Whole-exome sequencing reveals activating STAT1 mutations in some patients with autosomal dominant chronic mucocutaneous candidiasis disease.

Experimental autoimmune encephalomyelitis (EAE) is a T cell–mediated autoimmune disease of the central nervous system induced by antigen-specific effector Th17 and Th1 cells. We show that a key chemokine, CXCL12 (stromal cell–derived factor 1α), redirects the polarization of effector Th1 cells into CD4+CD25−Foxp3−interleukin (IL) 10high antigen-specific regulatory T cells in a CXCR4-dependent manner, and by doing so acts as a regulatory mediator restraining the autoimmune inflammatory process. In an attempt to explore the therapeutic implication of these findings, we have generated a CXCL12-immunoglobulin (Ig) fusion protein that, when administered during ongoing EAE, rapidly suppresses the disease in wild-type but not IL-10–deficient mice. Anti–IL-10 neutralizing antibodies could reverse this suppression. The beneficial effect included selection of antigen-specific T cells that were CD4+CD25−Foxp3−IL-10high, which could adoptively transfer disease resistance, and suppression of Th17 selection. However, in vitro functional analysis of these cells suggested that, even though CXCL12-Ig–induced tolerance is IL-10 dependent, IL-10–independent mechanisms may also contribute to their regulatory function. Collectively, our results not only demonstrate, for the first time, that a chemokine functions as a regulatory mediator, but also suggest a novel way for treating multiple sclerosis and possibly other inflammatory autoimmune diseases.

The current study identifies CCR8 regulatory T cells (T cells) as drivers of immunosuppression. We show that in human peripheral blood cells, more than 30% of T up-regulate CCR8 following activation in the presence of CCL1. This interaction induces STAT3-dependent up-regulation of FOXp3, CD39, IL-10, and granzyme B, resulting in enhanced suppressive activity of these cells. Of the four human CCR8 ligands, CCL1 is unique in potentiating T cells. The relevance of these observations has been extended using an experimental model of multiple sclerosis [experimental autoimmune encephalomyelitis, (EAE)] and a stabilized version of mouse CCL1 (CCL1-Ig). First, we identified a self-feeding mechanism by which CCL1 produced by T cells at an autoimmune site up-regulates the expression of its own receptor, CCR8, on these cells. Administration of CCL1-Ig during EAE enhanced the in vivo proliferation of these CCR8 regulatory cells while inducing the expression of CD39, granzyme B, and IL-10, resulting in the efficacious suppression of ongoing EAE. The critical role of the CCL1-CCR8 axis in T cells was further dissected through adoptive transfer studies using CCR8 mice. Collectively, we demonstrate the pivotal role of CCR8 T cells in restraining immunity and highlight the potential clinical implications of this discovery.

IFN-γ-inducible protein 10 (IP-10) is a CXC chemokine that is thought to manifest a proinflammatory role because it stimulates the directional migration of activated T cells, particularly Th1 cells. It is an open question whether this chemokine is also directly involved in T cell polarization. We show here that during the course of adjuvant-induced arthritis the immune system mounts a notable Ab titer against self-IP-10. Upon the administration of naked DNA encoding IP-10, this titer rapidly accelerates to provide protective immunity. Self-specific Ab to IP-10 developed in protected animals, as well as neutralizing Ab to IP-10 that we have generated in rabbits, could inhibit leukocyte migration, alter the in vivo and in vitro Th1/Th2 balance toward low IFN-γ, low TNF-α, high IL-4-producing T cells, and adoptively transfer disease suppression. This not only demonstrates the pivotal role of this chemokine in T cell polarization during experimentally induced arthritis but also suggests a practical way to interfere in the regulation of disease to provide protective immunity. From the basic science perspective, this study challenges the paradigm of in vivo redundancy. After all, we did not neutralize the activity of other chemokines that bind CXCR3 (i.e., macrophage-induced gene and IFN-inducible T cell α chemoattractant) and yet significantly blocked not only adjuvant-induced arthritis but also the in vivo competence to mount delayed-type hypersensitivity.

