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. 2011 Jan 1;17(1):77-88.
doi: 10.1158/1078-0432.CCR-10-1198. Epub 2010 Nov 3.

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

Isao Tawara  1 , Motoko Koyama, Chen Liu, Tomomi Toubai, Dafydd Thomas, Rebecca Evers, Peter Chockley, Evelyn Nieves, Yaping Sun, Kathleen P Lowler, Chelsea Malter, Norihiro Nishimoto, Geoffrey R Hill, Pavan Reddy
Affiliations

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

Isao Tawara et al. Clin Cancer Res. .

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 VSports手机版. 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 V体育安卓版. .

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 V体育ios版. 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 VSports最新版本. .

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"V体育官网" Figures

Figure 1
Figure 1. IL-6 elevation after BMT and involvement of donor IL-6 in GVHD
(A) B6 (H-2b) recipients were irradiated (10 Gy) on day −1 and transplanted with BALB/c(H-2d) donor 5×106 T cell-depleted bone marrow (TCDBM) and 2×106 whole T cells (top left). B6D2F1 (H-2b/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
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 (formula image, 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-2Kb-positive) CD4 T cells were shown.
Figure 3
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
Figure 4. Anti-IL-6R mAb treatment suppresses GVHD
(A) C3H.SW (H-2b) 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-2b) 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-2b/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-2b) 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
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-2Kb-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-2b) 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
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).
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