Kidney-infiltrating CD4+ T-cell clones promote nephritis in lupus-prone mice
2012; Elsevier BV; Volume: 82; Issue: 9 Linguagem: Inglês
10.1038/ki.2012.242
ISSN1523-1755
AutoresAkiko Okamoto, Keishi Fujio, Nelson H. Tsuno, Koki Takahashi, Kazuhiko Yamamoto,
Tópico(s)T-cell and B-cell Immunology
ResumoIn systemic lupus erythematosus, CD4+ T cells play key roles in the initiation and promotion of autoantigen-specific humoral immunity, and indirect evidence suggests that T cells are pathogenic effectors in lupus nephritis. The contribution of kidney-infiltrating T cells to nephritis, however, has not been verified because of the difficulty in directly analyzing organ-infiltrating T cells. Here, we examined the pathogenic roles of autoreactive cytokine-expressing CD4+ T cells from the kidneys of early nephritic MRL/lpr mice. Interferon (IFN)-γ-secreting cells were enriched among CD5highCD4+ T cells found in the inflamed kidneys. Using single-cell analysis of the T-cell receptor (TCR)highCD5highCD4+ T cells from the kidneys of early nephritic MRL/lpr mice, two IFN-γ-expressing CD4+ T cell clones, MLK2 and MLK3, were identified. CD4+ T cells transduced with the T-cell receptor genes from each clone responded to splenic dendritic cells in an MHC class II–dependent manner, but not to B cells or macrophages. MLK3-transduced CD4+ T cells proliferated in the spleens of prenephritic mice, promoted nephritis progression upon adoptive transfer, and enhanced the deposition of C3 without promoting anti-double-stranded DNA antibody production. Thus, CD4+ T cells in the inflamed kidneys of MRL/lpr mice contribute to nephritis progression. In systemic lupus erythematosus, CD4+ T cells play key roles in the initiation and promotion of autoantigen-specific humoral immunity, and indirect evidence suggests that T cells are pathogenic effectors in lupus nephritis. The contribution of kidney-infiltrating T cells to nephritis, however, has not been verified because of the difficulty in directly analyzing organ-infiltrating T cells. Here, we examined the pathogenic roles of autoreactive cytokine-expressing CD4+ T cells from the kidneys of early nephritic MRL/lpr mice. Interferon (IFN)-γ-secreting cells were enriched among CD5highCD4+ T cells found in the inflamed kidneys. Using single-cell analysis of the T-cell receptor (TCR)highCD5highCD4+ T cells from the kidneys of early nephritic MRL/lpr mice, two IFN-γ-expressing CD4+ T cell clones, MLK2 and MLK3, were identified. CD4+ T cells transduced with the T-cell receptor genes from each clone responded to splenic dendritic cells in an MHC class II–dependent manner, but not to B cells or macrophages. MLK3-transduced CD4+ T cells proliferated in the spleens of prenephritic mice, promoted nephritis progression upon adoptive transfer, and enhanced the deposition of C3 without promoting anti-double-stranded DNA antibody production. Thus, CD4+ T cells in the inflamed kidneys of MRL/lpr mice contribute to nephritis progression. Systemic lupus erythematosus is an autoimmune disease characterized by autoantibody production and multiorgan damage. As many autoantibodies in systemic lupus erythematosus exhibit high-affinity and somatic mutations,1.William J. Euler C. Christensen S. et al.Evolution of autoantibody responses via somatic hypermutation outside of germinal centers.Science. 