In primed allo-tolerance, TIM-3-Ig rapidly suppresses TGFbeta, but has no immediate effect on Foxp3.

T-cell immunoglobulin mucin-3 (TIM-3) is only expressed by differentiated TH1 cells following their proliferative response to antigen, functioning to terminate TH1-mediated immunity upon binding to the TIM-3 ligand, galectin-9. This critical regulatory process involves Treg cells via their stable expression of galectin-9. Soluble TIM-3-Ig blocks galectin-9 and prevents induction of peripheral tolerance. Here we have looked for evidence that TIM-3-Ig might also break established regulatory tolerance. Using allo-primed spleen cells cultured ex vivo and challenged with irradiated donor-type stimulator cells either alone or together with 20 microg/ml TIM-3-Ig, we measured daily cytokine release [IL2, inferon gamma (INFgamma), transforming growth factor beta (TGFbeta), IL6, IL10] and cellular Foxp3 protein. In allo-tolerance, a specific effect of TIM-3-Ig was some fourfold reduction in TGFbeta. Foxp3 was induced in the allo-tolerant response to donor and this was not altered by TIM-3-Ig over the 5-day culture period. No Foxp3 was detected in either rejected or donor stimulator cells at any time. Thus, in an ex vivo model of in vivo tolerance to heart allografts, TIM-3-Ig therapy appears to reduce the stable tolerogenic environment by a rapid and specific repression of TGFbeta release.

Regulatory transplantation tolerance and "stemness": evidence that Foxp3 may play a regulatory role in SOCS-3 gene transcription.

Immune self-tolerance is controlled by a subset of T lymphocytes that are regulatory (Treg) and epigenetically programmed to suppress autoreactive immune effector cells in vivo. Treg require expression of Foxp3, a transcription factor that not only represses the interleukin-2 gene promoter, but also sequesters key mediators of T-cell signal transduction by complexing with cytoplasmic NFAT and NFkappaB. We have discovered that expression of Foxp3 is linked to two stem cell-related factors, namely leukemia inhibitory factor (LIF) and axotrophin. Because both LIF and axotrophin each influence Foxp3, we now ask if reciprocal cross-talk occurs; for example, does Foxp3 in turn influence LIF and/or axotrophin? We compared the effect of wt-Foxp3 versus mutant DeltaE251-Foxp3, which lacks transcriptional activity, on transcript levels of axotrophin, LIF, and suppressor of cytokine signaling-3 (SOCS-3; a feedback inhibitor of LIF) in the Jurkat human T-cell line. Unexpectedly, a 50-fold increase in SOCS-3 transcripts occurred in the DeltaE251-Foxp3 cells, coincident with a dramatic decrease in LIF transcription. This implies that, either directly or indirectly, transcription of SOCS-3 is negatively regulated by wt-Foxp3. Suppression of SOCS-3 by Foxp3 would support a model wherein Foxp3 promotes LIF signaling in Treg and is further evidence of reciprocity between Foxp3, LIF, and axotrophin.

Evidence for functional inter-relationships between FOXP3, leukaemia inhibitory factor, and axotrophin/MARCH-7 in transplantation tolerance.

In an ex vivo mouse model, regulatory transplantation tolerance is not only linked to Foxp3, but also to release of leukaemia inhibitory factor (LIF) and to expression of axotrophin (also known as MARCH-7), a putative ubiquitin E3 ligase associated with feedback control of T cell activation and of T cell-derived LIF. Given this coordinate correlation with tolerance, we now ask if Foxp3 expression is influenced by LIF or by axotrophin. In spleen cells from allo-rejected mice we found that exogenous LIF reduced interferon gamma release in response to donor antigen by 50%, but LIF had no direct effect on levels of Foxp3 protein in allo-primed cells that were either tolerant, or aggressive, for donor antigen. However, we did find an effect of axotrophin on Foxp3: in the axotrophin null mouse, thymic Foxp3 transcripts were reduced compared to axotrophin wildtype littermates. To test whether these findings in the mouse were of potential significance in man we measured transcript levels of axotrophin and LIF in peripheral blood cell samples collected for a recently published clinical study concerning haematopoietic stem cell recipients. In controls, human peripheral blood CD4+CD25+cells contained significantly more FOXP3 and axotrophin than CD4+CD25-cells. In bone marrow autograft recipients, where peripheral blood cell samples directly represent both the grafted tissue and the immune response, both FOXP3 and axotrophin negatively correlated with graft versus host disease (GVHD). These data suggest that (i) thymic Foxp3+T cell development is influenced by axotrophin; and (ii) clinical auto-GVHD inversely correlates with axotrophin transcript expression as has been previously reported for FOXP3.

Leukaemia inhibitory factor (LIF) is functionally linked to axotrophin and both LIF and axotrophin are linked to regulatory immune tolerance.

Axotrophin (axot) is a newly characterised stem cell gene and mice that lack axotrophin are viable and fertile, but show premature neural degeneration and defective development of the corpus callosum. By comparing axot+/+, axot+/- and axot-/- littermates, we now show that axotrophin is also involved in immune regulation. Both T cell proliferation and T cell-derived leukaemia inhibitory factor (LIF) were suppressed by axotrophin in a gene-dose-dependent manner. Moreover, a role for axotrophin in the feedback regulation of LIF is implicated. This is the first evidence that fate determination mediated by LIF maybe qualified by axotrophin.

Transplantation tolerance: gene expression profiles comparing allotolerance vs. allorejection.

An understanding of the molecular basis of immune regulation will allow development of therapies for diseases caused by immune dysregulation and for therapeutic exploitation of the immune response in transplantation of organ grafts or stem cells. To identify critical regulatory factors in immunity, we have used a mouse model wherein infectious regulatory tolerance is inducible by CD4/CD8 blockade in recipients of vascularised heart grafts. Once established, this transplantation tolerance is robust and isolated "tolerant" spleen cells show powerful immune regulatory properties, being able to impose donor-specific allotolerance upon fully immune competent naive recipients. Here, we present a compound comparison of four gene arrays (tolerance vs. rejection, at 48 h, and at 123 h) where a relatively small number of differentially expressed genes occurred. In rejection, there was a strong progressive amplification of IFNgamma and granzyme B mRNAs. In tolerance, both ELKL motif kinase and axotrophin occurred in the group of upregulated genes. Mice lacking ELKL motif kinase develop autoimmune disease, whilst axotrophin is a newly discovered stem cell gene that has only been explored in the context of neural development. This gene expression data is the first to demonstrate a link between axotrophin and regulatory tolerance and, since axotrophin, LIF, STAT3 and c-kit each function in stem cells, we propose that common mechanisms play a central role both in developmental regulation of stem cells, and in immune regulation.