Jean-François Gauchat

Involvement of LOX-1 in Dendritic Cell-Mediated Antigen Cross-Presentation

Some exogenous antigens, such as heat shock proteins or apoptotic bodies, gain access to the MHC class I processing pathway and initiate CTL responses, a process called cross-priming. To be efficient in vivo, this process requires endocytosis of the antigen by dendritic cells via receptors which remain unidentified. Here, we report that scavenger receptors are the main HSP binding structures on human dendritic cells and identify LOX-1 as one of these molecules. A neutralizing anti-LOX-1 mAb inhibits Hsp70 binding to dendritic cells and Hsp70-induced antigen cross-presentation. In vivo, to target LOX-1 with a tumor antigen using an anti-LOX-1 mAb induces antitumor immunity. Thus, the scavenger receptor LOX-1 is certainly a promising target for cancer immunotherapy.

Cutting edge: IL-21 is a switch factor for the production of IgG1 and IgG3 by human B cells

IL-21 is a cytokine that regulates the activation of T and NK cells and promotes the proliferation of B cells activated via CD40. In this study, we show that rIL-21 strongly induces the production of all IgG isotypes by purified CD19+ human spleen or peripheral blood B cells stimulated with anti-CD40 mAb. Moreover, it was found to specifically induce the production of IgG1 and IgG3 by CD40-activated CD19+CD27− naive human B cells. Although stimulation of CD19+ B cells via CD40 alone induced γ1 and γ3 germline transcripts, as well as the expression of activation-induced cytidine deaminase, only stimulation with both anti-CD40 mAb and rIL-21 resulted in the production of Sγ/Sμ switch circular DNA. These results show that IL-21, in addition to promoting growth and differentiation of committed B cells, is a specific switch factor for the production of IgG1 and IgG3.

Type I interferon restricts type 2 immunopathology through the regulation of group 2 innate lymphoid cells

Viral respiratory tract infections are the main causative agents of the onset of infection-induced asthma and asthma exacerbations that remain mechanistically unexplained. Here we found that deficiency in signaling via type I interferon receptor led to deregulated activation of group 2 innate lymphoid cells (ILC2 cells) and infection-associated type 2 immunopathology. Type I interferons directly and negatively regulated mouse and human ILC2 cells in a manner dependent on the transcriptional activator ISGF3 that led to altered cytokine production, cell proliferation and increased cell death. In addition, interferon-γ (IFN-γ) and interleukin 27 (IL-27) altered ILC2 function dependent on the transcription factor STAT1. These results demonstrate that type I and type II interferons, together with IL-27, regulate ILC2 cells to restrict type 2 immunopathology.

IFN-γ-Mediated Inhibition of Human IgE Synthesis by IL-21 Is Associated with a Polymorphism in the IL-21R Gene

IL-21 is a cytokine produced by CD4+ T cells that has been reported to regulate human, as well as, mouse T and NK cell function and to inhibit Ag-induced IgE production by mouse B cells. In the present study, we show that human rIL-21 strongly enhances IgE production by both CD19+CD27− naive, and CD19+CD27+ memory B cells, stimulated with anti-CD40 mAb and rIL-4 and that it promotes the proliferative responses of these cells. However, rIL-21 does not significantly affect anti-CD40 mAb and rIL-4-induced Cε promoter activation in a gene reporter assay, nor germline Cε mRNA expression in purified human spleen or peripheral blood B cells. In contrast, rIL-21 inhibits rIL-4-induced IgE production in cultures of PBMC or total splenocytes by an IFN-γ-dependent mechanism. The presence of a polymorphism (T-83C), in donors heterozygous for this mutation was found to be associated not only with lower rIL-21-induced IFN-γ production levels, but also with a lower sensitivity to the inhibitory effects of IL-21 on the production of IgE, compared with those in donors expressing the wild-type IL-21R. Taken together, these results show that IL-21 differentially regulates IL-4-induced human IgE production, via its growth- and differentiation-promoting capacities on isotype-, including IgE-, committed B cells, as well as via its ability to induce IFN-γ production, most likely by T and NK cells, whereas the outcome of these IL-21-mediated effects is dependent on the presence of a polymorphism in the IL-21R.

