Nonapoptotic and Extracellular Activity of Granzyme B Mediates Resistance to Regulatory T Cell (Treg) Suppression by HLA-DR−CD25hiCD127lo Tregs in Multiple Sclerosis and in Response to IL-6

Abstract

In autoimmune patients, regulatory T cells are increasingly found to be unable to suppress patient-derived T cells, an outcome referred to as Treg resistance. Here we show that CD4 T cells from patients with MS resist suppression by patient derived- or healthy donor derived- ex vivo Tregs. Importantly, we report that Granzyme B (GzmB) contributes to this Treg resistance via a novel, apoptosis-independent mechanism. We show that memory CD4+CD127loFoxP3+ Treg subsets do not express GzmB, while activated, non-regulatory CD4 T cells isolated from patients with MS express higher levels of GzmB than cells from healthy donors. In contrast to the intracellular GzmB that mediates apoptosis, GzmB can be found in extracellular fluids where it is hypothesized to regulate other cellular processes. Here we show that providing extracellular GzmB strongly inhibits Treg suppression, without altering Treg viability. However, if GzmB and GzmB-specific inhibitor are both provided to the co-cultures, Treg suppression occurs. Thus, these data suggest that a novel activity of extracellular GzmB is to regulate Treg suppression. In addition, we find that the suppression-abrogating cytokine, IL-6, augments GzmB expression by human CD4 T cells, and inhibits Treg suppression via this non-apoptotic GzmB-mediated mechanism. Lastly, in examining the mechanism whereby GzmB inhibits Treg function, we show that extracellular GzmB reduces Treg expression of CD39 and PD-L1. Altogether, these data indicate that extracellular GzmB plays an unexpected, non-apoptotic role in regulating Treg suppression and suggest that inactivation of specifically the extracellular activity of GzmB may be an efficacious therapeutic in autoimmunity.

Expression of Treg/Th17 cells as well as related cytokines in patients with inflammatory bowel disease

“…The initial CD4 + cells could differentiate into Treg under the simple stimulation of TGF-β. This kind of cells mainly inhibited inflammatory reaction 17,19. Th17 and Treg could promote or inhibit the mutual effect of inflammation.…”

Enhanced T cell responses to IL-6 in type 1 diabetes are associated with early clinical disease and increased IL-6 receptor expression

“…It is likely that enhanced IL-6 responses promote pathogenic T cell function locally in the inflamed islet or pancreatic lymph node in concert with other proinflammatory cytokines such as TNF, IL-1β, IL-21 and IL-23. In this setting, enhanced IL-6 responses may alter the fate and function of islet specific T cells, resulting in increased pathogenicity through impaired T reg function, the resistance of T eff to suppression by T reg (15, 16), and enhanced cytotoxicity via the induction of granzyme B linked to IL-6 trans-signaling (41, 42). In addition, IL-6 has been linked to reduced apoptosis of antigen-specific CD4 T cells in mice (43); this may be a mechanism by which autoantigen-specific T cells from individuals with diabetes prolong their survival.…”

Immunopathology of multiple sclerosis

“…Another possibility is the skewing of T Reg cells towards an IFNγ-secreting T H 1 cell-like phenotype in patients, which is reversible upon IFNβ therapy 91 . An alternative but non-mutually exclusive explanation for the action of autoreactive effector T cells in multiple sclerosis is that, rather than being Neuroaxonal damage promotes a deficit in the highly energy-demanding axonal transport processes, and this deficit in turns contributes to further energy deficiency and metabolic stress as mitochondria and other motor protein cargo are not transported to distal parts of the axon Presynaptic energy failure and lipid peroxidation driven by ROS and RNS can lead to postsynaptic neuronal apoptosis by promoting excessive postsynaptic stimulation by neurotransmitters Injured axon the outcome of inadequate peripheral suppression, the effector T cells themselves are actively resistant to suppressive mechanisms, with the suggestion that IL-6-induced signal transducer and activator of transcription 3 (STAT3)-mediated signalling contributes to this resistance 92,93 . Such resistance mechanisms emphasize the putative caveat of studies that document patient T Reg cell dysfunction using autologous effector T cells, as these reports may in fact reflect increased effector T cell resistance.…”