Interleukin-6 modulates graft-versus-host responses after experimental allogeneic bone marrow transplantation

Abstract

Purpose: The graft-versus-tumor (GVT) effect is a potent form of immunotherapy against many hematologic malignancies and some solid tumors. The beneficial GVT effect after allogeneic bone marrow transplantation (BMT) is tightly linked to its most significant complication, graft-versus-host disease (GVHD). The role of interleukin-6 (IL-6) after allogeneic BMT is not well understood. This study used a series of complementary knockout and antibody blockade strategies to analyze the impact of IL-6 in multiple clinically relevant murine models of GVHD and GVT.

Experimental design: We examined the effect of the source of IL-6 by analyzing the role IL-6 deficiency in donor T cells, donor bone marrow or in host tissues. We confirmed and extended the relevance of IL-6 deficiency on GVHD and GVT by treating BMT recipients with anti-mouse IL-6 receptor (IL-6R), MR16-1.

Results: Deficiency of IL-6 in donor T cells led to prolongation of survival. Total inhibition of IL-6 with MR16-1 caused an even greater reduction in GVHD-induced mortality. The reduction in GVHD was independent of the direct effects on T effector cell expansion or donor regulatory T cells. GVT responses were preserved after treatment with MR16-1.

Conclusion: MR16-1 treatment reduced GVHD and preserved sufficient GVT. Tocilizumab, a humanized anti-IL-6R monoclonal antibody (mAb), is approved in several countries including the United States and European Union for the treatment of rheumatoid arthritis and other inflammatory diseases. Blockade of IL-6 with anti-IL-6R mAb therapy may be testable in clinical trials as an adjunct to prevent GVHD in BMT patients without a significant loss of GVT.

Figure 1. IL-6 elevation after BMT and involvement of donor IL-6 in GVHD

(A) B6 (H-2^b) recipients were irradiated (10 Gy) on day −1 and transplanted with BALB/c(H-2^d) donor 5×10⁶ T cell-depleted bone marrow (TCDBM) and 2×10⁶ whole T cells (top left). B6D2F1 (H-2^b/d) recipients were irradiated (10 Gy) on day −1 and transplanted with B6 5×10⁶ TCDBM and 2×10⁶ whole T cells (top, middle). C3H.SW recipients were irradiated (10 Gy) on day −1 and infused with B6 5×10⁶ TCDBM and 1×10⁶ whole T cells (top, right). Sera were collected from recipients (4–5 recipients/group) on day 4, 7 and 16. IL-6 levels of each sample were measured by ELISA. (B) BALB/c recipients were irradiated (9 Gy) on day 0 and received 10×10⁶ B6 TCDBM (◆, n=8), 10×10⁶ wild type (WT) B6 TCDBM and 5×10⁵ WT B6 whole T cells (■, n=17) or 10×10⁶ IL-6−/− B6 TCDBM and 5×10⁵ IL-6−/− whole T cells (○, n=17). Survival of the recipients was monitored daily. ■ vs. ○ P<0.05 (C) BALB/c recipients were irradiated (8 Gy) on day −1 and received 5×10⁶ synegeneic TCDBM (●, n=15), 5×10⁶ wild type (WT) B6 TCDBM and 1×10⁶ WT B6 whole T cells (■, n=23) or 5×10⁶ WT B6 TCDBM and 1×10⁶ IL-6−/− whole T cells (○, n=23). Survival of the recipients was monitored daily. ■ vs. ○ P=0.0013.

Figure 2. Serum cytokine levels in BMT recipients

(A). BALB/c recipients were irradiated (9 Gy) on day 0 and received 10×10⁶ B6 TCDBM (, n=6), 10×10⁶ wild type (WT) B6 TCDBM and 5×10⁵ WT B6 whole T cells (■, n=12–20) or 10×10⁶ IL-6−/− B6 TCDBM and 5×10⁵ IL-6−/− whole T cells (□, n=12–20). Sera were collected from recipients on day 7. Cytokines were determined by cytokine bead array. (B). Splenocytes from day 7 recipient (□, WT B6 donor, ■ IL-6−/− B6 donor, n=20 respectively) were stimulated with soluble anti-CD3 mAb (2 μg/ml) in the presence of brefeldin A (1/1000 dilution; BioLegend) for 4 hours. Cells were processed for intracellular cytokine staining (ICC), per the manufacturer’s protocol (BD Cytofix/Cytoperm Kit; BD Pharmingen). Representative plots of cytokine expression gated on donor (H-2K^b-positive) CD4 T cells were shown.

Figure 3. Donor bone marrow or host-derived IL-6 is not involved in GVHD

(A) BALB/c recipients were irradiated (9 Gy) on day 0 and received 10×10⁶ B6 TCDBM (◆, n=6), 10×10⁶ wild type (WT) B6 TCDBM and 5×10⁵ WT B6 whole T cells (■, n=12) or 10×10⁶ IL-6−/− B6 TCDBM and 5×10⁵ WT B6 whole T cells (◆, n=12). Survival of the recipients was monitored daily. (B) B6 recipients were irradiated (10 Gy) on day −1 and received syngeneic donor B6 5×10⁶ TCDBM (●, n=12), or allogeneic donor C3H.SW 5×10⁶ TCDBM and 2×10⁵ CD8 T cells (■, n=12). IL-6−/− recipients were irradiated (10 Gy) on day −1 and received donor C3H.SW 5×10⁶ TCDBM and 2×10⁵ CD8 T cells (○, n=14). Survival of the recipients was monitored daily.

