International Immunology, Vol. 26, No. 5, pp. 283–289 doi:10.1093/intimm/dxt066 Advance Access publication 26 December 2013
© The Japanese Society for Immunology. 2013. All rights reserved. For permissions, please e-mail: [email protected]
Loss of MHC II ubiquitination inhibits the activation and differentiation of CD4 T cells Rikiya Ishikawa1, Mizuho Kajikawa2 and Satoshi Ishido2 Division of Immunobiology, Department of Supramolecular Biology, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan 2 Laboratory of Integrative Infection Immunity, Showa Pharmaceutical University, Machida, Tokyo 194–8543, Japan Correspondence to: S. Ishido; E-mail: [email protected]
Peptide–MHC class II complexes (pMHC II) are degraded by MARCH-I-mediated ubiquitination, and the stabilization of pMHC II by loss of its ubiquitination is one phenotype defining the activation of conventional dendritic cells (cDCs). However, the role of such stabilization of pMHC II in the context of T-cell activation/differentiation remains unclear. Here, we show that loss of pMHC II ubiquitination inhibits the activation and differentiation of CD4 T cells, probably through down-regulation of CD18/ integrin β2 and their diminished IL-12 production in a cell intrinsic manner. The cDCs generated from mice whose pMHC II ubiquitination is inhibited had a decreased ability to activate naive CD4 T cells and induce Th1/Th17 differentiation. In addition, cDCs whose MHC II ubiquitination was inhibited showed down-regulation of CD18/integrin beta 2 and of IL-12 production. This unexpected finding suggests that loss of MHC II ubiquitination contributes to the negative feedback of CD4 T-cell immune responses. Keywords: CD86, dendritic cells
Introduction Antigen presentation is a critical process for the initiation of T-cell immunity (1). Invading pathogens are phagocytosed, and proteins derived from them are processed and presented through MHC class II (MHC II) by antigen-presenting cells (APCs) (1, 2). Peptide–MHC II complexes (pMHC II) are composed of pathogen-derived peptides that stimulate CD4 T cells through interaction with the TCR complex. Together with the maturation signals for APCs, pMHC II can initiate immunity against the pathogen (2, 3). Thus, it is important to understand how MHC II-mediated antigen presentation is regulated in vivo. In this regard, we and other groups have found that the expression level of pMHC II on the surface of APCs is regulated by ubiquitination (4, 5). In addition, by generating genetically modified mice, we identified MARCH-I as a physiological E3 ubiquitin ligase for MHC II (6). To understand the role of MARCH-I-mediated pMHC II ubiquitination, several groups including ours examined how this process is regulated in the context of immune responses. At present, it has been shown that several signals that induce the activation/maturation of dendritic cells (e.g. TLR stimuli) stabilize the cell surface pMHC II through inhibition of pMHC II ubiquitination by down-regulation of MARCH-I (7–10). Thus, the loss of pMHC II ubiquitination has been recognized as one of the hallmarks of activation
of DCs (5, 11). Based on these findings, a commonly held belief in the field is that loss of pMHC II ubiquitination promotes pathogen immunity through enhancement of antigen presentation (11). However, in reality, there are no findings supporting such an attractive hypothesis. On the contrary, we have previously reported the opposite: that splenic DCs in mice whose pMHC II is not ubiquitinated were functionally impaired, suggesting that loss of pMHC II ubiquitination delivers negative feedback to the immune response (11, 12). In this paper, we examined how loss of pMHC II ubiquitination influences the function of conventional DCs (cDCs) in a cell intrinsic manner by using in vitro generated bone marrow (BM)-derived cDCs, using Flt3L as the inducer. The cDCs whose pMHC II ubiquitination was inhibited showed a decreased ability to stimulate naive CD4 T cells and support their differentiation into Th1 and Th17 cells. This outcome appears to be specific for the stabilization of pMHC II, because cDCs whose CD86 is stabilized by loss of ubiquitination did not show impaired ability to stimulate naive CD4 T cells. Mechanistically, we found that expression of CD18/integrin β2 was significantly down-regulated in cDCs in which pMHC II ubiquitination was inhibited. Consistently, inhibition by a CD18-neutralizing antibody or genetic deletion of CD18 impaired cDC-mediated CD4 T-cell activation.
Downloaded from http://intimm.oxfordjournals.org/ at University of Ulster at Coleraine on June 9, 2015
Received 1 August 2013, accepted 20 November 2013
Featured Article
1
284 Inhibition by loss of MHC II ubiquitination Thus, our results highlight that the loss of pMHC II ubiquitination is an inhibitory inducer for cDC-mediated CD4 T-cell activation. We propose that stabilization of pMHC II contributes to negative feedback to CD4 T-cell activation/ differentiation. Methods Mice
Flow cytometry analysis To examine the expression of surface molecules, cells were incubated on ice for 45 min with fluorescein-conjugated antibodies in PBS containing 2% FCS and Fc blocker (antiCD16/32 antibody). Intracellular expression of IFN-γ and IL-17 was examined using a cytofix/cytoperm permeabilization kit (BD). All samples were analyzed on a FACSCalibur system. mAbs specific for MHC class II, CD4, CD11c, LFA-1, CD18, CD86, IFN-γ and IL-17 were obtained from BioLegend.
