A Motif in the V3 Domain of the Kinase PKC-U Determines Its Localization in the Immunological Synapse and Functions in T Cells Via Association with CD28

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A motif in the V3 domain of the kinase PKC-u determines its localization in the immunological synapse and functions in T cells via association with CD28

Kok-Fai Kong1, Tadashi Yokosuka2,3, Ann J Canonigo-Balancio1, Noah Isakov4, Takashi Saito2,5 & Amnon Altman1

Protein kinase C-u (PKC-u) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. Here we found that the V3 domain of PKC-u was necessary and sufficient for localization to the immunological synapse mediated by association with the coreceptor CD28 and dependent on the kinase Lck. We identified a conserved proline-rich motif in V3 required for association with CD28 and immunological synapse localization. We found association with CD28 to be essential for PKC-u-mediated downstream signaling and the differentiation of T helper type 2 cells (TH2 cells) and interleukin 17–producing helper T cells (TH17 cells) but not of T helper type 1 cells (TH1 cells). Ectopic expression of V3 sequestered PKC-u from the immunological synapse and interfered with its functions. Our results identify a unique mode of CD28 signaling, establish a molecular basis for the immunological synapse localization of PKC-u and indicate V3-based ‘decoys’ may be therapeutic modalities for T cell–mediated inflammatory diseases.

15–17 The induction of an immune response depends on effective communica- T cells (TH17 cells) require PKC-θ. In contrast, TH1 immune tion between antigen-specific T cells and antigen-presenting cells (APCs). responses to intracellular pathogens such as Leishmania major13, When a T cell expressing a cognate T cell antigen receptor (TCR) encounters as well as antiviral effector and memory cytotoxic T lymphocyte an activated APC, both cells actively redistribute their receptors and ligands responses, remain relatively intact in Prkcq−/− mice18–21. Consistent to the interface, creating a platform for effective signaling known as the with those in vivo findings, Prkcq−/− CD4+ T cells show impaired immunological synapse. At steady state, the mature immunological synapse in vitro differentiation into the TH2 and TH17 lineages, whereas TH1 is composed of concentric rings with a central core (the central supra differentiation is only slightly lower13–15. PKC-θ has been found to molecular activation cluster (cSMAC)) containing clusters of TCRs and cos- be required for allograft rejection and graft-versus-host disease but timulatory molecules, and a peripheral ring (the peripheral supramolecular not for the graft-versus-leukemia response in mice22. activation cluster (pSMAC)) of adhesion molecules1. The engagement of Although the antigen-induced localization of PKC-θ to the immuno these surface molecules triggers signaling cascades that result in the recruit- logical synapse and, more specifically, to the cSMAC is well established3,4, ment of intracellular proteins, including kinases, adaptors and cytoskeletal the molecular basis for this highly selective localization is not clear, nor is it proteins, to the immunological synapse2. One of the most prominent proteins known whether this localization is required for the signaling functions of to be recruited to the immunological synapse after antigen stimulation is the PKC-θ in T cells. Here we identified and characterized the hinge region of kinase PKC-θ, whose localization is limited to the cSMAC3,4. PKC-θ, known as the V3 domain, as having a critical role in determining PKC-θ is a member of the novel Ca2+-independent PKC subfamily its localization to the immunological synapse and cSMAC through binding expressed predominantly in T cells that has important and nonredundant to the coreceptor CD28 and, consequently, dictating its signaling from the roles in T cell activation and survival (but not in T cell development)5–7, immunological synapse. Fine mapping further identified an evolutionarily which reflects its unique ability to activate the transcription factors conserved proline-rich motif required for association with CD28, localiza- NF-κB, AP-1 and NFAT5,8–12. Studies of PKC-θ-deficient (Prkcq−/−) tion to the cSMAC and PKC-θ-mediated functions. Notably, the isolated mice have characterized the importance of PKC-θ in various dis- V3 domain of PKC-θ acted as a decoy in blocking PKC-θ-dependent func- ease models and have shown that its requirement in various forms of tions, including TH2 and TH17 differentiation, but not TH1 differentia- immunity is quite selective and not absolute. Thus, T helper type 2 tion, and inflammation. Our findings indicate a unique signaling mode 13,14 (TH2) responses to allergens or helminth infection and autoim- of CD28 and establish the molecular basis for the specialized localization mune diseases mediated by interleukin 17 (IL-17)-producing helper and function of PKC-θ in antigen-stimulated T cells.

1Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA. 2Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan. 3Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan. 4The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, Be’er Sheva, Israel. 5Laboratory of Cell Signaling, World Premiere International Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan. Correspondence should be addressed to A.A. ([email protected]).

Received 4 April; accepted 29 August; published online 2 October 2011; doi:10.1038/ni.2120

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– OVA + OVA a b IS No IS d Vector WT PKC-θ+δV3 PKC-GFP PKC-GFP * Talin + AF647 Talin + AF647 50 * APC (CMAC) DIC APC (CMAC) DIC No stim 10.3 11.6 13.0 40 30

20 200 α-CD3 + WT Conjugates 10 α-CD28 100 19.8 62.4 34.1 0 0 WT V3 V3 0 1 2 3 4 ∆ δ Events 10 10 10 10 10 – + θ θ CD69 PKC- PKC- e PKC-θ– ∆V3 No stim c 20 * No stim * 5.1 9.6 7.3 y + MCC ) 4 15 RLU

4 10 200 α-CD3 + PKC-θ+ 10 ×

( -CD28 100 V3 5 α 2.0 70.4 27.5

δ RE/AP activit 0 0 0 1 2 3 4 Vector WT PKC- + 10 10 10 10 10 PKC-θ– θ CD25 ∆V3 δV3 IB: Xpress 200 Requirement of PKC- V3 for localization to the immunological Figure 1 θ Cells IB: actin 100 synapse, cSMAC and signaling. (a) Immunofluorescence imaging of 4.3 53.1 54.8 Prkcq−/− OT-II CD4+ T cells infected with retrovirus expressing GFP-tagged 0 Events 0 1 2 3 4 wild-type (WT) PKC-θ, PKC-θ–∆V3 or PKC-θ+δV3 (PKC-GFP; green) and 10 10 10 10 10 PKC-θ mixed (1:1 ratio) with APCs labeled with the cell-tracking dye CMAC (APC (CMAC); blue), preincubated with (+OVA) or without (–OVA) OVA f KO WT g KO WT ) peptide; fixed conjugates were stained with anti-talin plus secondary 2 20 Vector PKC-θ+δV3 2.0 Vector PKC-θ+δV3 10 ×