2002; 297: 2066-2070Crossref PubMed Scopus (413) Google Scholar,2.Wellmann U. Letz M. Herrmann M. et al.The evolution of human anti-double-stranded DNA autoantibodies.Proc Natl Acad Sci USA. 2005; 102: 9258-9263Crossref PubMed Scopus (168) Google Scholar it is supposed that the activation of autoreactive B cells is preceded by the activation of autoreactive T cells, which supply help signals to B cells. Nucleosomes, ribonucleoprotein, and Sm are candidate targets for autoreactive T cells in systemic lupus erythematosus.3.Mohan C. Adams S. Stanik V. et al.Nucleosome: a major immunogen for pathogenic autoantibody-inducing T cells of lupus.J Exp Med. 1993; 177: 1367-1381Crossref PubMed Scopus (630) Google Scholar, 4.Monneaux F. Hoebeke J. Sordet C. et al.Selective modulation of CD4+ T cells from lupus patients by a promiscuous, protective peptide analog.J Immunol. 2005; 175: 5839-5847Crossref PubMed Scopus (59) Google Scholar, 5.Talken B.L. Holyst M.M. Lee D.R. et al.T cell receptor beta-chain third complementarity-determining region gene usage is highly restricted among Sm-B autoantigen-specific human T cell clones derived from patients with connective tissue disease.Arthritis Rheum. 1999; 42: 703-709Crossref PubMed Scopus (23) Google Scholar In a previous study, nucleosome-specific T-cell clones induced the development of lupus nephritis in lupus-prone mice.6.Adams S. Leblanc P. Datta S.K. Junctional region sequences of T-cell receptor beta-chain genes expressed by pathogenic anti-DNA autoantibody-inducing helper T cells from lupus mice: possible selection by cationic autoantigens.Proc Natl Acad Sci USA. 1991; 88: 11271-11275Crossref PubMed Scopus (91) Google Scholar Although these autoantibody-associated T cells are considered to exacerbate lupus nephritis by enhancing autoantibody production, it is unclear whether organ-infiltrating T cells directly contribute to kidney inflammation. T cells are pathogenic effectors in lupus glomerulonephritis.7.Bagavant H. Fu S.M. Pathogenesis of kidney disease in systemic lupus erythematosus.Curr Opin Rheumatol. 2009; 21: 489-494Crossref PubMed Scopus (130) Google Scholar JHD-MRL/lpr mouse strain, a strain that lacks circulating immunoglobulins but possess B cells expressing a B-cell receptor transgene, suffer from renal disease.8.Chan O.T. Hannum L.G. Haberman A.M. et al.A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus.J Exp Med. 1999; 189: 1639-1648Crossref PubMed Scopus (599) Google Scholar Treating (NZB × NZW) F1 mice with cytotoxic T-lymphocyte antigen-4Ig and a suboptimal dose of cyclophosphamide resulted in a significant delay in mortality without a reduction in glomerular immune complex deposits.9.Schiffer L. Sinha J. Wang X. et al.Short term administration of costimulatory blockade and cyclophosphamide induces remission of systemic lupus erythematosus nephritis in NZB/W F1 mice by a mechanism downstream of renal immune complex deposition.J Immunol. 2003; 171: 489-497Crossref PubMed Scopus (125) Google Scholar In NZM2328 mice, early immune complex deposition and acute cellular glomerulonephritis were found to be associated with glomerular and periglomerular T-cell infiltration.10.Bagavant H. Deshmukh U.S. Wang H. et al.Role for nephritogenic T cells in lupus glomerulonephritis: progression to renal failure is accompanied by T cell activation and expansion in regional lymph nodes.J Immunol. 2006; 177: 8258-8265Crossref PubMed Scopus (58) Google Scholar However, the pathogenicity of kidney-infiltrating T cells has not been verified because of the difficulty in directly analyzing organ-infiltrating T cells. In this study, we aimed to identify pathogenic autoreactive T-cell receptors (TCRs) in the kidneys of early nephritic MRL/lpr mice via single-cell analysis. We focused on interferon (IFN)-γ as a pathogenic cytokine because the deletion or inhibition of IFN-γ and its receptor ameliorated nephritis progression in MRL/lpr mice.11.Haas C. Ryffel B. Le Hir M. IFN-gamma is essential for the development of autoimmune glomerulonephritis in MRL/Ipr mice.J Immunol. 1997; 158: 5484-5491PubMed Google Scholar, 12.Balomenos D. Rumold R. Theofilopoulos A.N. Interferon-gamma is required for lupus-like disease and lymphoaccumulation in MRL-lpr mice.J Clin Invest. 