Figure 4. Anti-IL-6R mAb treatment suppresses GVHD

(A) C3H.SW (H-2^b) recipients were irradiated (10 Gy) on day −1 and injected with either syngeneic C3H.SW 5×10⁶ TCDBM and 1 ×10⁶ whole T cells were infused (●) or allogeneic B6 (H-2^b) 5×10⁶ T TCDBM and 1×10⁶ whole T cells were infused. Allogeneic BMT recipients were treated with control rat IgG on day −1 and 3 at 0.5 mg/dose (■, n=12) or with MR16-1 on day −1 and 3 at 0.5 mg/dose (○, n=9). GVHD clinical score was monitored weekly and survival of the recipients was monitored daily. ■ vs. ○ (survival P=0.0235 and clinical score P<0.05) (B) B6D2F1 (H-2^b/d) recipients were irradiated (10 Gy) on day −1 and injected with either syngeneic B6D2F1 5×10⁶ TCDBM and 2 ×10⁶ whole T cells were infused (●, n=6). or allogeneic B6-CD45.1 (H-2^b) 5×10⁶ T TCDBM and 2×10⁶ whole T cells were infused. Allogeneic BMT recipients were treated with control rat IgG on day −1 and 3 at 0.5 mg/dose (■, n=12) or with MR16-1 on day −1 and 3 at 0.5 mg/dose (○, n=12). GVHD clinical scores were monitored weekly and survival was monitored daily. ■ vs. ○ P=0.0139 (survival) and <0.05 (clinical score) (C) GVHD specific pathology on day +7: Histopathological score of GVHD severity was analyzed in from the recipients of in B6→B6D2F1 model 7 days post-BMT.

Figure 5. Effect of Anti-IL-6R mAb treatment on regulatory T cells and GVHD

(A) BALB/c mice were irradiated (8 Gy) on day −1 and received 5×10⁶ B6 TCDBM 5 ×10⁵ CD90 T cells. Recipients were treated with anti-IL-6R (MR16-1) or control rat IgG on day −1 and 3 (0.5 mg/dose) intravenously. On day 6 and 10 post-BMT, spleen cells were harvested, stained with anti-H-2Kb, CD4, CD8a and FoxP3 mAbs and analyzed by flow cytometry. H-2K^b-positive cells were defined as donor derived cells. (left). B6-CD45.1 mice were irradiated (10 Gy) on day −1 and received C3H.SW 5×10⁶ TCDBM 1 ×10⁶ whole T cells. Recipients were treated with anti-IL-6R (MR16-1) or control rat IgG on day −1 and 3 (0.5 mg/dose) intravenously. On day 5 and 29 post-BMT, spleen cells were harvested, stained with anti-CD229.1, CD4, CD8a and FoxP3 mAbs and analyzed by flow cytometry. CD229.1-positive cells were defined as donor derived cells. (right). (B) WT B6 recipients were irradiated (10 Gy) on day −1 and received synegeneicB6 5×10⁶ TCDBM (●, n=15). Allogeneic C3H.SW (H-2^b) 5×10⁶ TCDBM and 2×10⁵ CD8 T cells were infused. Allogeneic BMT recipients were treated with control rat IgG on day −1 and 3 at 0.5 mg/dose (■, n=18) or with MR16-1 on day −1 and 3 at 0.5 mg/dose (○, n=18). Survival of the recipients was monitored daily. ■ vs. ○ P=0.0075. (C):B6-CD45.1 mice were irradiated (10 Gy) and large intestines were harvested. Tissue was cut and incubated in the K-SFM media with or without of recombinant murine IL-6 (20 ng/ml) for 4h. Tissue was fixed with 10% formalin, embedded in paraffin. Paraffin-sections were stained using ApopTag Apoptosis Detection Kit. Apoptosis of epithelial cells were scored as 0~3+. Typical images of apoptosis score 1+, 3+ are shown. Bar graph shows mean ± SEM (n=4)

Figure 6. Anti-IL-6R treatment preserves GVT effect

(A) B6D2F1 recipients were irradiated (10 Gy) on day −1 and transplanted with syngeneic B6D2F1 5×10⁶ TCDBM and 2 ×10⁶ whole T cells (●, n=6) along with or without2×10³ P815 cells. Syngeneic recipients were treated with control rat IgG on day −1 and 3 at 0.5 mg/dose (▲, n=7) or with MR16-1 on day −1 and 3 at 0.5 mg/dose (▼, n=8). Survival of the recipients was monitored daily (top left). B6D2F1 recipients were irradiated and treated with either control IgG-(◆ and ■, n=7) or MR16-1 (◇ and ○, n=8) and injected with or without P815 tumor cells as above. All of the animals received allogeneic B6-CD45.1 5×10⁶ TCDBM and 2 ×10⁶ whole T cells (top right). Survival of the recipients was monitored daily. ■ vs. ○ P=0.0319, ◆ vs. ◇ P=0.0287. (B) BALB/c animals were irradiated (8 Gy, day −1) and received syngeneic BALB/c 5×10⁶ TCDBM and 5×10⁵ whole T cells along with or without J558 tumor cells (●, n=13). The animals were treated with either control rat IgG on day −1 and 3 at 0.5 mg/dose (▲, n =7) or with MR16-1 on day −1 and 3 at 0.5 mg/dose (▼, n=8). Survival was monitored daily (bottom, left). ■ vs ○ P=0.9816. BALB/c animals were irradiated and treated with either rat IgG-treated (◆, day−1 and 3 at 0.5 mg/dose, n=14) or MR16-1-treated (◇, day−1 and 3 at 0.5 mg/dose, n=14) as above and were infused with allogeneic B6 5×10⁶ TCDBM and 5×10⁵ whole T cells with or without 1 ×10⁵ J558 cells. Survival was monitored daily (bottom). ◆ vs ◇ P=0.0285 (bottom right).