Alexa Fluor 647–coupled antibody (Talin + AF647; red). DIC, differential ( 15 1.5 interference contrast. Original magnification, ×63. (b) Quantification of the localization of PKC-θ in (IS) or outside (No IS) the immunological synapse 10 1.0 poration and cSMAC in T cell–APC conjugates as described in a (n = ~40), limited -2 (ng/ml) 5 IL 0.5 to conjugates that had reorganized their talin and had detectable PKC-θ. H incor

3 0 0 *P < 0.05 (one-way analysis of variance (ANOVA)). (c) Luciferase activity in 0 1.25 2.5 5.0 0 1.25 2.5 5.0 MCC-specific T hybridoma cells transfected with empty vector or vector α-CD3 + α-CD28 (5 µg/ml) α-CD3 + α-CD28 (5 µg/ml) encoding PKC-θ tagged with the Xpress epitope, together with a luciferase reporter for the CD28 response element RE/AP and a β-galactosidase (β-Gal) reporter, then incubated for 6 h with DCEK fibroblasts expressing I-Ek and B7-1 in the presence (+ MCC) or absence (No stim) of MCC peptide. Results are presented in relative luciferase units (RLU), relative to β-Gal activity. Below, immunoblot analysis (IB) of PKC-θ expression, probed with anti-Xpress. *P < 0.05 (one-way ANOVA). (d,e) Expression of CD69 (d) or CD25 and PKC-θ (e) in GFP+ CD4+ T cells sorted from Rag1−/− mice reconstituted with Prkcq−/− bone marrow cells transduced with empty vector, wild-type PKC-θ or PKC-θ+δV3, left unstimulated or stimulated overnight with anti-CD3 plus anti-CD28. Numbers above bracketed lines indicate percent positive cells. (f,g) IL-2 production (f) and proliferation (g) of GFP+ CD4+ T cells isolated as in d, or T cells isolated from control, unreconstituted Prkcq−/− mice (KO), and left unstimulated or stimulated for 48 h with anti-CD3 plus anti-CD28. Data are representative of six experiments (a,b) or two experiments (d,e) or are from three (c) or two (f,g) experiments (mean and s.e.m. in c,f,g).

RESULTS Prkcq−/− CD4+ T cells with transgenic expression of the ovalbumin Importance of the V3 domain for PKC-u function (OVA)-specific OT-II TCR, wild-type PKC-θ localized to the center As a first step toward understanding the basis for unique antigen- of the immunological synapse (that is, the cSMAC) after stimulation induced PKC-θ localization to the immunological synapse and with APCs pulsed with OVA peptide (Fig. 1a,b), as evident from its cSMAC, we compared the amino acid sequences of PKC-θ and central localization relative to that of talin, a known pSMAC marker4. PKC-δ, its closest relative (with 62% identity and 75% homology) In contrast, PKC-θ–∆V3 did not translocate to the immunological syn- in the newly identified, Ca2+-independent PKC subfamily apse and instead remained largely cytosolic. Because V3 domain dele- (Supplementary Fig. 1). However, unlike PKC-θ, PKC-δ does not tion could cause a gross conformational change, we next generated an translocate to the immunological synapse after interaction between exchange mutant in which we replaced the native V3 domain of PKC-θ T cells and APCs3. Sequence alignment showed substantial diver- with the corresponding domain of PKC-δ (PKC-θ+δV3). Similar to gence between the V3 (hinge) domains of these two isoforms (amino PKC-θ–∆V3 mutants, this mutant also failed to translocate to the acids ~291–378 of human PKC-θ; Supplementary Fig. 1), which immunological synapse and the cSMAC (Fig. 1a,b). The localization of suggested that this region might have a role in targeting PKC-θ talin to the peripheral immunological synapse in T cells expressing both to the immunological synapse. This region has not been linked mutants indicated that the organization of a mature immunological before, to our knowledge, to the regulation of PKC-θ localization synapse was not grossly impaired in the absence of wild-type PKC-θ. or function other than its general role as a flexible hinge that allows These data suggest that the unique V3 domain of PKC-θ is required for PKC proteins to undergo a conformational change from a resting its selective localization to the immunological synapse and cSMAC. state into an ‘open’, active conformation23. The quality of T cell activation seems to correlate with the clustering To determine if the V3 domain is required for the localization of of PKC-θ in the immunological synapse4,24. However, direct evidence PKC-θ to the immunological synapse, we constructed mutant PKC-θ is missing that the localization of PKC-θ to the immunological synapse with deletion of V3 (PKC-θ–∆V3). When retrovirally transduced into and cSMAC is essential for its downstream functions. Therefore, we

