Gongxian Liao

Cutting Edge: The Natural Ligand for Glucocorticoid-Induced TNF Receptor-Related Protein Abrogates Regulatory T Cell Suppression

CD4+25+ regulatory T (Treg) cells maintain immunological self-tolerance through mechanisms that are only in part understood. Previous studies suggest that the glucocorticoid-induced TNFR-related protein (GITR), which is preferentially expressed on the surface of Treg cells, potentially provides a signal that abrogates Treg suppression. In this study, we show that a soluble form of mouse GITR ligand (sGITR-L) induces GITR-dependent NF-κB activation and blocks in vitro suppression mediated by both resting and preactivated polyclonal and Ag-specific Treg cells. Since sGITR-L along with rIL-2 induces proliferation of CD4+25+ cells, it appears that sGITR-L can break the anergic state of Treg cells. Because sGITR-L also up-regulates IL-2 secretion by activated CD4+25 −T cells, these two sGITR-L induced signals synergize to interfere with suppressor activity by CD4+25+ Treg cells.

SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages

Phagocytosis is a pivotal process by which macrophages eliminate microorganisms after recognition by pathogen sensors. Here we unexpectedly found that the self ligand and cell surface receptor SLAM functioned not only as a costimulatory molecule but also as a microbial sensor that controlled the killing of Gram-negative bacteria by macrophages. SLAM regulated activity of the NADPH oxidase NOX2 complex and phagolysosomal maturation after entering the phagosome, following interaction with the bacterial outer membrane proteins OmpC and OmpF. SLAM recruited a complex containing the intracellular class III phosphatidylinositol kinase Vps34, its regulatory protein kinase Vps15 and the autophagy-associated molecule beclin-1 to the phagosome, which was responsible for inducing the accumulation of phosphatidylinositol-3-phosphate, a regulator of both NOX2 function and phagosomal or endosomal fusion. Thus, SLAM connects the Gram-negative bacterial phagosome to ubiquitous cellular machinery responsible for the control of bacterial killing.

GITR engagement preferentially enhances proliferation of functionally competent CD4^+CD25^+FoxP3^+ regulatory T cells

Naturally occurring regulatory T cells (Treg) express high levels of glucocorticoid-induced tumour necrosis factor receptor (GITR). However, studies of the role of GITR in Treg biology has been complicated by the observation that upon activation effector CD4(+) T (Teff) cells also express the receptor. Here, we dissect the contribution of GITR-induced signaling networks in the expansion and function of FoxP3(+) Treg. We demonstrate that a high-affinity soluble Fc-GITR-L dimer, in conjugation with alphaCD3, specifically enhances in vitro proliferation of Treg, which retain their phenotypic markers (CD25 and FoxP3) and their suppressor function, while minimally affecting Teff cells. Furthermore, Fc-GITR-L does not impair Teff susceptibility to suppression, as judged by cocultures employing GITR-deficient and GITR-sufficient CD4(+) T-cell subsets. Notably, this expansion of Treg could also be seen in vivo, by injecting FoxP3-IRES-GFP mice with Fc-GITR-L even in the absence of antigenic stimulation. In order to test the efficacy of these findings therapeutically, we made use of a C3H/HeJ hemophilia B-prone mouse model. The use of liver-targeted human coagulation factor IX (hF.IX) gene therapy in this model has been shown to induce liver toxicity and the subsequent failure of hF.IX expression. Interestingly, injection of Fc-GITR-L into the hemophilia-prone mice that were undergoing liver-targeted hF.IX gene therapy increased the expression of F.IX and reduced the anticoagulation factors. We conclude that GITR engagement enhances Treg proliferation both in vitro and in vivo and that Fc-GITR-L may be a useful tool for in vivo tolerance induction.

A novel isoform of the Ly108 gene ameliorates murine lupus

The expression of the new Ly108 isoform H1 weakens lupus-like disease of C57BL/6.Sle1b mice.

Mechanism of oral tolerance induction to therapeutic proteins.

