signif- icant differences were observed between groups for indi- vidual organ involvement and severity or overall NIH 0-3
Table 2. Acute graft-versus-host disease (GvHD) organ staging and overall grade. Study arm
Overall acute GvHD grade Ustekinumab Placebo
096 I13 II 5 5 III 0 1
Skin stage
0 12 8 114 201 322
GI stage
0 12 10 134 201
Liver stage
0 15 15
P=0.51
P=0.35
P=0.57
GI: gastrointestinal tract.
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