Blockade of Interleukin 6 Signaling Attenuates Graft Versus Host Disease by Augmenting Regulatory T Cell Reconstitution

Graft-versus-host disease (GVHD) is characterized by a proinflammatory milieu that is attributable to conditioning regimen-induced host tissue damage as well as secretion of inflammatory cytokines by alloactivated donor T cells and other effector cell populations. Interleukin 6 (IL-6) is one of the cytokines that is a component of this proinflammatory environment and is of particular interest with respect to GVHD biology since presence or absence of IL-6 plays a pivotal role in determining the fate of naive T cells as they differentiate to become either proinflammatory or regulatory T cells, respectively. How IL-6 contributes to the pathophysiology of GVHD, however, is not well understood. To address this question, we employed a monoclonal antibody (Ab), MR16-1, that binds to both the soluble and membrane components of the IL-6 receptor to determine whether inhibition of IL-6 signaling affected GVHD severity and what impact antibody blockade had on the reconstitution and expansion of regulatory and effector T cells post transplantation. To identify regulatory T cells (Tregs) in vivo, we used transgenic donor mice in which the foxp3 gene is coupled to the enhanced green fluorescent protein (eGFP) so that eGFP would serve as a surrogate marker for Tregs. In initial studies, lethally irradiated Balb/c (H-2^d) mice were transplanted with T cell depleted (TCD) B6 eGFP-foxp3 (H-2^b) bone marrow plus splenocytes to induce GVHD. Cohorts of animals were then administered anti-IL-6R or isotype control Ab (0.5 mg/dose) once weekly. Mice treated with anti-IL-6R Ab had significantly less weight loss and reduced pathological damage in the liver, lung and colon when assessed 3–4 weeks post-BMT, demonstrating that protection was generalized and not restricted to a single target organ. Furthermore, antibody-treated animals also had a 12-fold increase in the number of donor-derived Tregs in the spleen compared to control mice (mean 9.5 × 10⁴ versus 0.8 × 10⁴, p=0.0001), indicating that blockade of IL-6 signaling was associated with increased Treg reconstitution. To determine if the increase in Tregs was operative under conditions where thymic function was absent, similar experiments were performed in thymectomized animals. These studies revealed that administration of anti-IL-6R Ab significantly reduced pathological damage in GVHD target organs and increased the absolute number of splenic Tregs. Since one of the mechanisms by which Tregs can be generated in the periphery in the absence of a thymus is through the peripheral conversion of CD4⁺ foxp3⁻ to CD4⁺ foxp3⁺ Tregs, we examined whether blockade of IL-6 enhanced Treg conversion during GVHD. Lethally irradiated Balb/c mice were transplanted with B6 Rag-1 BM cells and purified CD4⁺ eGFP⁻ T cells so that peripheral conversion could be determined by expression of eGFP. Anti-IL-6R antibody-treated mice had significantly reduced GVHD pathology that was associated with increased absolute numbers of converted Tregs in the spleen. Notably, we also observed a marked reduction in both T_H1 and T_H17 cells in these same animals, suggesting that IL-6 blockade might also affect expansion of alloreactive donor T cells. To address this question, we employed a murine model (B6® Balb.B) in which donor and recipient animals differ at multiple defined minor histocompatibility antigens. One of these minor antigens, termed H60, is preferentially recognized by donor CD8⁺ T cells during a GVH reaction. Mice administered anti-IL-6R antibody had a significant reduction in GVHD as assessed by weight loss and overall pathological score when compared to isotype-treated controls. Coincident with the reduction in pathology, there was also a significant decrease in the absolute number of CD8⁺ H60⁺ T cells, demonstrating that blockade of IL-6 signaling decreased the expansion of GVH-reactive donor T cells. In summary, we conclude that IL-6 plays a pivotal role in the pathophysiology of GVHD by driving the immune response in a proinflammatory direction. Conversely, blockade of IL-6/IL-6R interactions significantly attenuates GVHD by augmenting Treg reconstitution and decreasing expansion of alloreactive donor T cells which serves to re-balance the immune response. Given the challenges associated with the ex vivo expansion of Tregs for therapy in humans, these results also suggest that anti-IL-6R antibody treatment might represent a novel approach for the expansion of these cells in vivo.