The Functional and Structural Properties of MD-2 Required for Lipopolysaccharide Binding Are Absent in MD-1

MD-1 and MD-2 are secretory glycoproteins that exist on the cell surface in complexes with transmembrane proteins. MD-1 is anchored by radioprotective 105 (RP105), and MD-2 is associated with TLR4. In vivo studies revealed that MD-1 and MD-2 have roles in responses to LPS. Although the direct binding function of MD-2 to LPS has been observed, the physiological function of MD-1 remains unknown. In this study, we compared the LPS-binding functions of MD-1 and MD-2. LPS binding to cell surface complexes was detected for cells transfected with TLR4/MD-2. In contrast, binding was not observed for RP105/MD-1-transfected cells. When rMD-2 protein was expressed in Escherichia coli, it was purified in complexes containing LPS. In contrast, preparations of MD-1 did not contain LPS. When rMD-2 protein was prepared in a mutant strain lacking the lpxM gene, LPS binding disappeared. Therefore, the secondary myristoyl chain attached to the (R)-3-hydroxymyristoyl chain added by LpxM is required for LPS recognition by MD-2, under these conditions. An amphipathic cluster composed of basic and hydrophobic residues in MD-2 has been suggested to be the LPS-binding site. We specifically focused on two Phe residues (119 and 121), which can associate with fatty acids. A mutation at Phe191 or Phe121 strongly reduced binding activity, and a double mutation at these residues prevented any binding from occurring. The Phe residues are present in MD-2 and absent in MD-1. Therefore, the LPS recognition mechanism by RP105/MD-1 is distinct from that of TLR4/MD-2.

Janus kinase 2/signal transducer and activator of transcription 3 inhibitors attenuate the effect of cardiotrophin-like cytokine factor 1 and human focal segmental glomerulosclerosis serum on glomerular filtration barrier

Recurrence of idiopathic focal segmental glomerulosclerosis (FSGS) after renal transplantation is believed to be caused by a circulating factor(s). We detected cardiotrophin-like cytokine factor 1 (CLCF1), a member of the interleukin 6 family, in the plasma from patients with recurrent FSGS. We hypothesized that CLCF1 contributes to the effect of FSGS serum on the glomerular filtration barrier in vitro. Presently, we studied the effect of CLCF1 on isolated rat glomeruli using an in vitro assay of albumin permeability (P(alb)). CLCF1 (0.05-100 ng/mL) increased P(alb) and caused maximal effect at 5-10 ng/mL (P < 0.001). The increase in Palb was analogous to the effect of FSGS serum. Anti-CLCF1 monoclonal antibody blocked the CLCF1-induced increase in P(alb) and significantly attenuated the effect of FSGS serum (P < 0.001). The heterodimer composed of CLCF1 and cosecreted molecule cytokine receptor-like factor 1 (CRLF1) attenuated the increase in P(alb) caused by CLCF1 or FSGS serum. Western blot analysis showed that CLCF1 upregulated phosphorylation of signal transducer and activator of transcription 3 (STAT3) (Tyr705) in glomeruli. This effect was diminished by the heterodimer CLCF1-CRLF1. Janus kinase 2 (JAK2) inhibitor BMS-1119543 or STAT3 inhibitor Stattic significantly blocked the effect of CLCF1 or FSGS serum on P(alb) (P < 0.001). These novel findings suggest that when monomeric CLCF1 increases P(alb), the heterodimer CLCF1-CRLF1 may protect the glomerular filtration barrier. We speculate that albuminuria in FSGS is related to qualitative or quantitative changes in the CLCF1-CRLF1 complex, and that JAK2 or STAT3 inhibitors may be novel therapeutic agents to treat FSGS.

The Composite Cytokine p28/Cytokine-Like Factor 1 Sustains B Cell Proliferation and Promotes Plasma Cell Differentiation

IL-27 is an APC-derived IL-6/IL-12 family composite cytokine with multiple functions such as regulation of Th1, Th17, and regulatory T cell differentiation, B cell proliferation, and Ig class switching. The IL-27 complex is formed by the association of the cytokine p28 with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27 cytokine and soluble receptor subunits p28 and EBI3 can be secreted independently. The p28 subunit has been shown to have IL-27–independent biological activities. We previously demonstrated that p28 can form an alternative composite cytokine with the EBI3 homolog cytokine-like factor 1 (CLF; CRLF1). p28/CLF modulates NK cell activity and CD4 T cell cytokine production in vitro. In this study we used IL-6–dependent plasmacytoma cell line B9 and CD4 T cells from IL-27Rα–deficient mice to demonstrate that p28/CLF activates IL-27–unresponsive cells, indicating that p28/CLF and IL-27 signal through different receptors. The observation that p28/CLF, unlike IL-27, sustains B9 plasmacytoma cell proliferation prompted us to investigate the effects of p28/CLF on mouse B cells. We observed that p28/CLF induces IgM, IgG2c, and IgG1 production and plasma cell differentiation. p28/CLF therefore has the potential to contribute to B and plasma cell function, differentiation, and proliferation in normal and pathological conditions such as Castelman’s disease and multiple myeloma.

IL-6 activates STAT5 in T cells.