1998; 101: 364-371Crossref PubMed Scopus (325) Google Scholar, 13.Schwarting A. Wada T. Kinoshita K. et al.IFN-gamma receptor signaling is essential for the initiation, acceleration, and destruction of autoimmune kidney disease in MRL-Fas(lpr) mice.J Immunol. 1998; 161: 494-503PubMed Google Scholar IFN-γ also contributes to crescent formation and cell-mediated immune injury.12.Balomenos D. Rumold R. Theofilopoulos A.N. Interferon-gamma is required for lupus-like disease and lymphoaccumulation in MRL-lpr mice.J Clin Invest. 1998; 101: 364-371Crossref PubMed Scopus (325) Google Scholar, 13.Schwarting A. Wada T. Kinoshita K. et al.IFN-gamma receptor signaling is essential for the initiation, acceleration, and destruction of autoimmune kidney disease in MRL-Fas(lpr) mice.J Immunol. 1998; 161: 494-503PubMed Google Scholar, 14.Jacob C.O. van der Meide P.H. McDevitt H.O. In vivo treatment of (NZB X NZW)F1 lupus-like nephritis with monoclonal antibody to gamma interferon.J Exp Med. 1987; 166: 798-803Crossref PubMed Scopus (361) Google Scholar, 15.Ozmen L. Roman D. Fountoulakis M. et al.Experimental therapy of systemic lupus erythematosus: the treatment of NZB/W mice with mouse soluble interferon-gamma receptor inhibits the onset of glomerulonephritis.Eur J Immunol. 1995; 25: 6-12Crossref PubMed Scopus (168) Google Scholar In human lupus, some researchers have observed a skew toward T-helper (Th) 1 predominance in proliferative glomerulonephritis.16.Akahoshi M. Nakashima H. Tanaka Y. et al.Th1/Th2 balance of peripheral T helper cells in systemic lupus erythematosus.Arthritis Rheum. 1999; 42: 1644-1648Crossref PubMed Scopus (256) Google Scholar,17.Masutani K. Akahoshi M. Tsuruya K. et al.Predominance of Th1 immune response in diffuse proliferative lupus nephritis.Arthritis Rheum. 2001; 44: 2097-2106Crossref PubMed Scopus (206) Google Scholar Moreover, polymorphisms in IFN-γ or its receptor are associated with lupus in some patients.18.Nakashima H. Inoue H. Akahoshi M. et al.The combination of polymorphisms within interferon-gamma receptor 1 and receptor 2 associated with the risk of systemic lupus erythematosus.FEBS Lett. 1999; 453: 187-190Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar,19.Miyake K. Nakashima H. Akahoshi M. et al.Genetically determined interferon-gamma production influences the histological phenotype of lupus nephritis.Rheumatology (Oxford). 2002; 41: 518-524Crossref PubMed Google Scholar We identified two TCRα- and β-chain pairs in IFN-γ-expressing CD4+ T cells from inflamed kidneys, and CD4+ T cells that had been transduced with either TCR pair displayed autoreactivity to splenic dendritic cells (DCs). Upon adoptive transfer to prenephritic mice, CD4+ T cells that had been transduced with one of the identified TCR, MLK3, promoted nephritis without enhancing autoantibody titers. Our results suggest that CD4+ T cells in the kidneys of MRL/lpr mice contribute to nephritis progression. First, we selected the T-cell surface molecules that are associated with autoreactivity and IFN-γ expression. Previous studies of TCR transgenic mice reported that high CD5 and TCR expression are reflective of high-avidity interactions with self-peptide:major histocompatibility complex (MHC) and are able to predict the survival/homeostatic expansion capacity of CD4+ T cells.20.Azzam H.S. Grinberg A. Lui K. et al.CD5 expression is developmentally regulated by T cell receptor (TCR) signals and TCR avidity.J Exp Med. 1998; 188: 2301-2311Crossref PubMed Scopus (496) Google Scholar,21.Kassiotis G. Zamoyska R. Stockinger B. Involvement of avidity for major histocompatibility complex in homeostasis of naive and memory T cells.J Exp Med. 2003; 197: 1007-1016Crossref PubMed Scopus (143) Google Scholar In mice expressing influenza hemagglutinin and hemagglutinin determinant S1-specific TCR, CD5 upregulation was a marker of their interaction with the S1 self-peptide.22.Boesteanu A. Rankin A.L. Caton A.J. Impact of effector cell differentiation on CD4+ T cells that evade negative selection by a self-peptide.Int Immunol. 