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investigated whether loss or replacement of the PKC-θ V3 domain V3 ∆ V3 – δ a θ + impairs the activation of key transcription factors essential for pro- θ δ 5,8–12 Vector WT PKC- ductive T cell activation that are known targets of PKC-θ , specifi- WT PKC- PKC- Vector Myc-V3 PKC PKC Myc cally NF-κB, AP-1 and NFAT (Fig. 1c). Stimulation of cells transfected IP: CD28 IP: CD28 IP: CD28 CD28 CD28 with empty vector with peptide-pulsed APCs consistently resulted in CD28 PKC Myc minimal stimulation of reporter genes (discussed below), which most PKC Lysates GFP Lysates GFP probably reflected the weaker stimulus provided by peptide-pulsed Lysates Actin Actin GFP APCs than by saturating concentrations of antibody to (anti-CD3) Actin and anti-CD28, which are more commonly used in such reporter assays. T cells transfected with wild-type PKC-θ had significantly b V3 + AF488 CD28-CFP Merged DIC higher basal activity of a CD28 response element (RE/AP; Fig. 1c) and NFAT (Supplementary Fig. 2), which was even greater after stimulation with peptide-pulsed APCs. However, the activation of these reporter genes was completely abrogated in T cells transfected with PKC-θ–∆V3 or PKC-θ+δV3 (Fig. 1c and Supplementary Fig. 2). We further analyzed the ability of PKC-θ+δV3 to activate primary T cells by generating bone marrow chimeras in irradiated mice defi- Figure 2 The PKC-θ V3 domain is required and sufficient for CD28 −/− cient in recombination-activating gene 1 (Rag1 mice) reconstituted interaction. (a) Association of PKC-θ V3 with CD28 in Prkcq−/− CD4+ with Prkcq−/− bone marrow cells infected in vitro with bicistronic retro T cells infected with retrovirus expressing empty vector or vectors for viruses expressing green fluorescent protein (GFP) and wild-type PKC-θ or PKC-δ (above lanes), then restimulated for 5 min with anti- PKC-θ or PKC-θ+δV3. We analyzed the transduced (GFP+) T cells CD3 plus anti-CD28, followed by immunoblot analysis of lysates directly 8 weeks later. CD4+ T cells reconstituted with wild-type PKC-θ and (Lysates) or after immunoprecipitation (IP) with anti-CD28. (b) Imaging of Jurkat T cells cotransfected with Myc-tagged PKC-θ V3 plus cyan costimulated with anti-CD3 and anti-CD28 considerably upregulated fluorescent protein–tagged CD28 (CD28-CFP) incubated at a ratio of their expression of both CD69 and CD25, two activation markers 1:1 with Raji B cells pulsed with staphylococcus enterotoxin E; fixed 6 regulated by PKC-θ ; however, the ability of PKC-θ+δV3 to induce conjugates were stained with rabbit anti-Myc plus secondary Alexa Fluor the expression of CD69 or CD25 was ~50% lower (Fig. 1d,e). The 488–coupled antibody (AF488). Original magnification, ×63. Data are transduced cells had similar expression of both wild-type PKC-θ and representative of three experiments (a) or are from four experiments (b). PKC-θ+δV3 (Fig. 1e, bottom). Furthermore, in contrast to CD4+ T cells reconstituted with wild-type PKC-θ, which proliferated T cells that we transduced with various retroviruses expressing PKC-θ and produced IL-2 in response to stimulation with anti-CD3 and or PKC-δ. The ∆V3 mutant (Fig. 2a), as well as the PKC-θ+δV3 mutant anti-CD28 in a dose-dependent manner, T cells reconstituted with and wild-type PKC-δ (Fig. 2a, middle), did not immunoprecipitate PKC-θ+δV3 failed to proliferate and produce IL-2 (Fig. 1f,g), similar together with CD28. Thus, the V3 domain of PKC-θ is required for to CD4+ T cells from Prkcq−/− mice or T cells from bone marrow the inducible interaction with CD28. Moreover, the V3 domain was chimeras reconstituted with empty-vector. These data establish that sufficient for this interaction, as Myc-tagged V3 transduced via retro the V3 domain of PKC-θ is critical for the activation of the PKC-θ- virus immunoprecipitated together with endogenous CD28 from dependent TCR-CD28 signaling pathways that are important for T cell Prkcq−/− primary CD4+ T cells (Fig. 2a, right). The V3 domain also activation and, furthermore, that this is a nonredundant function that localized together with cyan fluorescent protein–tagged CD28 in cannot be replaced by the V3 domain of the closely related PKC-δ. the immunological synapse of cotransfected Jurkat T cells (Fig. 2b). Therefore, the PKC-θ V3 domain is necessary and sufficient for the The PKC-u V3 domain interacts with CD28 CD3-CD28–induced interaction of PKC-θ with CD28. We hypothesized that the importance of the PKC-θ V3 domain for the enzyme’s localization to the immunological synapse and function A proline-rich V3 motif is critical for recruitment of PKC-u reflects a critical association of the V3 domain with a ligand that recruits Inspection of the PKC-θ V3 domain showed a proline-rich motif it to the immunological synapse. Given the reported colocalization of that corresponded to amino acids 328–336 of human PKC-θ PKC-θ and CD28 in the immunological synapse25,26 and the phorbol (ARPPCLPTP; proline residues underlined). This motif was phylo- ester–induced association between the two26, we investigated whether genetically conserved, especially at the two internal proline residues, costimulation with anti-CD3 and anti-CD28 caused PKC-θ to associate in PKC-θ enzymes from many species (Supplementary Fig. 4) but with CD28. Indeed, PKC-θ immunoprecipitated together with CD28 was absent from the hinge domains of other PKC enzymes. Because from T cells stimulated with anti-CD3 and anti-CD28 (Supplementary proline-rich motifs are known to bind Src homology 3 (SH3) and Trp- Fig. 3a). We observed maximal association after 5 min of costimula- Trp (WW) domains28, we investigated whether this motif is important tion, and this association decreased thereafter but was still present after for the localization and function of PKC-θ by inserting it into the V3 up to 30 min of costimulation. To identify the PKC-θ region required domain of PKC-δ (which does not translocate to the immunologi- for this interaction, we transfected the Jurkat human T lymphocyte cell cal synapse). Stimulation of Prkcq−/− OT-II T cells with conjugated line with a series of PKC-θ deletion mutants (Supplementary Fig. 3b). OVA-pulsed APCs induced translocation of transduced wild-type After costimulation with anti-CD3 and anti-CD28, wild-type PKC-θ, PKC-θ and endogenous talin to the cSMAC and the pSMAC, respec- as well as a mutant with deletion of the N-terminal C2 domain shown tively, whereas transduced wild-type PKC-δ did not translocate to the before to negatively regulate the activation of PKC-θ27, immuno immunological synapse (Fig. 3a,b). However, when we introduced precipitated together with CD28 (Supplementary Fig. 3c). However, the proline-rich motif into PKC-δ (PKC-δ+θPR), it had localization deletion of the V3 domain abolished this interaction. Deletion of both to the immunological synapse similar to that of wild-type PKC-θ, the C2 and C1a domains of PKC-θ resulted in less interaction but did which suggested that the proline-rich motif in PKC-θ V3 can mediate not abolish it. We repeated this analysis with primary Prkcq−/− CD4+ localization to the immunological synapse and cSMAC.

