IL-6-mediated endothelial injury impairs antiviral humoral immunity after bone marrow transplantation

Abstract

Endothelial function and integrity are compromised after allogeneic bone marrow transplantation (BMT), but how this affects immune responses broadly remains unknown. Using a preclinical model of CMV reactivation after BMT, we found compromised antiviral humoral responses induced by IL-6 signaling. IL-6 signaling in T cells maintained Th1 cells, resulting in sustained IFN-γ secretion, which promoted endothelial cell (EC) injury, loss of the neonatal Fc receptor (FcRn) responsible for IgG recycling, and rapid IgG loss. T cell-specific deletion of IL-6R led to persistence of recipient-derived, CMV-specific IgG and inhibited CMV reactivation. Deletion of IFN-γ in donor T cells also eliminated EC injury and FcRn loss. In a phase III clinical trial, blockade of IL-6R with tocilizumab promoted CMV-specific IgG persistence and significantly attenuated early HCMV reactivation. In sum, IL-6 invoked IFN-γ-dependent EC injury and consequent IgG loss, leading to CMV reactivation. Hence, cytokine inhibition represents a logical strategy to prevent endothelial injury, thereby preserving humoral immunity after immunotherapy.

Keywords: Bone marrow transplantation; Cytokines; Immunoglobulins; Transplantation.

Figure 1. Inhibition of IL-6 signaling attenuates CMV reactivation in humans and mice.

(A) HCMV viremia within 100 days of BMT in a prospectively enrolled observational cohort (left) and a phase I/II clinical trial investigating the addition of tocilizumab to standard GVHD prophylaxis (right). Only samples with detectable and quantifiable HCMV DNA are plotted, representing 25 of 43 and 20 of 35 at-risk patients, respectively. Dashed lines indicate the institutional threshold for preemptive antiviral therapy at 600 copies/μL. At-risk patients were those who were HCMV-seropositive and/or received a graft from an HCMV-seropositive donor. (B) Proportion of at-risk patients with clinically significant HCMV viremia in the 2 cohorts (**P < 0.01, by Fisher’s exact test). (C) Experimental schema of the murine BMT and MCMV reactivation model (created with BioRender.com). Latently MCMV-infected (D, E, G, and H) or uninfected (F) B6D2F1 mice were transplanted with BM (5 × 10⁶) and T cells (2 × 10⁶) from B6.Cd4^Cre+ Il6r^fl/fl (Cre⁺) mice or littermate controls (Cre^–). TCD BM (5 × 10⁶) from Cre^– donors was used in the non-GVHD control groups (TCD). (D and E) Viral loads in target organs and plasma (viremia) at weeks 4–5 after BMT (spleen: n = 9–10 per group from 2 experiments; others: n = 14–15 per group from 3 experiments). (F) Plasma IL-6 levels at week 3 in MCMV-naive recipients (GVHD groups: n = 13–14 per group from 3 experiments; TCD group: n = 4 from 1 experiment) and (G) Latently MCMV-infected recipients (GVHD groups: n = 9–10 per group from 2 experiments; TCD group: n = 5 from 1 experiment). (H) Correlation between plasma IL-6 levels and MCMV viremia 5 weeks after BMT (n = 9–10 per group from 2 experiments). Dashed lines indicate the limit of detection. Data are presented as the median ± IQR and were analyzed with the Mann-Whitney U test (*P < 0.05 and **P < 0.01).

Figure 2. IL-6 signaling does not modify protective donor T cell or B cell responses to MCMV.

(A–C) Latently infected B6D2F1 mice were transplanted with BM (5 × 10⁶) and T cells (2 × 10⁶) from B6.Cd4^Cre+ Il6r^fl/fl (Cre⁺) mice or littermate controls (Cre^–) and analyzed 4–5 weeks after BMT. (A) Numbers of CD4⁺ and CD8⁺ T cells in spleens (n = 10–12 from 3 experiments). (B) m38 tetramer⁺ CD8⁺ T cells in spleens (n = 11–12 from 3 experiments) including representative flow cytometric plots. (C) CD19⁺ B cells in the blood, spleen or BM (n = 11–14 from 3 experiments; BM was from femur and tibia). (D–F) Latently infected B6D2F1 mice were transplanted with BM (5 × 10⁶) from C57BL/6J (WT) mice or with B6.μMt BM and T cells (2 × 10⁶) from B6.Cd4^Cre+ Il6r^fl/fl (Cre⁺) mice. TCD BM (5 × 10⁶) was administered to non-GVHD control groups (n = 10–11 per group from 2 experiments). (D) CD19⁺ B cells in the peripheral blood over time or in the spleen and BM (femur and tibia for BM) at week 6 after BMT. (E) MCMV viremia at weeks 5 and 6 after BMT. (F) Representative flow cytometric plots (CD90.2^–CD11b^–CD19^– gate) showing CD138⁺ plasma cells in the spleen and BM at week 6 without a discernible persistent recipient (H2D^d+) population in the BM+T settings. Number of CD19^–CD138⁺ plasma cells in the spleen and BM (femur and tibia) at week 6. Data are presented as the median ± IQR and were analyzed with the Mann-Whitney U test (*P < 0.05 and **P < 0.01).