2006; 18: 1017-1027Crossref PubMed Scopus (3) Google Scholar Therefore, we hypothesized that the autoreactive T cells in the early inflamed kidney would exhibit increased CD5 expression levels. We then compared the CD5 levels of CD4+ T cells from the spleens and kidneys of control BALB/c and early nephritic MRL/lpr mice. In the early nephritic MRL/lpr mice, the kidney CD4+ T cells exhibited increased CD5 levels (Figure 1a). This was consistent with previous studies that detected CD5 upregulation in activated T cells23.Carrera A.C. Cardenas L. Tugores A. et al.Activators of protein kinase C up-regulate the cell surface expression of CD2 and CD5 T cell glycoproteins.J Biol Chem. 1989; 264: 15650-15655Abstract Full Text PDF PubMed Google Scholar,24.Lozano F. Alberola-Ila J. Places L. et al.Protein kinase C-dependent up-regulation of CD5 surface expression on normal and lymphoblastoid T cells.Immunology. 1990; 70: 434-439PubMed Google Scholar and suggested that autoreactive T cells respond to self-antigens in the early inflamed kidney. When the association between CD5 expression and IFN-γ production was examined in kidney CD4+ T cells, an IFN-γ-positive subset was detected among the T cells with high CD5 levels in the kidneys of early nephritic MRL/lpr mice (Figure 1b). T-cell clonality was then compared according to the CD5 expression level in order to examine whether CD5highCD4+ T cells are clonally different from total CD4+ T cells and CD5lowCD4+ T cells. The reverse transcription PCR/single-strand conformational polymorphism (SSCP) method for TCR (TCR-SSCP) efficiently detects subtle nucleotide changes in the complementarity determining region (CDR) 3 sequences of clonally expanded T cells in vivo.25.Yamamoto K. Sakoda H. Nakajima T. et al.Accumulation of multiple T cell clonotypes in the synovial lesions of patients with rheumatoid arthritis revealed by a novel clonality analysis.Int Immunol. 1992; 4: 1219-1223Crossref PubMed Scopus (151) Google Scholar,26.Tahara H. Fujio K. Araki Y. et al.Reconstitution of CD8+ T cells by retroviral transfer of the TCR alpha beta-chain genes isolated from a clonally expanded P815-infiltrating lymphocyte.J Immunol. 2003; 171: 2154-2160Crossref PubMed Scopus (38) Google Scholar When total CD4+ T cells, CD5lowCD4+ T cells, and CD5highCD4+ T cells from the nephritic kidneys of MRL/lpr mice were sorted and had their T-cell clonality compared using TCR-SSCP analysis, most of the dominant CD5highCD4+ T cells clonotypes were different from those of the total CD4+ T cells and CD5lowCD4+ T-cell populations (Figure 2). This indicated that CD5highCD4+ T cells constitute a distinct clonal population that is different from those of total CD4+ T cells and CD5lowCD4+ T cells. Although the CD5highCD4+ T-cell population contained IFN-γ-secreting cells, not all of the CD5highCD4+ T cells secreted IFN-γ. We used the TCR expression level as an additional indicator of autoreactivity because TCR expression levels are also associated with avidity for the self-peptide:MHC complex.21.Kassiotis G. Zamoyska R. Stockinger B. Involvement of avidity for major histocompatibility complex in homeostasis of naive and memory T cells.J Exp Med. 2003; 197: 1007-1016Crossref PubMed Scopus (143) Google Scholar To efficiently identify the TCRα chain gene in single-cell analysis, we focused on CD4+ T cells that express Vα2, which is one of the