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a – OVA + OVA b IS d No stim * No IS + MCC PKC-GFP PKC-GFP 40 * * Talin + AF647 Talin + AF647 10 * * APC (CMAC) DIC APC (CMAC) DIC 30 8 6 20 RLU) 4 4 10 × (

Conjugates 10 2 WT RE/AP activity 0 0 PKC-θ PKC-δ+ PKC-δ Vector WT PKC-δ PKC-δ θPR +θPR c α-CD3 + α-CD28 No stim + + δ -δ δ -δ PKC-δ+ L PR PR WT PKC- θ PKC WT PKC- θ PKC WC θPR PKC PKC IP: CD28 IP: CD28 CD28 * CD28 PKC PKC-δ PKC Lysates GFP Lysates GFP

Actin Actin

Figure 3 A proline-rich motif in the V3 domain of PKC-θ determines its localization to the immunological synapse and interacts with CD28. (a) PKC-θ localization in CD4+ T cells from OT-II transgenic mice infected with retrovirus expressing GFP-tagged wild-type PKC-θ, PKC-δ+θPR or wild-type PKC-δ (green), then stimulated, fixed, stained and analyzed as in Figure 1a. Original magnification, ×63. (b) Quantitative analysis of the results in a (as in Fig. 1a). *P < 0.05 (one-way ANOVA). (c) PKC-CD28 association in Prkcq−/− CD4+ T cells infected with retrovirus expressing wild-type PKC-θ, PKC-δ+θPR or wild-type PKC-δ, then restimulated for 5 min with anti-CD3 plus anti-CD28 (left) or left unstimulated (right). * indicates position of the immunoprecipitating antibody heavy chain. (d) Luciferase activity in MCC-specific T hybridoma cells cotransfected with the vectors in a together with reporter plasmids for RE/AP and β-Gal, then stimulated and assessed in Figure 1c. *P < 0.05 (one-way ANOVA). Data are representative of six experiments (a,b; mean and s.e.m. in b) or three experiments (c) or are from two experiments (d; mean and s.e.m. of triplicates).

Similar to wild-type PKC-θ, but unlike wild-type PKC-δ, PKC- Prkcq−/− background reconstituted with the proline mutants studied δ+θPR also immunoprecipitated together with CD28 in transduced above. Unstimulated T cells from mice reconstituted with PKC-θ had Prkcq−/− CD4+ T cells costimulated with anti-CD3 and anti-CD28 very low expression of CD69 and CD25 (Fig. 4d). T cells reconstituted (Fig. 3c, left). This association was dependent on T cell stimulation, with wild-type PKC-θ or P330-6A and stimulated with anti-CD3 and as we did not observe it in similar immunoprecipitates from unstimu- anti-CD28 considerably upregulated their expression of these activa- lated T cells (Fig. 3c, right). Similarly, the PKC-δ+θPR mutant was tion markers. However, in cells reconstituted with the P331-4A or able to stimulate the activity of reporters for RE/AP (Fig. 3d) and 4PA mutant, upregulation of CD69 and CD25 was ~40–50% lower NFAT (Supplementary Fig. 5) in a stimulated moth cytochrome c (Fig. 4d,e). We obtained parallel results when we measured prolifera- (MCC)-specific T cell hybridoma to a degree approaching that of tion (Fig. 4e) and IL-2 production (Fig. 4f) in the same cells, although wild-type PKC-θ (~70–80%) but significantly higher than that of CD4+ T cells expressing the P331-4A or 4PA mutant did produce wild-type PKC-δ. Therefore, the proline-rich motif in PKC-θ V3 is some IL-2 (Fig. 4f). The transduced T cells had similar expression of required for the inducible association with CD28 and the activation the wild-type or mutant PKC-θ proteins (data not shown and Fig. 1e). of PKC-θ-dependent signaling. Therefore, activation of primary T cells also critically depended on To determine which proline residues in the proline-rich motif are the Pro331 and Pro334 residues of PKC-θ. important for localization to the immunological synapse, association with CD28 and reporter activation, we substituted the two external pro- The kinase Lck mediates the PKC-u–CD28 interaction line residues (P330-6A), two internal proline residues (P331-4A) or all The identification of the Pro-X-X-Pro motif, a potential SH3-binding four proline residues (4PA) with alanine in the PKC-θ proline-rich motif motif, in the PKC-θ V3 domain as being essential for association with (amino acids 330–336, where the numbering refers to residues of com- CD28 was notable because our mapping analysis of the CD28 cyto- plete human PKC-θ, with the sequence PPXXPXP, where ‘X’ is any amino plasmic tail showed that a C-terminal proline-rich motif in mouse acid). Localization of the transduced P330-6A PKC-θ mutant to the CD28 (amino acids 206–209 (Pro-Tyr-Ala-Pro)) was required for the immunological synapse and cSMAC in OVA-stimulated Prkcq−/− OT-II CD28–PKC-θ interaction (data not shown). That is the same CD28 T cells was intact and was similar to that of wild-type PKC-θ (Fig. 4a motif required for colocalization of PKC-θ–CD28 to the cSMAC, and Supplementary Fig. 6). In contrast, the P331-4A and 4PA PKC-θ for stabilization of IL-2 mRNA and for the reorganization of lipid 26,29–31 mutants failed to localize to the immunological synapse and remained rafts , as well as for PKC-θ-dependent TH2- and TH17-mediated largely cytosolic, which suggested that Pro331 and Pro334 are essential inflammatory responses13–17,21. Because direct association between for the antigen-induced recruitment of PKC-θ to the immunological syn- the proline-rich motifs in PKC-θ and CD28 is unlikely, we sur- apse. We obtained similar results when we analyzed these PKC-θ mutants mised that this interaction requires an intermediary molecule, with for their ability to associate with CD28 and activate reporter genes. Thus, the kinase Lck being a strong candidate. Indeed, we found that the the P331-4A and 4PA mutants, but not the P330-6A mutant, did not interaction between CD28 and V3 was absent in Lck-deficient Jurkat coimmunoprecipitate with CD28 (Fig. 4b) and were less able to activate (JCam1.6) cells and was restored after transfection of an expression reporters for RE/AP (Fig. 4c) or NFAT (Supplementary Fig. 7). plasmid for wild-type Lck, which was also included in the V3-CD28 We further established the importance of the proline-rich (Pro- complex (Fig. 5). The V3 domain associated with Lck in which the Src X-X-Pro) motif in CD4+ T cells from bone marrow chimeras on the homology 2 (SH2) domain was inactivated, but CD28 was not present

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a b c d e 20 ) IS No IS No stim + MCC No stim α-CD3 + α-CD28 2 10

100 100 ×

* ( 15 10 * 40 * VectorWTP330-6AP331-4A4PA 80 80 * PKC 8 30 IP: CD28 60 60 10 CD28 6 cells (%) cells (%) RLU) + + 20 4 40 40 PKC 4 5 10 × Conjugates 10 GFP ( 2 20 20

