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Signaling and Promote Autoimmune Myocarditis1
* Molecular Biomedicine, Swiss Federal Institute of Technology, Zurich-Schlieren, Switzerland;
Institute of Virology and Antiviral Therapy, Klinikum, Friedrich Schiller University Jena, Jena, Germany; and
Department of Pathology, University Hospital, Zurich, Switzerland
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Abstract |
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(PKC-), plays a central role in the activation and differentiation of Th2 cells while being redundant in CD4+ and CD8+ antiviral responses. Recent evidence indicates that PKC- may however be required for some T cell-driven autoimmune responses. We have investigated the role of PKC- in the induction of autoimmune myocarditis induced by either Coxsackie B3 virus infection or immunization with 
-myosin/CFA (experimental autoimmune myocarditis (EAM)). PKC--deficient mice did not develop EAM as shown by impaired inflammatory cell infiltration into the heart, reduced CD4+ T cell IL-17 production, and the absence of a myosin-specific Ab response. Comparatively, PKC- was not essential for both early and late-phase Coxsackie virus-induced myocarditis. We sought to find alternate pathways of immune stimulation that might reconcile the differential requirements for PKC- in these two disease models. We found systemic administration of the TLR ligand CpG restored EAM in PKC--deficient mice. CpG could act directly upon TLR9-expressing T cells to restore proliferation and up-regulation of Bcl-xL, but exogenous IL-6 and TGF-
was required for Th17 cell differentiation. Taken together, these results indicate that TLR-mediated activation of T cells can directly overcome the requirement for PKC- signaling and, combined with the dendritic cell-derived cytokine milieu, can promote the development of autoimmunity. |
Introduction |
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28 days and is followed by a second T cell-driven (10) chronic phase of the disease, which occurs around day 35 p.i. (11).
A number of investigations have examined the roles of inflammatory cytokines in the development of myocarditis (12, 13, 14, 15, 16), and similarly, the respective roles of CD4+ and CD8+ T cells in disease pathogenesis have been assessed (8, 17, 18, 19). However, to date, the important signaling cascades underlying T cell function in this disease remain unclear. One key molecule involved in the development of T cell proliferation and effector function is protein kinase C- (PKC-) (20, 21). PKC- is a serine/threonine-specific protein kinase, which is primarily expressed in T cells and is recruited to the centre of the immunological synapse upon TCR and CD28 signaling (22, 23, 24). T cells genetically deficient in PKC- have impaired IL-2 production and as a result proliferate poorly in vitro (20). However, the in vivo implications of the absence of PKC- are less clear. Protective Th1 responses against Leishmania major develop normally in PKC--deficient mice, so too can CD8+ T cell effector and memory responses (25), however, Th2 immune responses against infection with the helminth parasite Nippostrongylus brasiliensis (26) or allergic responses against inhaled OVA protein are strikingly impaired (26, 27). A series of recent studies have shown that PKC- is required for experimental autoimmune encephalomyelitis (EAE) and arthritis (28, 29), indicating that PKC- may have differential roles in the induction of Th1, Th2, and Th17 immune responses.
We have investigated the importance of PKC- during the development of EAM and CVB3 virus-induced myocarditis. Surprisingly, while the disease pathogenesis is similar in these two models, the respective requirements for PKC- signaling in its development were distinct. When EAM was induced by immunizing mice with myosin peptide in CFA, mice deficient in PKC- failed to develop IL-17-producing CD4+ T cells and, consequently, disease. Comparatively, both acute and chronic myocarditis developed after CVB3 infection, irrespective of the absence of PKC-. We show that the presence of systemic TLR ligands was sufficient to overcome the absence of PKC-, as concurrent administration of CpG restored EAM induction in vivo. Notably, CpG could act directly upon T cells to increase proliferation and expression of Bcl-xL in the absence of PKC-. These data show that TLR signaling directly upon T cells can overcome what would otherwise be a critical requirement for PKC- in T cell activation and the development of autoimmunity.