Quantitative RT–PCR Total RNAs were isolated from OT-II CD4 T cells, which were co-cultivated with cDCs for 16 h by using TRIzol (Invitrogen). The first-strand cDNA was generated by using the SuperScript III system (Invitrogen) using oligo dT as primers. Quantitative PCR (qPCR) was performed by the SYBR Green method (Eppendorf realplex system) for IL-2 mRNA. Statistics Data were analyzed with the unpaired, two-tailed Student’s t-test. Values with P
© The Japanese Society for Immunology. 2013. All rights reserved. For permissions, please e-mail: [email protected]
Loss of MHC II ubiquitination inhibits the activation and differentiation of CD4 T cells Rikiya Ishikawa1, Mizuho Kajikawa2 and Satoshi Ishido2 Division of Immunobiology, Department of Supramolecular Biology, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan 2 Laboratory of Integrative Infection Immunity, Showa Pharmaceutical University, Machida, Tokyo 194–8543, Japan Correspondence to: S. Ishido; E-mail: [email protected]
Peptide–MHC class II complexes (pMHC II) are degraded by MARCH-I-mediated ubiquitination, and the stabilization of pMHC II by loss of its ubiquitination is one phenotype defining the activation of conventional dendritic cells (cDCs). However, the role of such stabilization of pMHC II in the context of T-cell activation/differentiation remains unclear. Here, we show that loss of pMHC II ubiquitination inhibits the activation and differentiation of CD4 T cells, probably through down-regulation of CD18/ integrin β2 and their diminished IL-12 production in a cell intrinsic manner. The cDCs generated from mice whose pMHC II ubiquitination is inhibited had a decreased ability to activate naive CD4 T cells and induce Th1/Th17 differentiation. In addition, cDCs whose MHC II ubiquitination was inhibited showed down-regulation of CD18/integrin beta 2 and of IL-12 production. This unexpected finding suggests that loss of MHC II ubiquitination contributes to the negative feedback of CD4 T-cell immune responses. Keywords: CD86, dendritic cells
Introduction Antigen presentation is a critical process for the initiation of T-cell immunity (1). Invading pathogens are phagocytosed, and proteins derived from them are processed and presented through MHC class II (MHC II) by antigen-presenting cells (APCs) (1, 2). Peptide–MHC II complexes (pMHC II) are composed of pathogen-derived peptides that stimulate CD4 T cells through interaction with the TCR complex. Together with the maturation signals for APCs, pMHC II can initiate immunity against the pathogen (2, 3). Thus, it is important to understand how MHC II-mediated antigen presentation is regulated in vivo. In this regard, we and other groups have found that the expression level of pMHC II on the surface of APCs is regulated by ubiquitination (4, 5). In addition, by generating genetically modified mice, we identified MARCH-I as a physiological E3 ubiquitin ligase for MHC II (6). To understand the role of MARCH-I-mediated pMHC II ubiquitination, several groups including ours examined how this process is regulated in the context of immune responses. At present, it has been shown that several signals that induce the activation/maturation of dendritic cells (e.g. TLR stimuli) stabilize the cell surface pMHC II through inhibition of pMHC II ubiquitination by down-regulation of MARCH-I (7–10). Thus, the loss of pMHC II ubiquitination has been recognized as one of the hallmarks of activation
of DCs (5, 11). Based on these findings, a commonly held belief in the field is that loss of pMHC II ubiquitination promotes pathogen immunity through enhancement of antigen presentation (11). However, in reality, there are no findings supporting such an attractive hypothesis. On the contrary, we have previously reported the opposite: that splenic DCs in mice whose pMHC II is not ubiquitinated were functionally impaired, suggesting that loss of pMHC II ubiquitination delivers negative feedback to the immune response (11, 12). In this paper, we examined how loss of pMHC II ubiquitination influences the function of conventional DCs (cDCs) in a cell intrinsic manner by using in vitro generated bone marrow (BM)-derived cDCs, using Flt3L as the inducer. The cDCs whose pMHC II ubiquitination was inhibited showed a decreased ability to stimulate naive CD4 T cells and support their differentiation into Th1 and Th17 cells. This outcome appears to be specific for the stabilization of pMHC II, because cDCs whose CD86 is stabilized by loss of ubiquitination did not show impaired ability to stimulate naive CD4 T cells. Mechanistically, we found that expression of CD18/integrin β2 was significantly down-regulated in cDCs in which pMHC II ubiquitination was inhibited. Consistently, inhibition by a CD18-neutralizing antibody or genetic deletion of CD18 impaired cDC-mediated CD4 T-cell activation.
Downloaded from http://intimm.oxfordjournals.org/ at University of Ulster at Coleraine on June 9, 2015
Received 1 August 2013, accepted 20 November 2013
Featured Article
1
284 Inhibition by loss of MHC II ubiquitination Thus, our results highlight that the loss of pMHC II ubiquitination is an inhibitory inducer for cDC-mediated CD4 T-cell activation. We propose that stabilization of pMHC II contributes to negative feedback to CD4 T-cell activation/ differentiation. Methods Mice
Flow cytometry analysis To examine the expression of surface molecules, cells were incubated on ice for 45 min with fluorescein-conjugated antibodies in PBS containing 2% FCS and Fc blocker (antiCD16/32 antibody). Intracellular expression of IFN-γ and IL-17 was examined using a cytofix/cytoperm permeabilization kit (BD). All samples were analyzed on a FACSCalibur system. mAbs specific for MHC class II, CD4, CD11c, LFA-1, CD18, CD86, IFN-γ and IL-17 were obtained from BioLegend.
Quantitative RT–PCR Total RNAs were isolated from OT-II CD4 T cells, which were co-cultivated with cDCs for 16 h by using TRIzol (Invitrogen). The first-strand cDNA was generated by using the SuperScript III system (Invitrogen) using oligo dT as primers. Quantitative PCR (qPCR) was performed by the SYBR Green method (Eppendorf realplex system) for IL-2 mRNA. Statistics Data were analyzed with the unpaired, two-tailed Student’s t-test. Values with P
