Josée P. A. Wagenaar

ANERGIC T CELLS ACTIVELY SUPPRESS T CELL RESPONSES VIA THE ANTIGEN-PRESENTING CELL

We here show that anergic T cells are active mediators of T cell suppression. In co‐culture experiments, we found that anergic T cells, derived from established rat T cell clones and rendered anergic via T cell presentation of the specific antigen (Ag), were active inhibitors of T cell responses. Anergic T cells inhibited not only the responses of T cells with the same Ag specificity as the anergic T cells, but were also capable of efficiently inhibiting polyclonal T cell responses directed to other epitopes. This suppression required close cell‐cell contact between antigen‐presenting cells (APC), anergic T cells and responder T cells, and only occurred when the epitope recognized by the anergic T cell was present. The suppression was not caused by passive competition for ligands on the APC surface, IL‐2 consumption, or cytolysis, and was not mediated by soluble factors derived from anergic T cells that were stimulated with their specific Ag. When responder T cells were added 24 h after co‐culturing anergic cells in the presence of Ag and APC, T cell responses were still suppressed, indicating that the suppressive effect was persistently present. However, anergic T cells were not able to suppress responder T cells that had already received a full activation signal. We propose that suppression by anergic T cells is mediated via the APC, either through modulation of the T cell‐activating capacity of the APC (APC/T cell interaction), or by inhibition of T cells recognizing their ligand in close proximity on the same APC (T/T cell interaction).

Cell stress induced HSP are targets of regulatory T cells: a role for HSP inducing compounds as anti-inflammatory immuno-modulators?

T cell responses to heat shock proteins (HSP) have disease suppressive activities through production of anti‐inflammatory cytokines in patients and in models of inflammatory diseases. There is evidence that the anti‐inflammatory activity of HSP‐specific T cells depends on their recognition of endogenous HSP epitopes as expressed by stressed cells at sites of inflammation. Previously, we have demonstrated that such T cells can be induced by conserved sequences of microbial HSP. Now we propose that drug induced up‐regulation of endogenous HSP can contribute to anti‐inflammatory T cell regulation.

T cell-stimulatory Fragments of Foot-and-mouth Disease Virus Released by Mild Treatment With Cathepsin D

Cathepsin D and cathepsin B are endosomal/lysosomal proteases that are thought to play a role during in vivo antigen processing, releasing fragments for binding to major histocompatibility complex class II products and subsequent presentation to T cells. Here we treated purified foot-and-mouth disease virus (FMDV) strain A10Holland with both enzymes. Cathepsin D, but not cathepsin B, was shown to release fragments from reduced or non-reduced FMDV under mild conditions in vitro. Twenty-eight predominant cathepsin D-released fragments were purified by HPLC and identified by amino acid composition analysis and sequencing. The unseparated set of fragments produced (the digest) was able to stimulate T cells from eight vaccinated cattle. With respect to the response to intact virus the extent of the response to the digest differed between animals: four animals could be classified as good responders, three as intermediate responders and one as a low responder. Subsequently, we investigated the proliferative T cell response to a large set of synthetic peptides in detail for two animals, one belonging to the group of good responders, the other being the low responder. The peptides covered all 28 cathepsin D-released fragments analysed and also several sequences not recovered from the digest. In this way seven T cell sites could be identified, five of which coincided with cathepsin D-released fragments. The other two T cell sites were VP2[54-72], being a homologue of a T cell site identified for FMDV strain O1K and the N terminus of VP4. Whether the most dominantly recognized T cell site was recovered from the digest or not was shown to be related to the good or low response to the digest. These findings suggest a role for cathepsin D in the release of some but not all T cell-stimulatory fragments from FMDV.

Stress proteins are used by the immune system for cognate interactions with anti‐inflammatory regulatory T cells

Since the initial discovery of the protective role of heat shock protein (HSP) 60 in arthritis, T cell recognition of endogenous HSP was found to be one of the possible underlying mechanisms. Recently we have uncovered potent disease‐suppressive Tregs (anti‐inflammatory immunosuppressive T cells) recognizing HSP70 self‐antigens, and enabling selective targeting of such Tregs to inflamed tissues. HSP70 is a major contributor to the major histocompatibility complex (MHC) Class II ligandome and we have shown that a conserved HSP70‐epitope (B29) is abundantly present in murine MHC Class II. Upon transfer, B29‐induced CD4+CD25+Foxp3+T cells suppressed established proteoglycan‐induced arthritis (PGIA) in mice. These self‐antigen specific Tregs were activated in vivo and as little as 4.000 cells sufficed to fully inhibit arthritis. Furthermore, in vivo depletion of transferred Tregs abrogated disease suppression. Given that B29 can be presented by most human MHC class II molecules and that B29 inhibited arthritis in HLA‐DQ8 (human MHC) transgenic mice, we feel that therapeutic vaccination with selected HSP peptides can be an effective route for induction of anti‐inflammatory Tregs as a novel intervention in chronic inflammatory diseases.

Tolerogenic Dendritic Cells That Inhibit Autoimmune Arthritis Can Be Induced by a Combination of Carvacrol and Thermal Stress

Tolerogenic dendritic cells (DCs) can induce regulatory T cells and dampen pathogenic T cell responses. Therefore, they are possible therapeutic targets in autoimmune diseases. In this study we investigated whether mouse tolerogenic DCs are induced by the phytonutrient carvacrol, a molecule with known anti-inflammatory properties, in combination with a physiological stress. We show that treatment of DCs with carvacrol and thermal stress led to the mRNA expression of both pro- and anti-inflammatory mediators. Interestingly, treated DCs with this mixed gene expression profile had a reduced ability to activate pro-inflammatory T cells. Furthermore, these DCs increased the proportion of FoxP3+ regulatory T cells. In vivo, prophylactic injection of carvacrol-thermal stress treated DCs pulsed with the disease inducing antigen was able to suppress disease in a mouse model of arthritis. These findings suggest that treatment of mouse bone marrow derived DCs with carvacrol and thermal stress induce a functionally tolerogenic DC that can suppress autoimmune arthritis. Herewith carvacrol seems to offer novel opportunities for the development of a dietary based intervention in chronic inflammatory diseases.