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Materials and Methods |
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C57BL/6 and BALB/c wild-type mice were obtained from Charles River Laboratories or Laboratory Animal Center, Friedrich Schiller University (Jena, Germany). PKC--deficient mice were generated as described (20) and backcrossed >15 generations onto the C57BL/6 background and more than six generations onto the BALB/c background. Mice were maintained specific pathogen-free in BioSupport and Laborhaus Virologie animal facilities in isolated ventilated cages. Animals used in experiments were between 8 and 10 wk of age. CVB3 (Nancy strain) was provided by Prof. R. Kandolf (Department of Molecular Pathology, University Hospital, Tuebingen, Germany) and propagated six times in HeLa cells. All animal experimental procedures were approved by local animal committees (Zurich and Jena).
Induction of EAM and histological assessment
A murine heart muscle specific peptide derived from -myosin H chain (Ac-RSLKLMATLFSTYASADR-OH) was used as Ag. Bold letters indicate the arginine that was included at both ends of the -myosin H chain peptide to increase solubility. The peptide (purity 85%; ANAWA Biochemical Services and Products) was dissolved in CFA (Difco) and emulsified 1/1 with PBS. Mice were immunized s.c. with 100 µg/0.2 ml on days 0 and 7. Sham-immunized controls were injected with CFA emulsified with PBS alone. In the indicated experiments, 10 nM 1668pt CpG oligonucleotides (5'-TCC ATG ACG TTC CTG AAT AAT-3'; Microsynth) were injected i.p. on days –1, 0, 1 and 6, 7, 8. Fourteen days after the second immunization, mice were sacrificed and their hearts were removed, fixed in 4% neutral buffered formalin, and processed for H&E staining. The glass slides were coded and evaluated by a pathologist. For diagnosis of myocarditis, an inflammatory infiltrate forming foci between muscle fibers or surrounding individual myocytes, with or without associated myocyte necrosis or apoptosis, was considered essential. A vague increase in interstitial cellularity was not considered sufficient for diagnosis. Myocarditis was scored on a semiquantitative scale using grades from 0 to 4 (0, no inflammatory infiltrates; 1, small foci of inflammatory cells between myocytes or inflammatory cells surrounding individual myocytes; 2, larger foci of 100 inflammatory cells or involving 30 myocytes; 3, 10% of a myocardial cross-section involved; and 4, 30% of a myocardial cross-section involved).
Induction of CVB3-induced myocarditis, histological assessment, and determination of organ virus titer
Adult 8- to 13-wk-old male BALB/c and PKC--deficient mice were inoculated i.p. with 104 PFU of CVB3 and sacrificed under ether narcosis as indicated at day 7, 10, or 35 p.i. For histological staining, halves of murine hearts were treated as reported previously (30). For each myocardial sample, histological indicators of myocarditis were semiquantitatively graded according to the extent of cellular infiltration/fibrosis on a four-point scale ranging from 0 (no infiltration, no fibrosis) to 3 (strong infiltration, strong fibrosis) (30). A zero score indicated no presence of infiltrated cells or lesions/no fibrosis in the myocardium. A score 1 described a limited focal distribution of myocardial damage (two small or one large lesion)/mild fibrosis. Scores 2 and 3 described intermediate severity with multiple lesions or disseminated infiltrations over the entire examined heart tissue/moderate or diffuse fibrosis. The CVB3 titer of the heart tissue was determined on HeLa cells (30) by the TCID50 assay method according to Reed and Muench (31). The resulting titers were expressed as log TCID50 per 100 mg of wet heart tissue.
ELISA
Serum levels of cardiac myosin-specific IgG Abs were measured by ELISA on plates coated with 5 µg/ml total cardiac myosin (Sigma-Aldrich). Sera were serially diluted on coated plates as indicated. After 2 h, plates were washed extensively and incubated with HRP-labeled anti-mouse IgG1 (BD Pharmingen) or anti-mouse IgG2a (Jackson ImmunoResearch Laboratories) Abs for 2 h until addition of substrate. OD was measured at 450 nm.