Accumulation of myeloid-derived suppressor cells (MDSC) in melanoma microenvironment is supported by chemokine receptor/chemokine signaling. Although different chemokines were suggested to be involved in this process, the role of CCR5 and its ligands is not established. Using a transgenic mouse melanoma model, we found an accumulation of CCR5 MDSCs in melanoma lesions associated with both increased concentrations of CCR5 ligands and tumor progression. Tumor-infiltrating CCR5 MDSCs displayed higher immunosuppressive activity than their CCR5 counterparts. Upregulation of CCR5 expression on CD11bGr1 myeloid cells was induced by CCR5 ligands and other inflammatory factors. In melanoma patients, CCR5 MDSCs were enriched at the tumor site and correlated with enhanced production of CCR5 ligands. Moreover, they exhibited a stronger immunosuppressive pattern compared with CCR5 MDSCs. Blocking CCR5/CCR5 ligand interactions increased survival of tumor-bearing mice and was associated with reduced migration and immunosuppressive potential of MDSCs in tumor lesions. Our findings define a critical role for CCR5 in recruitment and activation of MDSCs, suggesting a novel strategy for melanoma treatment. These findings validate the importance of the CCR5/CCR5 ligand axis not only for MDSC recruitment but also for further activation of their immunosuppressive functions in the tumor microenvironment, with potentially broad therapeutic implications, given existing clinically available inhibitors of this axis. .

IFN-γ-inducible protein 10 (IP-10) is a CXC chemokine that stimulates the directional migration of activated T cells, particularly Th1 cells. We demonstrate in this work that during activation this chemokine drives naive CD4+ T cells into Th1 polarization. Administration of plasmid DNA encoding self IP-10 was found capable of breaking down immunological tolerance to IP-10, resulting in the generation of self-specific immunity to the gene product of the vaccine. Despite the CpG motif that drives T cells into Th1, the vaccine redirected the polarization of myelin basic protein-specific T cells into Th2 and conferred the vaccinated recipients a high state of resistance against experimental autoimmune encephalomyelitis, a T cell-mediated autoimmune disease of the CNS. The vaccine also suppressed full-blown ongoing disease in a mouse model of multiple sclerosis. Self-specific Ab to IP-10 developed in protected animals could inhibit leukocyte migration, alter the in vitro Th1/Th2 balance of autoimmune T cells, and adoptively transfer disease suppression. This demonstrates not only the pivotal role of a chemokine in T cell polarization and function but also its potential implications for plasmid DNA gene therapy.

In order to explore the mechanism(s) underlying the pro-tumorigenic capacity of heparanase we established an inducible Tet-on system. Heparanase expression was markedly increased following addition of doxycycline (Dox) to the culture medium of CAG human myeloma cells infected with the inducible heparanase gene construct, resulting in increased colony number and size in soft agar. Moreover, tumor xenografts produced by CAG-heparanase cells were markedly increased in mice supplemented with Dox in their drinking water compared with control mice maintained without Dox. Consistently, we found that heparanase induction is associated with decreased levels of CXCL10, suggesting that this chemokine exerts tumor suppressor properties in myeloma. Indeed, recombinant CXCL10 attenuated the proliferation of CAG, U266 and RPMI-8266 myeloma cells. Similarly, CXCL10 attenuated the proliferation of human umbilical vein endothelial cells (HUVEC), implying that CXCL10 exhibits anti-angiogenic capacity. Strikingly, development of tumor xenografts produced by CAG-heparanase cells over expressing CXCL10 was markedly reduced compared with control cells. Moreover, tumor growth was significantly attenuated in mice inoculated with human or mouse myeloma cells and treated with CXCL10-Ig fusion protein, indicating that CXCL10 functions as a potent anti-myeloma cytokine.

A single G protein-coupled receptor (GPCR) can activate multiple signaling cascades based on the binding of different ligands. The biological relevance of this feature in immune regulation has not been evaluated. The chemokine-binding GPCR CXCR3 is preferentially expressed on CD4+ T cells, and canonically binds 3 structurally related chemokines: CXCL9, CXCL10, and CXCL11. Here we have shown that CXCL10/CXCR3 interactions drive effector Th1 polarization via STAT1, STAT4, and STAT5 phosphorylation, while CXCL11/ CXCR3 binding induces an immunotolerizing state that is characterized by IL-10 hi (Tr1) and IL-4 hi (Th2) cells, mediated via p70 kinase/mTOR in STAT3-and STAT6-dependent pathways. CXCL11 binds CXCR3 with a higher affinity than CXCL10, suggesting that CXCL11 has the potential to restrain inflammatory autoimmunity. We generated a CXCL11-Ig fusion molecule and evaluated its use in the EAE model of inflammatory autoimmune disease. Administration of CXCL11-Ig during the first episode of relapsing EAE in SJL/J mice not only led to rapid remission, but also prevented subsequent relapse. Using GFP-expressing effector CD4 + T cells, we observed that successful therapy was associated with reduced accumulation of these cells at the autoimmune site. Finally, we showed that very low doses of CXCL11 rapidly suppress signs of EAE in C57BL/6 mice lacking functional CXCL11.