BACKGROUND IL-6 is a pleiotropic cytokine which emerged recently as a key regulator of CD4 T cell function. IL-6 alone or in combination with other cytokines promotes T helper 1, T helper 17 and T follicular helper cell differentiation whilst inhibiting the induction of regulatory T cell generation. IL-6 activates multiple pathways among which JAK/STAT3 is the most clearly validated in the control of CD4 T helper differentiation. Activation of STAT5 by cytokines such as IL-2 can counteract IL-6-induced T helper 17 and T follicular helper cell differentiation and promote the induction of regulatory T cell generation. STAT5 and STAT3 are known to compete for promoter binding sites in CD4 T cells and the two transcription factors are believed to have opposite functions in the control of CD4 T cell differentiation. METHODS We analyzed IL-6-induced STAT1, 3 and 5 activation by flow cytometry (phosflow) in mouse mononuclear cells and its effect on the level of the mRNA coding for cytokine-inducible SH2-containing protein (CIS). RESULTS The results show that IL-6 also induces STAT5 activation in both CD4 and CD8 T as well as NK cells. Analysis of STAT5 phosphorylation in CD4 T cells indicates that it is transient and requires higher cytokine concentrations than that of STAT3. CD4 T cell stimulation with IL-6 induces the synthesis of CIS, which is encoded by a gene known to be regulated by STAT5. CONCLUSIONS Thus, IL-6 at concentrations corresponding to levels observed in the serum during inflammation may activate, in CD4 T cells, a STAT5-negative feedback loop which alters the balance between STAT3-dependent pro-inflammatory helper T cells and STAT5-induced T regulatory cells. STAT5 activation may modulate the differentiation of T helper cells through attenuation of TGF-β stability and production. Since STAT5 is directly activated by Janus kinases, therapeutic approaches designed to inhibit STAT3 activation or to recruit STAT3 phosphatases may be useful in altering the balance of activated STAT3 and STAT5 in favor a profile that would be beneficial in pathologies involving IL-6.

Comparison of Lipopolysaccharide-Binding Functions of CD14 and MD-2

ABSTRACT Prior to being recognized by the cell surface Toll-like receptor 4/MD-2 complex, lipopolysaccharide (LPS) in the bacterial outer membrane has to be processed by LPS-binding protein and CD14. CD14 forms a complex with monomeric LPS extracted by LPS-binding protein and transfers LPS to the cell surface signaling complex. In a previous study, we prepared a functional recombinant MD-2 using a bacterial expression system. We expressed the recombinant protein in Escherichia coli as a fusion protein with thioredoxin and demonstrated specific binding to LPS. In this study, we prepared recombinant CD14 fusion proteins using the same approach. Specific binding of LPS was demonstrated with a recombinant protein containing 151 amino-terminal residues. The region contained a hydrophilic region and the first three leucine-rich repeats (LRRs). The LRRs appeared to contribute to the binding because removal of the region resulted in a reduction in the binding function. LPS binding to the recombinant MD-2 was resistant to detergents. On the other hand, the binding to CD14 was prevented in the presence of low concentrations of detergents. In the case of human MD-2, the secondary myristoyl chain of LPS added by LpxM was required for the binding. A nonpathogenic penta-acyl LPS mutant lacking the myristoyl chain did not bind to MD-2 but did so normally to CD14. The broader LPS-binding spectrum of CD14 may allow recognition of multiple pathogens, and the lower affinity for LPS binding of CD14 allows transmission of captured materials to MD-2.

A Polyglutamic Acid Motif Confers IL-27 Hydroxyapatite and Bone-Binding Properties

The p28 subunit of the composite cytokine IL-27 comprises a polyglutamic acid domain, which is unique among type I cytokines. This domain is very similar to the acidic domain known to confer hydroxyapatite (HA)-binding properties and bone tropism to bone sialoprotein. We observed IL-27 binding to HA, in accordance with previous studies reporting successful p28 HA chromatography. The IL-27 polyglutamic acid domain is located in a flexible inter-α helix loop, and HA-bound IL-27 retained biological activity. Using IL-27 alanine mutants, we observed that the p28 polyglutamic acid domain confers HA- and bone-binding properties to IL-27 in vitro and bone tropism in vivo. Because IL-27 is a potent regulator of cells residing in endosteal bone marrow niches such as osteoclasts, T regulatory, memory T, plasma, and stem cells, this specific property could be beneficial for therapeutic applications. IL-27 has potent antitumoral and antiosteoclastogenic activities. It could therefore also be useful for therapies targeting hematologic cancer or solid tumors metastasis with bone tropism. Furthermore, these observations suggest that polyglutamic motifs could be grafted onto other type I cytokine inter-α helix loops to modify their pharmacological properties.