Oral tolerance is defined as the specific suppression of humoral and/or cellular immune responses to an antigen by administration of the same antigen through the oral route. Due to its absence of toxicity, easy administration, and antigen specificity, oral tolerance is a very attractive approach to prevent unwanted immune responses that cause a variety of diseases or that complicate treatment of a disease. Many researchers have induced oral tolerance to efficiently treat autoimmune and inflammatory diseases in different animal models. However, clinical trials yielded limited success. Thus, understanding the mechanisms of oral tolerance induction to therapeutic proteins is critical for paving the way for clinical development of oral tolerance protocols. This review will summarize progress on understanding the major underlying tolerance mechanisms and contributors, including antigen presenting cells, regulatory T cells, cytokines, and signaling pathways. Potential applications, examples for therapeutic proteins and disease targets, and recent developments in delivery methods are discussed.

Tolerance Induction to Cytoplasmic β-Galactosidase by Hepatic AAV Gene Transfer — Implications for Antigen Presentation and Immunotoxicity

Background Hepatic gene transfer, in particular using adeno-associated viral (AAV) vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein. Major Findings AAV-2 vector-mediated hepatic gene transfer for expression of cytoplasmic β-galactosidase (β-gal) was performed in immune competent mice, followed by a secondary β-gal gene transfer with E1/E3-deleted adenoviral Ad-LacZ vector to provoke a severe immunotoxic response. Transgene expression from the AAV-2 vector in ∼2% of hepatocytes almost completely protected from inflammatory T cell responses against β-gal, eliminated antibody formation, and significantly reduced adenovirus-induced hepatotoxicity. Consequently, ∼10% of hepatocytes continued to express β-gal 45 days after secondary Ad-LacZ gene transfer, a time point when control mice had lost all Ad-LacZ derived expression. Suppression of inflammatory T cell infiltration in the liver and liver damage was linked to specific transgene expression and was not seen for secondary gene transfer with Ad-GFP. A combination of adoptive transfer studies and flow cytometric analyses demonstrated induction of Treg that actively suppressed CD8+ T cell responses to β-gal and that was amplified in liver and spleen upon secondary Ad-LacZ gene transfer. Conclusions These data demonstrate that tolerance induction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that expression of a cytoplasmic neo-antigen in few hepatocytes can induce Treg and provide long-term suppression of inflammatory responses and immunotoxicity.

Nonredundant Roles of IL-10 and TGF-β in Suppression of Immune Responses to Hepatic AAV-Factor IX Gene Transfer

Hepatic gene transfer using adeno-associated viral (AAV) vectors has been shown to efficiently induce immunological tolerance to a variety of proteins. Regulatory T-cells (Treg) induced by this route suppress humoral and cellular immune responses against the transgene product. In this study, we examined the roles of immune suppressive cytokines interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in the development of tolerance to human coagulation factor IX (hF.IX). Interestingly, IL-10 deficient C57BL/6 mice receiving gene transfer remained tolerant to hF.IX and generated Treg that suppressed anti-hF.IX formation. Effects of TGF-β blockade were also minor in this strain. In contrast, in C3H/HeJ mice, a strain known to have stronger T-cell responses against hF.IX, IL-10 was specifically required for the suppression of CD8(+) T-cell infiltration of the liver. Furthermore, TGF-β was critical for tipping the balance toward an regulatory immune response. TGF-β was required for CD4(+)CD25(+)FoxP3(+) Treg induction, which was necessary for suppression of effector CD4(+) and CD8(+) T-cell responses as well as antibody formation. These results demonstrate the crucial, nonredundant roles of IL-10 and TGF-β in prevention of immune responses against AAV-F.IX-transduced hepatocytes.