Lysates H incorporation CD69 CD25 RE/AP activity 3 0 Actin 0 0 0 0 0 1.25 2.50 5.00 WT 4PA WT 4PA WT 4PA WT 4PA α-CD3 + α-CD28 (5 µg/ml) Vector Vector Vector P330-6AP331-4A P330-6AP331-4A P330-6AP331-4A P330-6AP331-4A f 4 KO Vector 3 WT Figure 4 Importance of the proline-rich motif in the V3 domain of PKC-θ for localization to the immunological P330-6A synapse and CD28 interaction. (a) Quantification of PKC-θ localization in Prkcq−/− OT-II CD4+ T cells infected 2 P331-4A 4PA

with retrovirus expressing GFP-tagged wild-type or mutant PKC-θ (images, Supplementary Fig. 5). *P < 0.05 IL-2 (ng/ml) 1 (one-way ANOVA). (b) PKC-θ–CD28 association in Prkcq−/− CD4+ T cells infected with retrovirus expressing wild-type or mutant PKC-θ and restimulated for 5 min with anti-CD3 plus anti-CD28. (c) Luciferase activity in MCC-specific 0 0 1.25 2.50 5.00 T hybridoma cells transfected with empty vector or expression vector for wild-type or mutant PKC-θ, together with α-CD3 + α-CD28 (5 µg/ml) reporter plasmids for RE/AP and β-Gal, then stimulated and assessed as in Figure 1c. *P < 0.05 (one-way ANOVA). (d–f) Expression of CD69 and CD25 (d) and proliferation (e) and production of IL-2 (f) in T cells isolated from control, unreconstituted Prkcq−/− mice (KO) or from Rag1−/− bone marrow chimeras reconstituted with Prkcq−/− bone marrow cells transduced with retrovirus expressing empty vector or expression vector for wild-type or mutant PKC-θ; T cells from Prkcq−/− mice or sorted GFP+ CD4+ T cells from chimeras were left unstimulated or stimulated overnight (d) or for 48 h (e,f) with anti-CD3 plus anti-CD28. Data are representative of five (a), four (b) or two (c) experiments or are from two experiments (d–f; mean and s.e.m. in c,e,f). in this complex. When we reconstituted JCam1.6 cells with Lck in OVA. The V3 domain translocated to the immunological synapse, but which SH3 was inactivated, PKC-θ V3 failed to precipitate together endogenous PKC-θ was sequestered from the immunological synapse, with CD28 or Lck (Fig. 5). These findings suggested that Lck medi- and we found it mostly in the cytoplasm (Fig. 6a,b). However, when ates the interaction between PKC-θ and CD28, with its SH3 domain we transduced cells with a mutant V3 domain in which all four proline binding the proline-rich motif in PKC-θ V3 and its SH2 domain residues were replaced by alanine or a V3 domain with deletion of the binding phosphorylated Tyr207 in the CD28 motif at amino acids entire proline-rich motif, they did not localize in the immunological 206–209 (Pro-Tyr-Ala-Pro)30,32. This mode of a tri-partite interaction synapse and, moreover, they did not interfere with the localization of (Supplementary Fig. 8) was consistent with findings that the SH2 endogenous PKC-θ to the immunological synapse (Fig. 6a,b). domain of Lck has a much higher affinity than the SH3 domain for We next studied the effects of V3 on the activation of reporter the phosphorylated Pro-Tyr-Ala-Pro motif of CD28 (ref. 33) and, genes. As expected, expression of wild-type PKC-θ alone resulted conversely, that in stimulated T cells, the Lck SH3 domain is consider- in much more antigen-induced activity of reporter genes for both ably more effective than the SH2 domain in binding PKC-θ34. RE/AP (Fig. 6c) and NFAT (Supplementary Fig. 9) than that in control transfectants. Expression of the V3 domain alone did not Ectopic expression of V3 interferes with T cell activation activate these reporters. However, coexpression of the V3 domain Given the critical role of the V3 domain in the association of PKC-θ together with wild-type PKC-θ resulted in dose-dependent inhibition with CD28, localization to the immunological synapse and T cell acti- of the activity of the PKC-θ-dependent reporter gene. This inhibi- vation, we investigated whether ectopic expression of the isolated V3 tory activity was eliminated when the critical proline-rich motif was domain would interfere with the localization and function of PKC-θ by altered or deleted (Fig. 6c and Supplementary Fig. 9), which resulted functioning as a CD28-associated ‘decoy’ (Fig. 2a, right) and sequester- in intact PKC-θ-induced reporter activity. −/− ing endogenous PKC-θ from CD28 and the immunological synapse. TH2 and TH17 immune responses are compromised in Prkcq + 13–15 We infected OT-II CD4 T cells with a bicistronic retrovirus expressing mice, whereas the TH1 response remains relatively intact . GFP and Myc-tagged V3 alone and examined the localization of the Therefore, we determined whether ectopic V3 expression would transduced V3 protein and endogenous PKC-θ after stimulation with similarly inhibit helper T cell differentiation. We infected preactivated C57BL/6 (B6) CD4+ T cells with retrovirus expressing wild-type or W97A R154K mutant V3 domains and cultured the cells in vitro under TH1, TH2 * * Lck SH3 SH2 Cat domain or TH17 differentiation conditions. Consistent with published find- 13–15 ings , differentiation into the TH1 lineage was unaffected by any Myc-V3 V3 Myc of the ectopically expressed V3 vectors. In contrast, the non-mutant V3 domain inhibited TH17 and TH2 differentiation by ~75%. This Lck : Vector WT W97A R154K W97A, R154K V3-Myc : + + + + + inhibition was completely reversed (for TH17) or partially reversed Lck Figure 5 The interaction between CD28 and the V3 domain of PKC-θ IP: Myc CD28 is Lck dependent. Immunoassay (bottom) of the PKC-θ–Lck–CD28 association in Lck-deficient JCam1.6 Jurkat cells cotransfected with Myc Myc-tagged PKC-θ V3 plus expression vector for wild-type or mutant Lck Lck (top), then stimulated for 5 min with anti-CD3 and anti-CD28, followed by immunoblot analysis of Lck and endogenous CD28 in whole-cell lysates WCL CD28 (WCL) or lysates immunoprecipitated with anti-Myc. W97A, inactivated

Myc SH3 domain; R154K, inactivated SH2 domain; Cat, catalytic. Asterisks (top) indicate point substitutions. Data are from three experiments.

