Neutralizing IL-6 Reduces Human Arterial Allograft Rejection by Allowing Emergence of CD161+ CD4+ Regulatory T Cells

Abstract

Peri-operative injuries to an allograft exacerbate graft rejection, which, in humans, is primarily mediated by effector memory T cells. IL-6 transcripts are elevated in human coronary artery segments rapidly increase post-transplantation into immunodeficient mouse hosts compared to pre-transplant specimens and fall dramatically by 30 days. Adoptive transfer of human PBMCs allogeneic to the artery two days post-operatively results in T cell infiltrates and intimal expansion four weeks later. Antibody neutralization of human IL-6 reduces the magnitude of intimal expansion and total T cell infiltration, but increases the relative expression of CD161 while decreasing other Th17 markers. Co-culture of class II MHC-expressing human endothelial cells (ECs) with allogeneic CD4(+) memory T cells results in T cell activation and EC secretion of IL-6. Neutralizing IL-6 in primary allogeneic T cell-EC co-cultures results in enhanced T cell proliferation of CD161(+) CD4(+) T cells, reduces total T cell proliferation upon restimulation in secondary cultures, an effect dependent on CD161(+) T cells, increases expression of FoxP3 in CD161(+) T cells, and generates T cells that suppress proliferation of freshly isolated T cells. These data suggest that IL-6 released from injured allograft vessels enhances allogeneic T cell infiltration and intimal expansion in a model of human allograft rejection by inhibiting an increase in CD161(+) regulatory T cells.

Rapamycin-treated human endothelial cells preferentially activate allogeneic regulatory T cells

“…IL-6 is known to inhibit the function of natural Tregs (71), as well as the induction of iTregs (52). Given that neutralizing IL-6 in such cocultures increases Treg formation (5) and that rapa-ECs poorly secrete IL-6, we were surprised to find that addition of recombinant IL-6 into cocultures did not alter the proportions of FoxP3 + cells. We suspect that the explanation resides in the observation that rapamycin treatment did not completely eliminate IL-6 production by the ECs in our culture system.…”

“…In addition to cytokines, formation of iTregs can also be driven by cell surface signals provided by APCs (35), such as PD-L1, which promotes iTreg differentiation even in the absence of TGF-β (36). Although these observations are based on studies of naive T cell commitment, human memory CD4 + T cells display plasticity and are capable of acquiring regulatory activity following antigenic restimulation (5,(37)(38)(39). The relationship between human ECs and Tregs is incompletely understood, and it is unknown whether human ECs can be induced to express molecules that favor activation or differentiation of tolerance-promoting Tregs instead of pathogenic inflammatory effectors T cells.…”

“…Based on our data with cultured ECs, we speculate that this induced expression of PD-1 ligands on graft ECs acts to reduce their alloimmunogenicity and may lead to preferential activation of Tregs in vivo. In addition, we have recently shown that serological neutralization of IL-6 diminishes intimal expansion in a similar model of artery allograft rejection (5). Given that rapamycin reduces IL-6 production by cultured ECs, it is possible that such a phenome-surface-stained cells were then permeabilized and restained using a FoxP3 Staining Kit (eBioscience), according to the manufacturer's instructions, with anti-human FoxP3-APC (clone PCH101; eBioscience).…”

“…APC function requires expression of MHC-peptide complexes (the antigen or signal 1), expression of antigen-independent costimulators (signal 2), and, often, production of activating cytokines (signal 3). Human vascular ECs in situ basally express MHC class I and class II molecules (2,3); costimulatory molecules such as LFA-3 (CD58), ICOS ligand (CD275), OX40 ligand (CD252), 41BB ligand (CD137L), CD40, and GITR ligand (4); and cytokines such as IL-1α, IL-6, IL-15, and IL-18 (5)(6)(7)(8) that can contribute to T cell activation and differentiation. This panoply of molecular signals enables human ECs to function as APCs and activate allogeneic memory, but not naive, CD4 + T cells to proliferate, secrete effector cytokines, and reject human allografts in immunodeficient mouse hosts (4,(9)(10)(11).…”

Inflammatory and Immune Responses in the Arterial Media

“…48 Administration of biological antagonists in this model does not differentiate between VSMC versus endothelial sources of cytokine (though VSMC effects may predominate because they constitute the most frequent cell type of the vessel wall), and T cells are minor producers of the cytokines tested. 32,49 Blocking IL-1 selectively inhibits IL-17 production by artery-infiltrating T cells, 50 neutralization of IL-6 leads to the emergence of a regulatory T cell subset, 51 whereas inhibition of TGF-β increases IFN-γ production by alloreactive T cells. 52 These data support the concept that early nonimmune injury of organ grafts, for example, by ischemia-reperfusion, can influence the later process of immune-mediated arteriosclerosis.…”