Blocking CD27-CD70 Costimulatory Pathway Suppresses Experimental Colitis

The pathogenesis of human inflammatory bowel disease (IBD) and most experimental models of IBD is dependent on the activation and expansion of CD4+ T cells via interaction with mucosal APCs. The costimulatory receptor CD70 is transiently expressed on the surface of conventional dendritic cells, but is constitutively expressed by a unique APC population in the intestinal lamina propria. We used two experimental IBD models to evaluate whether interfering the interaction between CD70 and its T cell ligand CD27 would affect the development of colitis. Adoptive transfer of naive CD27-deficient CD45RBhigh CD4+ T cells into Rag-1−/− mice resulted in significantly less disease than when wild-type CD45RBhighCD4+ T cells were used. Moreover, a monoclonal anti-CD70 Ab prevented the disease caused by the transfer of wild-type CD45RBhigh CD4+ T cells into Rag-1−/− mice and the same Ab also ameliorated an established disease. The colitis associated proinflammatory cytokines IL-6, TNF-α and IFN-γ were significantly reduced after anti-CD70 Ab treatment, suggesting an overall reduction in inflammation due to blockade of pathogenic T cell expansion. Anti-CD70 Ab treatment also suppressed trinitrobenzene sulfonic acid-induced colitis in SJL/J mice. Because anti-CD70 Ab treatment suppressed multiple proinflammatory cytokines, this may be a more potent therapeutic approach for IBD than blockade of individual cytokines.

Identification and characterization of two related murine genes, Eat2a and Eat2b, encoding single SH2-domain adapters

Human EAT-2 (SH2D1B) and SLAM-associated protein (SAP) (SH2D1A) are single SH2-domain adapters, which bind to specific tyrosine residues in the cytoplasmic tail of six signaling lymphocytic activation molecule (SLAM) (SLAMF1)-related receptors. Here we report that, unlike in humans, the mouse and rat Eat2 genes are duplicated with an identical genomic organization. The coding regions of the mouse Eat2a and Eat2b genes share 91% identity at the nucleotide level and 84% at the protein level; similarly, segments of introns are highly conserved. Whereas expression of mouse Eat2a mRNA was detected in multiple tissues, Eat2b was only detectable in mouse natural killer cells, CD8+ T cells, and ovaries, suggesting a very restricted tissue expression of the latter. Both the EAT-2A and EAT-2B coimmunoprecipitated with mouse SLAM in transfected cells and augmented tyrosine phosphorylation of the cytoplasmic tail of SLAM. Both EAT-2A and EAT-2B bind to the Src-like kinases Fyn, Hck, Lyn, Lck, and Fgr, as determined by a yeast two-hybrid assay. However, unlike SAP, the EAT-2 proteins bind to their kinase domains and not to the SH3 domain of these kinases. Taken together, the data suggest that both EAT-2A and EAT-2B are adapters that recruit Src kinases to SLAM family receptors using a mechanism that is distinct from that of SAP.

Synergy between rapamycin and FLT3 ligand enhances plasmacytoid dendritic cell–dependent induction of CD4+CD25+FoxP3+ Treg

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Treg) are critical elements for maintaining immune tolerance, for instance to exogenous antigens that are introduced during therapeutic interventions such as cell/organ transplant or gene/protein replacement therapy. Coadministration of antigen with rapamycin simultaneously promotes deletion of conventional CD4(+) T cells and induction of Treg. Here, we report that the cytokine FMS-like receptor tyrosine kinase ligand (Flt3L) enhances the in vivo effect of rapamycin. This occurs via selective expansion of plasmacytoid dendritic cells (pDCs), which further augments the number of Treg. Whereas in conventional DCs, rapamycin effectively blocks mammalian target of rapamycin (mTOR) 1 signaling induced by Flt3L, increased mTOR1 activity renders pDCs more resistant to inhibition by rapamycin. Consequently, Flt3L and rapamycin synergistically promote induction of antigen-specific Treg via selective expansion of pDCs. This concept is supported by the finding that Treg induction is abrogated upon pDC depletion. The combination with pDCs and rapamycin is requisite for Flt3L/antigen-induced Treg induction because Flt3L/antigen by itself fails to induce Treg. As co-administering Flt3L, rapamycin, and antigen blocked CD8(+) T-cell and antibody responses in models of gene and protein therapy, we conclude that the differential effect of rapamycin on DC subsets can be exploited for improved tolerance induction.