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50 * a α-Myc+AF555 α-PKC+AF647 GFP + CMAC Merged DIC b * 40 IS No IS 30 Myc-V3 20

Conjugates 10 0 V3 PR ∆ c V3-4PAV3- Myc–V3-4PA 20 No stim + MCC 15 RLU)

4 10

× 10 ( RE/AP 5

Myc–V3-∆PR activity 0 Vector + –– –– + + – + – WT – + + + + – ––+ + V3 –– + 4PA 4PA ∆PR ∆PR No stim Stim 1 40 d H

Vector Vector V3 V3-4PA V3-∆PR T + 3.2 0.05 38.4 0.04 39.5 0.02 34.7 0.0 33.7 0.02 γ 30 20 IFN- +

T 1 cells (%) H 10

GFP 0 0.03 0.02 0.0 0.06 0.06 Vector V3 V3-4PA V3-∆PR 4 10 0.04 0.00 0.06 0.04 0.07 0.02 0.08 0.06 0.05 0.05 25 17

3 H

10 T 20 + 2 TH17 10 15

101 10 IL-17A cells (%)

1.61 9.40 6.56 13.0 8.23 + 100 5 100 101 102 103 104 GFP 0 IL-17A Vector V3 V3-4PA V3-∆PR 4 30 10

4.03 0.06 1.11 0.40 1.26 0.09 0.68 0.13 1.50 0.30 2 3 H 10 T

+ 20 2 TH2 10 IL-4

1 + γ 10 cells (%) 10

0 1.16 24.0 3.76 13.9 17.5 GFP

IFN- 10 100 101 102 103 104 0 IL-4 Vector V3 V3-4PA V3-∆PR

Figure 6 The V3 domain interferes with PKC-θ-mediated signaling and T cell differentiation. (a) Localization of PKC-θ and V3 in OT-II CD4+ T cells infected with retrovirus expressing Myc-tagged V3, V3 with substitution of all four proline residues with alanine (V3-4PA) or V3 with deletion of the entire proline-rich motif (V3-∆PR; green), stimulated with OVA-pulsed, CMAC-labeled APCs (blue), and fixed; conjugates were stained with anti-Myc plus a secondary Alexa Fluor 555–coupled antibody (AF555; orange) and with anti–PKC-θ plus a secondary Alexa Fluor 647–coupled antibody (AF647; red) and analyzed as in Figure 1a. Original magnification, ×63. (b) Quantitative analysis of the results in a (as in Fig. 1b). *P < 0.05 (one-way ANOVA). (c) Luciferase activity in MCC-specific T hybridoma cells transfected with PKC-θ and/or V3 vectors (below graph) plus reporter plasmids for RE/AP- luciferase and β-Gal, then stimulated and analyzed as in Figure 1c. (d) Helper T cell differentiation of naive CD4+ T cells from B6 mice stimulated with anti-CD3 plus anti-CD28, cultured under TH1-, TH2- or TH17-polarizing conditions (left margin) and transduced by retrovirus with empty vector or vectors for PKC-θ V3 (above plots), analyzed by intracellular staining 8 h after restimulation with anti-CD3 plus anti-CD28. Numbers in quadrants indicate percent cells in each. Right, frequency of cytokine-producing cells (cumulative data); each symbol represents an individual replicate, and small horizontal lines indicate the mean. Data are representative of five experiments (a,b) or three experiments (c; mean and s.e.m.) or are from six experiments (d).

(~60–75%, for TH2) when the protein-rich motif was altered PKC-u V3 inhibits TH2- but not TH1-mediated airway inflammation or deleted (Fig. 6d). Hence, the V3 domain can act as a decoy in We further analyzed the effect of PKC-θ V3 in vivo in an airway blocking the localization of endogenous PKC-θ to the immunologi- inflammation model using a T cell adoptive-transfer system. Mice that cal synapse and thus attenuate its associated signaling and helper received OT-II TH2 cells transduced with an empty vector developed an T cell differentiation. inflammatory response much larger than that of PBS-challenged control

Figure 7 V3 inhibits TH2- but not TH1-mediated a 50 b 15 c 40 d 60 e 50 ) lung inflammation. (a–c) Infiltration of ) 4 4 40 40 30 10 mononuclear cells into bronchoalveolar lavage 10 × × 10 40 ( fluid (a) and cytokine expression by cells (b,c) ( 30 30

20 (pg/ml)

+ γ from naive B6 mice given OT-II CD4 T cells 20 - 20 5 20 IL-4 (pg/ml) IL-5 (pg/ml) stimulated with anti-CD3 plus anti-CD28 and 10 IFN Total cells Total cells 10 10 cultured under TH2-polarizing conditions, then 0 0 0 0 0 transduced with PKC- V3 vectors via retrovirus θ V3 PR V3 PR V3 PR V3 PR V3 PR PBS ∆ PBS ∆ PBS ∆ PBS ∆ PBS ∆ + Vector Vector Vector Vector Vector (as in Fig. 6), sorted as GFP populations V3-4PAV3- V3-4PAV3- V3-4PAV3- V3-4PAV3- V3-4PAV3- and adoptively transferred into recipient mice that were challenged with OVA and analyzed 1 d later. (d,e) Infiltration (d) and cytokine expression (e) by naive B6 mice treated as in a–c, except the transferred cells were cultured under TH1-polarizing conditions. Data are from two experiments (mean and s.e.m.).