major subfamilies of the murine TCR Vα chain. Interestingly, the frequency of Vα2highCD5highCD4+ T cells was increased in the kidneys of the early nephritic mice, but not in the spleens or kidneys of the prenephritic mice (Figure 3a). Single-cell sorting of Vα2highCD5highCD4+ T cells was performed to obtain pairs of TCRα and β chains from single cells that had expanded in the early inflamed kidney. In parallel, the total kidney Vα2+CD5+CD4+ T cell population was also sorted as a reference group (Figure 3a). The α-chain sequences of 85% of the sorted cells were successfully determined in our single-cell analysis (Figure 3b). The dominant Vα2highCD5highCD4+ T-cell clones were distinct from those of the total CD4+ T cells in the early inflamed kidney. Although most of the Vα2highCD5highCD4+ T-cell clones were identified among the Vα2+CD5+CD4+ T cells, they were not the dominant clones in this population, suggesting that the Vα2highCD5highCD4+ T cells found in the kidneys of MRL/lpr mice are a distinct T-cell population. The cytokine and Foxp3 gene expression levels of identified single-cell clones were examined by two-step nested PCR. Among the identified single cell clones obtained from Vα2highCD5highCD4+ T cells, two clones exclusively expressed IFN-γ, and these clones were designated MLK2 and MLK3 (Figure 4a and b). The Vα2-CDR3 sequence of MLK2 was identified in two single cell–sorted Vα2highCD5highCD4+ T cell clones, and the MLK3 Vα2-CDR3 sequence was identified in one single cell–sorted Vα2highCD5highCD4+ T-cell clone and the total kidney Vα2+CD5+CD4+ T-cell population. The CDR3 sequences of the TCRβ chains of MLK2 and MLK3 were determined by three-step semi-nested PCR using a series of Vβ1–19 primers (Figure 4a). The same TCRβ CDR3 sequence was identified in the two MLK2 clones. Although the TCRβ CDR3 sequence that paired with the MLK3 Vα2-CDR3 sequence from the total kidney Vα2+CD5+CD4+ T-cell population was not identified, the identity of the Vα-CDR3 sequence in different sources strongly suggested the in vivo clonal expansion of MLK3.27.Lathrop S.K. Bloom S.M. Rao S.M. et al.Peripheral education of the immune system by colonic commensal microbiota.Nature. 2011; 478: 250-254Crossref PubMed Scopus (765) Google Scholar The full-length sequences of the MLK2 and MLK3 TCRα and β chains were synthesized from a combination of V, CDR3, and C region sequences using heteroduplex PCR, as described previously.28.Fujio K. Okamoto A. Tahara H. et al.Nucleosome-specific regulatory T cells engineered by triple gene transfer suppress a systemic autoimmune disease.J Immunol. 2004; 173: 2118-2125Crossref PubMed Scopus (35) Google Scholar Furthermore, the phenotypes of MLK2 and MLK3 were consistent with Th1 because they did not express interleukin (IL)-4, IL-17, IL-17F, IL-10, or the Foxp3 gene (Figure 4b). On the other hand, there were differences in the distributions of these clones. Although the Vα2 CDR3 sequence of MLK2 was only identified in Vα2highCD5highCD4+ T cells, the Vα2 CDR3 sequence of MLK3 was identified in both Vα2+CD5+CD4+ T cells and Vα2highCD5highCD4+ T cells. The complementary DNA (cDNA) of the MLK2α- and β-chain, or the MLK3α- and β-chain, were subcloned into a bicistronic retrovirus vector. After retroviral infection, the transduction efficiency of the MLK3 clonotype was determined using anti-Vα2 and Vβ10 antibodies (mean clonotypic transduction efficiency: 20–30%; Figure 4c). Although we could not determine the transduction efficiency of the MLK2 clonotype owing to a lack of anti-mouse Vβ1 antibody, the transduction efficiency of Vα2 in MLK2 was similar to that in MLK3 (data not shown). The MLK2- and MLK3-transduced CD4+ T cells displayed a proliferative response to the splenic DCs of prenephritic MRL/lpr