Assessment of TLR expression
CD4+ T cells were sorted by MACS (Miltenyi Biotec) followed by sorting for CD4+CD62Lhigh cells by flow cytometry. RNA was prepared with TriReagent (Molecular Research Center) and treated with DNase (Invitrogen Life Technologies) to avoid genomic DNA contamination before RNA was reverse transcribed to cDNA (Invitrogen Life Technologies). TLR expression was quantified by real-time PCR (I-cycler; Bio-Rad) and samples were normalized to RNA polymerase II (RPII) expression levels. Primers used were: RPII forward (f), 5'-GCT TGG TTT AAT CCC CCT CA-3'; RPII reverse (r), 5'-CTT CAT TGC ACC TCA CAT CG-3'; TLR3f, 5'-GCG TTG CGA AGT GAA GAA CT-3'; TLR3r, 5'-AGG GCG AAT AAC TTG CCA AT-3'; TLR4f, 5'-TCA GAA CTT CAG TGG CTG GA-3'; TLR4r, 5'-CCT GGG GAA AAA CTC TGG AT-3'; TLR7f, 5'-CCA CCA GAC CTC TTG ATT CC-3'; TLR7r, 5'-CCA TCG AAA CCC AAA GAC TC-3'; TLR9f, 5'-CAT GGA CGG GAA CTG CTA CT-3'; TLR9r, 5'-GGC ACC TTT GTG AGG TTG TT-3'.
T cell proliferation and cytokine production
Total CD4+ T cells were isolated from spleen single-cell suspensions by MACS (Miltenyi Biotec) following the manufacturer’s instructions, and incubated with a final concentration of 2.5 mM CFSE (Molecular Probes) for 7 min, followed by extensive washing in medium. Cells were cultured in anti-CD3 coated 96-well flat-bottom plates at a concentration of 2 x 105 cells/well for 3 days. In the indicated samples, 100 nM CpG, 20 ng/ml IL-6, 5 ng/ml recombinant human TGF- (Sigma-Aldrich), or 1 x 105 CD11c+ splenic DC were included. For cytokine analysis, cells from the in vitro culture or cells isolated from perfused and collagenase-digested hearts were stimulated with 10–7 M PMA and 1 µg/ml ionomycin for 4 h at 37°C in IMDM medium. For the final 2 h, 10 µg/ml brefeldin A was added to the cultures to retain cytokines in the cytoplasm.
FACS analysis
Cells were stained as described previously (32). Abs used were Bcl-xL-PE (Abcam), TNF--FITC (eBioscience), TLR9-biotin (eBioscience), and IL-17-biotin (BD Pharmingen) followed by a secondary step with either streptavidin-PerCP or streptavidin-allophycocyanin. Cells were washed and analyzed by flow cytometry (FACSCalibur; BD Biosciences) and Flowjo software (Tree Star).
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Results |
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-deficient mice fail to develop EAM
It has previously been shown that PKC--deficient mice, on either BALB/c or C57BL/6 strain backgrounds, develop normal Th1 immune responses, and are protected against chronic disease development following infection with L. major (26). More recently, autoimmune diseases including EAE (28, 29) and arthritis (33) have been shown to require PKC- for their development, although these diseases now appear to be more dependent upon Th17 as opposed to Th1 cells for their progression. We sought to assess whether these findings were applicable to the development of EAM–a disease similarly dependent upon Th17 cells (12, 34). Accordingly, mice were immunized with myosin peptide in CFA on days 1 and 7, and on day 21 were sacrificed and hearts taken for histological analysis. Although disease development occurred in wild-type BALB/c mice, negligible disease induction was evident in PKC--deficient mice (Fig. 1A). The absence of disease was reflected both in the significantly reduced prevalence and severity grade of the clinical score (Fig. 1, A and B). Histological examination revealed the infiltrate in BALB/c mice consisted primarily of macrophages and lymphocytes, with some granulocytes also present (Fig. 1C), whereas the infiltrate in the PKC--deficient mice was strikingly reduced (Fig. 1D). We next assessed the development of myosin-specific Ab isotypes. Myosin-specific IgG2a and IgG1 were both evident in serum of wild-type mice, however, Ab levels were below the limits of detection in the PKC--deficient mice (Fig. 1E). The absence of a robust Ab response in the PKC--deficient mice is supportive of an overall impairment in the CD4+ Th cell response as opposed to a Th1/Th2 switch in cytokines profiles. Taken together, these data show that PKC- signaling is critical for the development of EAM.