Figure 3. IL-6 signaling promotes the loss of recipient IgG.

(A–D) Latently infected B6D2F1 recipient mice were transplanted with BM (5 × 10⁶) and T cells (2 × 10⁶) from uninfected B6.Cd4^Cre+ Il6r^fl/fl (Cre⁺) mice or littermate controls (Cre^–) and analyzed 4–5 weeks after BMT (n = 14 per group from 3 experiments). (A) MCMV-specific IgG titers. (B) Correlation between MCMV-specific IgG titers and viral load in plasma and liver. (C and D) Titers of MCMV-specific IgG1 and IgG3 (C) and IgG2 allotypes (D). Dashed lines indicate limit of detection. (E) Experimental schema for the quantification of IgG clearance by flow cytometry and (F) representative standard curve. (G) Noninfected B6D2F1 mice were transplanted with Cd4^Cre+ Il6r^fl/fl BM (5 × 10⁶) plus T cells (2 × 10⁶) (Cre⁺, n = 10, orange) or with Cd4^Cre– Il6r^fl/fl (Cre^–) BM plus T cells from littermate control mice, the latter with or without CSA to limit GVHD (Cre^–, n = 12, blue; or Cre^– plus CSA, n = 8, green). Mice transplanted with TCD Cd4^Cre– Il6r^fl/fl BM alone (TCD, n = 5, black) and nontransplanted mice (non-BMT, n = 6, red) were used as controls. The half-life (from days 7–21) of administered IgG2b was calculated for each individual mouse and compared between the indicated groups. Conc., concentration. Data were combined from 2 experiments. Data re presented as the median ± IQR and were analyzed with the Mann-Whitney U test (*P < 0.05 and **P < 0.01).

Figure 4. EC injury impairs FcRn recycling and promotes IgG loss.

(A and B) Noninfected B6D2F1 were transplanted with BM (5 × 10⁶) and T cells (2 × 10⁶) from Cd4^Cre– Il6r^fl/fl donors (n = 10 from 2 experiments). On day 14, liver cells were analyzed for (A) the expression of FcRn (i.e., representing IgG recycling) and (B) endocytosis (i.e., representing IgG uptake); representative histograms are shown. (C) Noninfected B6D2F1 mice were transplanted with BM (5 × 10⁶) with or without T cells (2 × 10⁶), and the half-life of infused IgG2b was compared between the indicated groups (n = 5, 5, 8, and 8). (D) Uninfected WT or Fcrn^–/– mice were transplanted with BM (10 × 10⁶) with or without T cells (5 × 10⁶) from BALB/c donors. The kinetics of administered IgG2b is shown (left), and the half-life was compared between the indicated groups (n = 4, 4, 8, and 7). Liver ECs were analyzed for the expression (MFI) of FcRn (right) on day 21. (E) MCMV latently infected Fcrn^–/– or WT B6 mice were transplanted with BM (10 × 10⁶) plus T cells (3 × 10⁶) from BALB/c donors, and MCMV viremia was quantified on day 19 (n = 13–14 per group from 2 experiments). (F) Latently MCMV-infected B6D2F1 mice were transplanted with BM (5 × 10⁶) plus T cells (2 × 10⁶) from B6 donors and treated with a FcRn inhibitor or a control antibody. MCMV viremia was quantified on day 21 (n = 9 per group from 2 experiments). (G–J) Noninfected B6D2F1 mice were transplanted with BM (5 × 10⁶) with or without T cells (2 × 10⁶) from Cd4^Cre Il6r^fl/fl donors, and livers were analyzed on day 14 (n = 8–12 from 2 experiments). (G) Representative flow cytometric plots showing frequencies and viability of CD31⁺ ECs. Max, maximum. (H) Viability and number of ECs per liver. (I) FcRn expression (MFI). (J) MFI of pHrodo dextran (left) with normalized expression relative to the non-BMT group (right). (K) Noninfected B6D2F1 mice were transplanted with BM (5 × 10⁶) with or without T cells (2 × 10⁶) from Cd4^Cre Il6r^fl/fl (Cre⁺) or littermate control (Cre^–) donors. GVHD target organs were taken for histological analysis on day 14. Shown are representative images of liver sections (left) and pathology scores in liver, skin, and ileum (right). Scale bar: 100 μm. Data are presented as the median ± IQR and were analyzed with the Mann-Whitney U test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 5. IL-6 maintains Th1 responses and IFN-γ secretion which mediates EC injury.