mice, whereas neither the splenic B cells nor macrophages induced the proliferation of MLK2- or MLK3-transduced CD4+ T cells (Figure 4d). When the MHC class II restriction of autoreactivity was examined, it was found that anti-I-Ek monoclonal antibody (mAb) blocked the proliferative response of the MLK2-transduced CD4+ T cells to splenic DCs. In contrast, the proliferative response of the MLK3-transduced CD4+ T cells to splenic DCs was inhibited by anti I-Ak mAb (Figure 4e). Therefore, the I-Ek molecule presents the cognate antigen to MLK2, and the I-Ak molecule presents the cognate antigen to MLK3. Although systemic autoantigens were candidates for the antigen recognized by inflammatory T cells in MRL/lpr mice, both the MLK2- and MLK3-transduced CD4+ T cells failed to display enhanced proliferation in response to the addition of nucleosomes in the presence of DCs (Figure 4f). We then examined the in vivo dynamics of MLK3-transduced CD4+ T cells in prenephritic mice, as we were able to track the MLK3-transduced cells using anti-Vα2 and Vβ10 antibodies. Mock- or MLK3-transduced CD4+ T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and transferred to prenephritic mice via the tail vein. Seven days after the transfer, the Vα2+Vβ10+CFSE+CD4+ T cells in the spleens of the MLK3-transferred mice displayed lower CFSE fluorescence than the Vα2+CFSE+CD4+ T cells in the spleens of the mock-transferred mice (Figure 5a). The Vα2+Vβ10+CFSE+CD4+ T cells in the spleens of the MLK3 group displayed significantly higher IFN-γ expression than the Vα2+CFSE+CD4+ T cells in the spleens of the mock-transferred mice (Figure 5b). Although the difference in IFN-γ expression between the groups was relatively small, the strong T-cell activation upon retroviral gene transfer might have been associated with elevated background IFN-γ expression in the mock-transduced cells. Furthermore, the migration of MLK2- or MLK3-transduced CD4+ T cells to the kidney was confirmed by the detection of the TCRα chain sequence of MLK2 or MLK3 by PCR in the spleens and kidneys of the MLK2- or MLK3-transferred mice (Figure 5c). Next, the pathogenicity of the MLK2- and MLK3-transduced cells was investigated in an in vivo transfer experiment. Two million mock-, MLK2-, or MLK3-transduced CD4+ T cells were intravenously transferred to 12-week-old prenephritic MRL/lpr mice. Intriguingly, the MLK3-transferred mice developed nephritis earliest and displayed the shortest survival among the three groups (Figure 6a). The MLK3-transferred mice displayed severe glomerulonephritis, mesangial proliferation, and thickening of the capillary walls at 26 weeks of age (Figure 6b). C3 deposition was significantly enhanced in the kidneys of the MLK3-transferred mice (Figure 6c). Although the difference in IgG deposition between the mock- and MLK3-transferred mice was not significant, there was a tendency toward increased IgG deposition in the MLK3-transferred mice. In accordance with the previous findings that Th1 cells activate macrophages29.Mosser D.M. Edwards J.P. Exploring the full spectrum of macrophage activation.Nat Rev Immunol. 2008; 8: 958-969Crossref PubMed Scopus (6176) Google Scholar and macrophages have pathogenic roles in murine lupus nephritis,30.Kikawada E. Lenda D.M. Kelley V.R. IL-12 deficiency in MRL-Fas(lpr) mice delays nephritis and intrarenal IFN-gamma expression, and diminishes systemic pathology.J Immunol. 