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is not required for CVB3-induced myocarditis and cardiac fibrosis
The data in Fig. 1 was supportive of recent studies showing a requirement for PKC- in EAE and arthritis (28, 29, 33), however, was surprising given that immune responses against L. major and viruses are normal in the absence of PKC- (25, 26). To assess whether the importance of PKC- was a general characteristic of autoimmune disease induction, we infected mice with CVB3, a virus commonly used to induce myocarditis in susceptible mouse strains. Although the technique of inducing myocarditis in EAM and CVB3 infection is distinct, the disease pathogeneses share many similarities (35, 36), and importantly, are T cell mediated (10). Susceptible BALB/c wild-type mice and PKC--deficient mice on a BALB/c background were infected with CVB3 virus and the development of acute myocarditis was assessed on day 7 p.i. In contrast to the EAM model, wild-type and PKC--deficient mice exhibited comparable CVB3 virus titers (Fig. 2A) and disease severity (Fig. 2B) indicating PKC- signaling was not required for initial disease induction. We next assessed the chronic phase of CVB3-induced myocarditis, and in line with the acute phase disease, both PKC--deficient and wild-type mice exhibited comparable severity of myocarditis (Fig. 2C) and cardiac fibrosis at day 35 p.i. (Fig. 2D). Taken together, these data indicate that the crucial role of PKC- in peptide/adjuvant induced EAM, can be overcome during CVB3 virus infection leading to the development of myocarditis.
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in the development of EAM
Considering responses against L. major (26), influenza virus, vaccinia virus, virus-like particles, lymphocytic choriomeningitis virus (25), and CVB3-induced autoimmune responses do not appear to require PKC- for their development, an additional factor specific to such infections must play a role. We hypothesized that TLR ligands present during all of these infections may bypass the central requirement for PKC- in T cell responses. Accordingly, we immunized mice with myosin peptide in CFA, and gave CpG i.p. on the day before, of and after each immunization. Two weeks after the final immunization, hearts were taken and assessed for myocarditis. Myocarditis was evident in wild-type mice that received myosin in CFA, in the presence or absence of CpG, and in line with prior data PKC--deficient mice failed to develop disease given the standard immunization protocol (Fig. 3, A–C). Interestingly, however, when CpG was administered during the myosin/CFA immunization period, PKC--deficient mice exhibited a comparable disease severity and prevalence to wild-type mice (Fig. 3, A and B). Histological analysis showed that administration of CpG concurrently with myosin in CFA induced a comparable macrophage and lymphocyte infiltrate in BALB/c and PKC--deficient mice (Fig. 3D). Cells isolated from perfused and digested hearts were restimulated in vitro and cytokine production was assessed by FACS. The proportion of CD4+ T cells producing IL-17 (Fig. 3E) and TNF- (Fig. 3F) was strikingly reduced in PKC--deficient mice, consistent with the reduced cellular infiltrate overall. Similar to the disease score and prevalence, both IL-17 and TNF- production were restored to wild-type levels when CpG had been administered in vivo (Fig. 3, E and F). No myocarditis was evident in mice administered with CpG in the absence of myosin/CFA at the time points analyzed in this study, nor was disease restored when CpG was included in the CFA/myosin emulsion (data not shown). Taken together, these data indicate that TLR ligands provide signals in vivo which bypass the otherwise critical role for PKC- in T cell activation and thus the development of autoimmune myocarditis.
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in T cell proliferation and survival
The myosin-CFA adjuvant emulsion is known to contain TLR ligands, but even when we supplemented this emulsion with CpG disease development was not restored in PKC--deficient mice. Considering that the CpG had to be given systemically, it was plausible that it might act directly on T cells in addition to its well-established role in driving DC maturation. We first examined whether wild-type and PKC--deficient CD4+ T cells expressed TLRs. CD4+CD62Lhigh cells were sorted from spleens of naive mice by flow cytometry, and TLR3, 4, 7, and 9 expression was determined by quantitative real-time PCR. Both wild-type and PKC--deficient T cells expressed TLR9 RNA transcripts (Fig. 4A) which was confirmed at a protein level by FACS analysis (Fig. 4B). We next isolated CD4+ T cells from naive wild-type or PKC--deficient mice by magnetic bead sorting and stimulated them with anti-CD3 in the presence or absence of CpG. As previously described, wild-type CD4+ T cells proliferated upon anti-CD3 stimulation, whereas PKC--deficient CD4+ T cells exhibited a striking impairment in proliferation (Fig. 4C). However, when the culture was supplemented with CpG, PKC--deficient T cells underwent extensive proliferation (Fig. 4C). These data were validated using highly purified CD4+CD62Lhigh cells supporting the conclusion that the CpG was acting upon T cells directly, rather than mediating its effect through contaminating DCs (data not shown). To further test this possibility, we supplemented the cultures with either wild-type or MyD88-deficient DCs and found that cultures containing DCs from either of these sources did not significantly influence CD4+ T cell proliferation upon addition of CpG (Fig. 4D).