(A and B) Noninfected B6D2F1 mice were transplanted with BM (5 × 10⁶), with or without T cells (2 × 10⁶), from Cd4^Cre Il6r^fl/fl (Cre⁺) or littermate control (Cre^–) donors. (A) Expression of IFN-γ and TNF in splenic T cells on day 14 (n = 6, 8, 10, and 13 from 2 experiments), as shown by representative flow plots and MFI. (B) Plasma levels of IFN-γ and TNF on day 14 (n = 12, 9 per group from 2 experiments). (C and D) Noninfected B6D2F1 were transplanted with TCD BM (5 × 10⁶) from C57BL6J (WT) ± T cells (2 × 10⁶) from WT, Ifng^–/–, or Tnf^–/– donors (n = 6–12 from 2 experiments). On day 12, liver ECs were analyzed for (C) viability and numbers per liver and (D) expression of FcRn (MFI) relative to WT T group. (E) Noninfected B6D2F1 were transplanted with TCD BM (5 × 10⁶) from C57BL6J (WT) mice with or without T cells (2 × 10⁶) from WT or Ifng^–/– donors, together with mouse anti–human CD4 IgG2b. Plasma levels of infused Abs were determined on day 12 (n = 4, 4, 7, 7 per group); non-BMT controls were included for comparison. (F and G) Experiments were conducted as described in C and D and analyzed for the expression of (F) VCAM-1 and (G) MHC-II on liver ECs. Data are presented as the median ± IQR and were analyzed with the Mann-Whitney U test (**P < 0.01, ***P < 0.001, and ****P < 0.0001).

Figure 6. Endothelial injury during GVHD is characterized by an IFN-γ and JAK/STAT-dependent inflammatory signature.

Noninfected B6D2F1 recipient mice were transplanted with WT TCD BM (5 × 10⁶) with or without T cells (2 × 10⁶) from WT or Ifng^–/– donors. ECs from 3 mice per group were isolated from the liver on day 7 and processed for single-cell RNA-Seq. (A) Uniform manifold approximation and projection (UMAP) of ECs colored by groups. (B) Expression of genes across groups. (C) SCpubr pathway activity scores (z score scaled) across groups. (D) Violin plots of pathway activity scores (JAK/STAT, NF-κB, and TNF), with each group’s mean compared using a Wilcoxon test (*P < 0.001).

Figure 7. IL-6R inhibition with tocilizumab maintains CMV-specific humoral immunity and protection from early HCMV reactivation.

Participants of a phase III clinical trial who were enrolled at Royal Brisbane and Women’s Hospital and at risk for HCMV reactivation were included for analysis (n = 85). (A and B) Cumulative incidence of HCMV viremia in all at-risk patients (n = 85) and a subset of patients who received unrelated donor graft (n = 54). Broken lines represent the placebo arm, and solid lines represent the tocilizumab arm. (B) Cumulative incidence of HCMV viremia among patients who had grade 0–1 acute GVHD (n = 55) and a subset of patient who received an unrelated donor graft (n = 31). (C–E) Plasma HCMV-specific IgG levels were determined at day 30 after BMT in at-risk patients (n = 82; 1 patient was excluded because of HCMV viremia prior to day 0, and 2 patients did not have a plasma sample stored). (C) Correlation between day 30 HCMV IgG levels and donor (D) and recipient (R) serostatus. (D) Correlation between day 30 HCMV IgG levels and HCMV viremia among HCMV-seropositive recipients. Patients were categorized according to early viremia (within the first 5 weeks after BMT), late viremia (from weeks 5–14 after BMT), and no viremia (no detectable viremia within the first 14 weeks, after which HCMV monitoring was no longer routinely performed. (E) Correlation between day 30 HCMV IgG levels and the treatment arm among HCMV-seropositive recipients and the subset receiving an unrelated donor graft (right). (F) CD19⁺ B cell counts in the peripheral blood 30 and 60 days after BMT in HCMV-seropositive recipients. Dashed line indicates the lower limit of normal for B cell counts at 80 × 10⁶/mL. (G) Proportions of IgD⁺CD27^– naive B cells, IgD^–CD27⁺ mature B cells, and CD38^hi plasmablasts within the CD19⁺ B cell compartment in the peripheral blood at day 60. (H) Concentration of albumin in the plasma at day 14 in all the participants from the RBWH cohort. Data are presented as the median ± IQR and were analyzed with the Mann-Whitney U test (*P < 0.05, **P < 0.01, and ****P < 0.0001).