2003; 170: 3915-3925Crossref PubMed Scopus (135) Google Scholar,31.Tesch G.H. Maifert S. Schwarting A. et al.Monocyte chemoattractant protein 1-dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Fas(lpr) mice.J Exp Med. 1999; 190: 1813-1824Crossref PubMed Scopus (273) Google Scholar macrophage infiltration was enhanced in the kidneys of the MLK3-transferred mice (Figure 6d). Although it was difficult to identify the TCRα chain of MLK3 by flow cytometry or immunofluorescent staining, the TCRα chain sequence of MLK3 was detected by PCR in the spleens and kidneys of MLK3-transferred mice at 26 weeks of age (Figure 6e). The TCRα chain sequence of MLK3 was not detected in the lungs or inguinal lymph nodes of the MLK3-transferred mice, which was suggestive of the kidney-specific accumulation of MLK3-transduced cells. In contrast to the progression of nephritis, the IgG anti-dsDNA antibody titers of the three groups were not significantly different (Figure 6f). Moreover, the total IgG, IgG2a, and IgG3 levels of the three groups were not significantly different (Figure 6g). These results suggest that MLK3-expressing CD4+ T cells significantly enhance nephritis progression without affecting anti-dsDNA antibody production. We demonstrated the nephritis-promoting effect of a kidney-infiltrating CD4+ T-cell clone in lupus-prone mice. Although it is difficult to isolate and culture cytokine-expressing clones that have infiltrated a parenchymatous organ, a combination of single-cell sorting and TCR reconstitution could verify the pathological role of kidney-infiltrating CD4+ T cells in lupus-prone mice. In experimental autoimmune encephalomyelitis, different levels of autoreactivity were produced by activating autoreactive T cells in the central nervous system.32.Kawakami N. Lassmann S. Li Z. et al.The activation status of neuroantigen-specific T cells in the target organ determines the clinical outcome of autoimmune encephalomyelitis.J Exp Med. 2004; 199: 185-197Crossref PubMed Scopus (151) Google Scholar In the aforementioned study, highly pathogenic T cells were significantly activated within the central nervous system to express IFN-γ, and our result is consistent with this observation. However, autoreactivity and IFN-γ expression are not sufficient for pathogenicity, because only MLK3 was found to be pathogenic in our analysis. The reasons for the differential effects of MLK2 and MLK3 are not clear. Both the MLK2- and MLK3-transduced CD4+ T cells failed to recognize nucleosomes and displayed autoreactivity that was not related to autoimmune inflammation because they exhibited similar degrees of proliferation to DCs from MRL/lpr, MRL/+, and haplotype-matched non-lupus-prone C3H mice (data not shown). MLK2 and MLK3 might recognize autoantigens that are irrelevant to humoral autoimmunity, because the transfer of MLK2- and MLK3-expressing T cells did not increase anti-dsDNA antibody titers or the amount of total IgG. Our observation that MLK3-transduced CD4+ T cells enhanced the deposition of C3 without enhancing humoral immunity is worth noting. IFN-γ was suggested to contribute to enhanced immune complex deposition in previous studies, which found that the deposition of IgG and C3 was decreased in the glomeruli of MRL/lpr mice lacking the IFN-γ receptor11.Haas C. Ryffel B. Le Hir M. IFN-gamma is essential for the development of autoimmune glomerulonephritis in MRL/Ipr mice.J Immunol. 1997; 158: 5484-5491PubMed Google Scholar or IL-12p40.30.Kikawada E. Lenda D.M. Kelley V.R. IL-12 deficiency in MRL-Fas(lpr) mice delays nephritis and intrarenal IFN-gamma expression, and diminishes systemic pathology.J Immunol. 