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-deficient cells have been shown to exhibit impaired survival. We thus assessed survival by propidium iodide and annexin staining and found that CpG-treated PKC--deficient cells which had entered cell cycle exhibited comparable levels of apoptosis as untreated wild-type cells (Fig. 5A). In addition, CpG treatment enhanced expression of the antiapoptotic molecule Bcl-xL, which was recently shown to be regulated by PKC- signaling (Fig. 5B) (37). As IL-17 has been described to be the primary effector cytokine in EAM and EAE, and is restored by CpG treatment in vivo, we assessed its production under these in vitro culture conditions. Wild-type CD4+ T cells produced IL-17 upon addition of exogenous IL-6 and TGF-, whereas PKC--deficient cells required IL-6, TGF- and CpG in combination (Fig. 5C). The addition of CpG, IL-6, or TGF- independently resulted in nominal IL-17 production by wild-type and PKC--deficient cells. Overall, while TLR ligand stimulation of PKC--deficient T cells can restore survival and proliferation, differentiation into Th17 cells requires additional signals including IL-6 and TGF- (38).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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plays in the activation of T cells in vitro, however, mounting in vivo evidence now shows that although PKC- may take center stage in the immunological synapse, other T cell activation pathways can circumvent its role. In the current study, we have assessed the requirements for PKC- signaling in the development of autoimmune myocarditis. Surprisingly, we found that PKC- was essential for T cell activation when specific Ag was administered in adjuvant, however, in the presence of soluble CpG or a viral infection, PKC- signaling was no longer required.
It was recently shown that administration of an OX40 agonistic Ab failed to restore the induction of EAE in PKC--deficient mice (29), indicating that at least this pathway of costimulation is not likely to be involved in our system. A further study has shown that PKC--deficient CD4+ T cells exhibit reduced up-regulation of LFA-1, which may influence the ability of these cells to enter sites of inflammation (28). Indeed, such impaired maturation of CD4+ T cells may be linked to the reduced activation and proliferation exhibited by PKC--deficient T cells in the absence of direct TLR stimulus (Fig. 4). Although in our experimental system, high Ag load provided by a replicating virus did not appear to be the central mechanism to bypass PKC- signaling, such a mechanism may be important in some systems. Evidence supporting this comes from in vitro proliferation assays, where PKC--deficient T cells were shown to proliferate to a similar degree to wild-type T cells upon stimulation with a 10- to 100-fold higher Ag concentration. We consider the most likely mechanism restoring PKC--deficient T cell responses in vivo to be direct TLR stimulation on T cells (overcoming the proliferation and survival defects), and the presence of DC-derived inflammatory cytokines (ensuring appropriate differentiation e.g., Th17). Surprisingly, CFA, generally considered a strong TLR ligand-containing adjuvant, was not adequate to overcome the defect in PKC- signaling during the induction of EAM. Given that CpG can act directly on T cells, the s.c. location of the CFA/peptide may limit the exposure of T cells to TLR ligands directly, thus not overcoming the PKC- defect. Notably, when CpG was emulsified in the CFA/myosin mixture, disease was not restored in the PKC--deficient mice, supporting the conclusion that the ligand acts on T cells directly. In line with this, Yang et al. (39) have shown that persistent TLR signals are required to overcome T cell tolerance. Lang et al. (40) recently demonstrated that TLR ligands could act directly on islet cells to up-regulate MHC I and enhance autoimmunity. Given that in our system the defect appears to be T cell proliferation in the absence of PKC- and that CpG can restore this defect when given directly to T cells, up-regulation of MHC class I in the heart by CpG is unlikely to be the primary mechanism in our system.