aDVANCE ONLINE PUBLICATION nature immunology A rt i c l e s mice, as shown by the greater number of infiltrating leukocytes in the However, the requirement for the proline-rich motif was not absolute, bronchoalveolar lavage fluid from mice that received OT-II TH2 cells as PKC-θ with mutant proline residues retained the ability to partially (Fig. 7a) and their augmented expression of the signature TH2 cytokines induce CD69 and CD25 upregulation. That probably reflected the fact IL-4 (Fig. 7b) and IL-5 (Fig. 7c). The introduction of V3 into the trans- that some aspects of T cell activation, such as Ca2+ signaling12 and ferred TH2 cells ameliorated the disease by diminishing the amount CD69 upregulation, are only partially dependent on PKC-θ. Indeed, 40 of infiltrating cells and TH2 cytokines to basal amounts (Fig. 7a–c). although PKC-θ has a role in CD69 upregulation , CD69 expression However, expression of the V3 domain with deletion of the proline-rich is also strongly dependent on signaling by the GTPase Ras41. motif failed to inhibit the inflammatory response. The 4PA mutant As there is no known precedent for direct interaction between two partially ‘rescued’ the inhibition, most probably because the surround- proline-rich motifs, our finding that proline-rich motifs in both PKC-θ ing amino acid residues in addition to the critical proline residues also and CD28 were required for their interaction led us to postulate that an contribute to the regulatory function of the V3 domain. intermediary protein mediates this interaction. Published studies30,32 Adoptive transfer of transduced TH1 effector cells similarly induced and our results here support the idea that Lck mediates this interaction lung inflammation manifested by leukocyte infiltration (Fig. 7d) and through two distinct domains, with its SH2 and SH3 domains bind- higher expression of interferon-γ (Fig. 7e). The native V3 domain (as ing the phosphorylated motif at amino acids 206–209 (Pro-Tyr-Ala- well as its mutant variants) did not inhibit the inflammatory response Pro) of CD28 or the motif at amino acids 331–334 (Pro-X-X-Pro) (Fig. 7d,e), consistent with the fact that TH1-mediated lung inflam- in PKC-θ V3, respectively. This idea is consistent with the reported mation is relatively independent of PKC-θ13–15. Thus, the V3 domain differences between the SH2 and SH3 domains of Lck in the efficiency of PKC-θ can also function in vivo in a dominant-negative manner to of their binding to phosphorylated CD28 relative to their binding to block PKC-θ-dependent inflammation. PKC-θ33,34. However, this model does not exclude the possibility that some other protein(s) act(s) as a bridge between CD28 and PKC-θ. DISCUSSION The importance of the V3 domain in targeting PKC-θ to CD28 PKC-θ is a critical mediator of TCR-CD28 cosignaling in T cells. and the immunological synapse is not inconsistent with the estab- Although the catalytic activity of PKC-θ is undoubtedly required for lished importance of the C1 domain in recruiting PKC-θ to the its downstream signaling functions, its localization to the immuno- plasma membrane or the immunological synapse. The PKC-θ C1 logical synapse3 and lipid rafts35, which is mediated by its regulatory domain is reported to localize to the center of the immunological region, is also critical. However, despite circumstantial evidence on this synapse42 (although that study did not formally distinguish between subject4,36, the relationship between the localization of PKC-θ to the the cSMAC and the pSMAC), which probably reflects the substantial cSMAC and its signaling functions, and whether the former is required accumulation of diacylglycerol (DAG), the PKC-mobilizing second for the latter, are not known, nor have the structural determinants that messenger, at the immunological synapse. However, accumulation of dictate this unique localization been identified. Here, we have shown DAG does not sufficiently explain the unique localization of PKC-θ that the V3 (hinge) domain of PKC-θ and, specifically, a proline-rich to the cSMAC, as other PKC proteins that contain a functional DAG- motif in V3, were required for this localization through its physical binding C1 domain do not stably localize to the immunological association with CD28 and, consequently, for PKC-θ-dependent T cell synapse. Hence, there must be some other PKC-θ-specific feature that activation and TH2- or TH17-mediated inflammation. This represents is responsible for its localization to the immunological synapse and direct evidence that the localization of PKC-θ to the cSMAC and its cSMAC. We propose that the V3 domain, through its CD28 binding, is ability to activate downstream targets are functionally linked, with both responsible for this highly selective, sustained and high-stoichiometry3 residing in a defined structural motif. The signaling events associated localization of PKC-θ after the initial binding of the C1 domain to with CD28 are not entirely understood, and it remains controver- membrane DAG, an event that by itself may be highly dynamic and sial whether CD28 induces unique signals or simply amplifies TCR of low stoichiometry. Indeed, PKC-θ–∆V3, despite containing an signals37,38. Hence, our finding of an inducible association between intact C1 domain, showed cytosolic localization in stimulated T cells. PKC-θ and CD28 is notable, as it indicates a previously unidentified, CD28- This stable, long-term recruitment to the cSMAC allows PKC-θ to specific signaling module that is not shared with TCR signals per se. mediate its functions. In contrast, other PKC proteins that contain a Published studies have circumstantially linked the localization functional C1 domain, such as the PKC-θ-related PKC-δ, which has of PKC-θ to the immunological synapse and its function to CD28. an affinity for DAG similar to that of PKC-θ27,43, may also localize at First, efficient PKC-θ-mediated activation of transcription factors DAG-rich membrane sites but will fail to activate sustained signaling depends on CD28 costimulation8,10. Second, CD28 is necessary for because of transient low-stoichiometry recruitment to the membrane the localization of PKC-θ to the cSMAC36, and this requirement has and/or substrate specificity distinct from those of PKC-θ. In support been mapped to a C-terminal Pro-Tyr-Ala-Pro-Pro motif starting of that idea, the C1 domain of PKC-θ localizes only transiently at the at Pro206 in CD28 (ref. 39). Third, the colocalization of PKC-θ and immunological synapse, whereas the localization of full-length PKC-θ CD28 to immunological-synapse–resident microclusters has been to the immunological synapse is prolonged and stable44. Similarly, documented25,26. Furthermore, in the mature immunological synapse, protein kinase D, which also contains a DAG-binding C1 domain, PKC-θ localizes together with CD28 in a newly defined peripheral translocates transiently to the T cell immunological synapse42. In subdomain of the cSMAC in a manner dependent on the same Pro- addition to the physical PKC-θ–CD28 association reported here, Tyr-Ala-Pro-Pro motif starting at Pro206 and immunoprecipitates other regulatory events that may contribute to the selective localiza- together with CD28 in T cells stimulated with phorbol ester26. Our tion of PKC-θ to the immunological synapse and cSMAC and its results here have established that this association was also induced functions include its tyrosine phosphorylation in the N-terminal by physiological stimulation with peptide-pulsed APCs and was C2 domain, which relieves C2-mediated negative regulation10,27,34, dependent on a highly conserved proline-rich motif present in the autophosphorylation at Thr219 in the C1 domain45 and/or specific V3 domain of PKC-θ but not in other PKC proteins. This unique C1 domain–mediated protein-protein interactions46. motif defines a newly identified function of PKC-θ V3: costimulation- Given the requirement of PKC-θ in TH2- and TH17-mediated dependent recruitment of the enzyme to the immunological synapse. inflammation and graft-versus-host disease but not in TH1 antiviral