2003; 170: 3915-3925Crossref PubMed Scopus (135) Google Scholar The more marked decrease in C3 deposition than in IgG deposition observed in these mice might be in accordance with our results. It was reported that IFN-γ induces the expression of the high-affinity receptor for IgG.33.Cassatella M.A. Bazzoni F. Calzetti F. et al.Interferon-gamma transcriptionally modulates the expression of the genes for the high affinity IgG-Fc receptor and the 47-kDa cytosolic component of NADPH oxidase in human polymorphonuclear leukocytes.J Biol Chem. 1991; 266: 22079-22082Abstract Full Text PDF PubMed Google Scholar Therefore, we speculate that IFN-γ produced by MLK3-transduced cells might promote kidney inflammation and Fc-receptor expression to augment immune complex deposition. However, the precise mechanism responsible for the MLK3-induced augmentation of C3 deposition should be investigated further. We selected CD5 and TCR expression levels as indicators of autoreactive cytokine-secreting T cells. However, several major T-cell clones from the MRL/lpr kidney were not detected in the single cell–sorted Vα2highCD5highCD4+ T-cell populations. It is not clear whether the major clones in the MRL/lpr kidney are stimulated in situ and express pathogenic cytokines. The high expression levels of CD5 and TCRs did not correlate with strong clonal expansion in the kidney (Figure 3b), and there is a possibility that another combination of markers could be used to identify the T-cell clones that expand most and display the highest cytokine expression. Nevertheless, our results indicate that CD5 and TCRs are useful markers for identifying autoreactive pathogenic clones. In previous studies, several antigen-specific TCRs were obtained from hybridomas stimulated with antigen in vitro, such as myelin basic protein-specific TCR,34.Gold D.P. Offner H. Sun D. et al.Analysis of T cell receptor beta chains in Lewis rats with experimental allergic encephalomyelitis: conserved complementarity determining region 3.J Exp Med. 1991; 174: 1467-1476Crossref PubMed Scopus (142) Google Scholar nucleosome-specific TCR,3.Mohan C. Adams S. Stanik V. et al.Nucleosome: a major immunogen for pathogenic autoantibody-inducing T cells of lupus.J Exp Med. 1993; 177: 1367-1381Crossref PubMed Scopus (630) Google Scholar and Type II collagen-specific TCR.35.Osman G.E. Toda M. Kanagawa O. et al.Characterization of the T cell receptor repertoire causing collagen arthritis in mice.J Exp Med. 1993; 177: 387-395Crossref PubMed Scopus (119) Google Scholar Although T cells with these TCR accumulate at inflammatory sites upon adoptive transfer,36.Nakajima A. Seroogy C.M. Sandora M.R. et al.Antigen-specific T cell-mediated gene therapy in collagen-induced arthritis.J Clin Invest. 2001; 107: 1293-1301Crossref PubMed Scopus (169) Google Scholar,37.Costa G.L. Sandora M.R. Nakajima A. et al.Adoptive immunotherapy of experimental autoimmune encephalomyelitis via T cell delivery of the IL-12 p40 subunit.J Immunol. 2001; 167: 2379-2387Crossref PubMed Scopus (160) Google Scholar it is not known whether they reflect the true nature of autoantigen-specific TCRs at in vivo inflammatory sites. Using our method, we were able to address the pathological contribution of organ-infiltrating T cells to inflammatory diseases. In summary, we have identified pathogenic autoreactive TCRs from the kidneys of early nephritic MRL/lpr mice via single-cell analysis. Our results suggest that IFN-γ-expressing CD4+ T cells in the inflamed kidneys of MRL/lpr mice contribute to nephritis progression. Our methods will enable us to analyze the characteristics of inflammatory T cells that have infiltrated parenchymatous organs. MRL/lpr and MRL/+ mice were purchased from Japan SLC (Shizuoka, Japan). All animal experiments were conducted in accordance with the relevant institutional and national guidelines. Fc block (anti-CD16/CD32) and the following purified mAbs were purchased from BD Bioscience (San Jose, CA): FITC-conjugated anti-CD4 (L3T4), allophycocyanin-conjugated anti-CD4, PE-conjugated anti-Vα2 (B20.1), FITC-conjugated anti-Vβ10(B21.5), FITC-conjugated anti-CD5 (53-7.3), PE-conjugated anti-CD
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