Myocarditis cannot strictly be defined as a Th1 disease, in fact, in some mouse strains the disease has been shown to exhibit Th2 characteristics. Afanasyeva et al. (41) showed that EAM development in A/J mice was dependent on IL-4 production and exacerbated upon blockade of IFN-
. Comparatively, IL-4R–/– mice on a BALB/c background have no defect in the development of EAM (14). As PKC--deficient mice exhibit impaired Th2 immune responses in vivo (26, 27), one possibility was that this defective Th2 cell development was responsible for the requirement for PKC- in EAM. However, considering PKC--deficient T cells have impaired proliferation in vivo upon immunization with CFA/peptide (data not shown), it is more likely that reduced expansion of myosin-specific cells is the mechanism of protection. Two recent studies have shown that in fact IL-17 is the major effector cytokine in EAM (12, 34) and indeed we found impaired IL-17 production in the absence of PKC-, as has recently been reported in models of EAE (28, 29). Thus far, a role for IL-4 in Th17 differentiation has not been described, but rather IL-6 and TGF- appear to be important (38).
TLR-mediated activation of DCs has become a central paradigm in immunology. The importance of TLR signaling has now been extended to B cells, as direct TLR stimulation was shown to induce B cell proliferation and IgG production (42). Culminating evidence also shows a role for TLR stimulation of T cells (43, 44). Gelman et al. (45) recently showed that CpG acts through a MyD88 and PI3K-dependent mechanism, which when considered in light of our current data, is the most likely PKC--independent pathway underlying the restoration of myocarditis by CpG.
Taken together with other recent investigations, these data highlight a further level of complexity and redundancy that has evolved for T cell activation. In the absence of PKC- signaling, Th2 and Th17 immune responses fail to develop while upon stimulation with replicating viruses or intracellular parasites, PKC- is rendered superfluous. One central parameter overcoming the absence of PKC- appears to be activation of both T cells and DCs by TLR ligands. Notably, although TLR ligands may support T cell proliferation and survival, the nature of the T cell response is still controlled by the local cytokine milieu. The differential requirements for PKC- in inducing T cell responses make it an attractive target for therapy. Although inhibiting PKC- signaling is unlikely to markedly influence the development of strong Th1/Tc1 reactions, such as antiviral responses, it may influence the induction of responses such as Th2 responses/allergy and Th17-mediated diseases. The therapeutic potential of targeting PKC- in autoimmune diseases is unclear. In this study, we show that induction of autoimmune myocarditis by Ag/adjuvant immunization requires PKC-; however, interfering with PKC- signaling during CVB3 infection is unlikely to influence the outcome of disease. Considering CVB3 infection is strongly associated with the development of myocarditis and dilated cardiomyopathy in humans, targeting of PKC-, in this case, may not be a valid therapeutic option.
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Disclosures |
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1 This work was supported by Swiss National Foundation Grant 3100A0-100233/1. 
2 Address correspondence and reprint requests to Dr. Manfred Kopf, Molecular Biomedicine, Swiss Federal Institute of Technology, Wagistrasse 27, CH8952 Zurich-Schlieren, Switzerland; E-mail address: Manfred.Kopf{at}ethz.ch or Dr. Benjamin J. Marsland, Molecular Biomedicine, Swiss Federal Institute of Technology, Wagistrasse 27, CH8952 Zurich-Schlieren, Switzerland; E-mail address: marsland{at}env.ethz.ch
3 Abbreviations used in this paper: CVB3, Coxsackie virus B3; EAM, experimental autoimmune myocarditis; EAE, experimental autoimmune encephalomyelitis; PKC-,protein kinase C-; p.i., postinfection; DC, dendritic cell; RPII, RNA polymerase.
Received for publication August 22, 2006. Accepted for publication January 1, 2007.
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B. C. Mercier, A. Cottalorda, C.-A. Coupet, J. Marvel, and N. Bonnefoy-Berard TLR2 Engagement on CD8 T Cells Enables Generation of Functional Memory Cells in Response to a Suboptimal TCR Signal J. Immunol., February 15, 2009; 182(4): 1860 - 1867. [Abstract] [Full Text] [PDF]
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M. Fukata, K. Breglio, A. Chen, A. S. Vamadevan, T. Goo, D. Hsu, D. Conduah, R. Xu, and M. T. Abreu The Myeloid Differentiation Factor 88 (MyD88) Is Required for CD4+ T Cell Effector Function in a Murine Model of Inflammatory Bowel Disease J. Immunol., February 1, 2008; 180(3): 1886 - 1894. [Abstract] [Full Text] [PDF]
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1) in PKC-