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or graft-versus-leukemia responses, PKC-θ is a likely drug target for a 14. Salek-Ardakani, S., So, T., Halteman, B.S., Altman, A. & Croft, M. Differential plethora of diseases. Small-molecule selective inhibitors of the catalytic regulation of Th2 and Th1 lung inflammatory responses by protein kinase C θ. J. Immunol. 173, 6440–6447 (2004). 47 activity of PKC-θ have been reported . However, the catalytic domains 15. Salek-Ardakani, S., So, T., Halteman, B.S., Altman, A. & Croft, M. Protein kinase of PKC proteins are highly conserved and, furthermore, kinase inhibi- Cθ controls Th1 cells in experimental autoimmune encephalomyelitis. J. Immunol. 175, 7635–7641 (2005). tors generally suffer from a lack of sufficient specificity and hence have 16. Anderson, K. et al. Mice deficient in PKC θ demonstrate impaired in vivo T cell potential toxic side effects. Our report of a new approach with which to activation and protection from T cell-mediated inflammatory diseases.Autoimmunity 39, attenuate the function of PKC-θ by blocking its obligatory interaction 469–478 (2006). 17. Tan, S.L. et al. Resistance to experimental autoimmune encephalomyelitis and with CD28 suggests that such blockade could serve as a basis for the impaired IL-17 production in protein kinase C θ-deficient mice. J. Immunol. 176, development of new therapeutic agents that would selectively suppress 2872–2879 (2006). undesired T cell–mediated inflammation while at the same time 18. Berg-Brown, N.N. et al. PKCθ signals activation versus tolerance in vivo. J. Exp. Med. 199, 743–752 (2004). preserving desired immunity, such as antiviral responses. 19. Giannoni, F., Lyon, A.B., Wareing, M.D., Dias, P.B. & Sarawar, S.R. Protein kinase C θ is not essential for T-cell-mediated clearance of murine gammaherpesvirus 68. Methods J. Virol. 79, 6808–6813 (2005). 20. Marsland, B.J. et al. Innate signals compensate for the absence of PKCθ during Methods and any associated references are available in the online in vivo CD8+ T cell effector and memory responses. Proc. Natl. Acad. Sci. USA 102, version of the paper at http://www.nature.com/natureimmunology/. 14374–14379 (2005). 21. Marsland, B.J. et al. TLR ligands act directly upon T cells to restore proliferation in the absence of protein kinase C- signaling and promote autoimmune myocarditis. Note: Supplementary information is available on the Nature Immunology website. θ J. Immunol. 178, 3466–3473 (2007). 22. Valenzuela, J.O. PKC is required for alloreactivity and GVHD but not for Acknowledgments et al. θ −/− immune responses toward leukemia and infection in mice. J. Clin. Invest. 119, We thank D. Littman (New York University) for Prkcq mice; T. So, W. Duan and 3774–3786 (2009). M. Croft for the MCC-specific T hybridoma DCEK fibroblast system and advice 23. Keranen, L.M. & Newton, A.C. Ca2+ differentially regulates conventional protein kinase on the allergic airway inflammation model; S. Becart, C. Fos and Y. Harada for Cs′ membrane interaction and activation. J. Biol. Chem. 272, 25959–25967 (1997). discussions and suggestions; and K. Hayashi for some of the PKC-θ constructs. 24. Huang, J., Sugie, K., La Face, D.M., Altman, A. & Grey, H.M. TCR antagonist Supported by the US National Institutes of Health (CA35299), by The Ministry peptides induce formation of APC-T cell conjugates and activate a Rac signaling of Education, Culture, Sports, Science, and Technology, Japan (Grant-in-Aid for pathway. Eur. J. Immunol. 30, 50–58 (2000). Scientific Research on Innovative Areas ‘Fluorescence Live imaging’ 23113521) and 25. Tseng, S.Y., Waite, J.C., Liu, M., Vardhana, S. & Dustin, M.L. T cell-dendritic cell immunological synapses contain TCR-dependent CD28–CD80 clusters that recruit the United States-Israel Binational Science Foundation. This is manuscript number protein kinase C θ. J. Immunol. 181, 4852–4863 (2008). 1392 from the La Jolla Institute for Allergy and Immunology. 26. Yokosuka, T. et al. Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C θ translocation. Immunity 29, 589–601 (2008). AUTHOR CONTRIBUTIONS 27. Melowic, H.R. et al. Mechanism of diacylglycerol-induced membrane targeting and K.-F.K. and A.A. designed the experiments and wrote the manuscript; K.-F.K. activation of protein kinase Cθ. J. Biol. Chem. 282, 21467–21476 (2007). generated and analyzed data; T.S. and T.Y. provided expertise in cell imaging; 28. Kay, B.K., Williamson, M.P. & Sudol, M. The importance of being proline: the T.S. participated in discussion of the data; T.Y. generated the PKC-GFP and interaction of proline-rich motifs in signaling proteins with their cognate domains. CD28–cyan fluorescent protein fusion vectors; N.I. participated actively in FASEB J. 14, 231–241 (2000). discussions leading to this work and in experimental design; and A.J.C.-B. did 29. Dodson, L.F. et al. Targeted knock-in mice expressing mutations of CD28 reveal an various experiments and animal work. essential pathway for costimulation. Mol. Cell. Biol. 29, 3710–3721 (2009). 30. Miller, J. et al. Two pathways of costimulation through CD28. Immunol. Res. 45, COMPETING FINANCIAL INTERESTS 159–172 (2009). 31. Sanchez-Lockhart, M. Cutting edge: CD28-mediated transcriptional and The authors declare no competing financial interests. et al. posttranscriptional regulation of IL-2 expression are controlled through different signaling pathways. 173, 7120–7124 (2004). Published online at http://www.nature.com/natureimmunology/. J. Immunol. 32. Sadra, A. et al. Identification of tyrosine phosphorylation sites in the CD28 Reprints and permissions information is available online at http://www.nature.com/ cytoplasmic domain and their role in the costimulation of Jurkat T cells. J. Immunol. 162, reprints/index.html. 1966–1973 (1999). 33. Hofinger, E. & Sticht, H. Multiple modes of interaction between Lck and CD28. 1. Dustin, M.L. The cellular context of T cell signaling. Immunity 30, 482–492 (2009). J. Immunol. 174, 3839–3840 (2005). 2. Dustin, M.L., Chakraborty, A.K. & Shaw, A.S. Understanding the structure and function 34. Liu, Y. et al. Regulation of protein kinase Cθ function during T cell activation by of the immunological synapse. Cold Spring Harb. Perspect. Biol. 2, a002311 (2010). Lck-mediated tyrosine phosphorylation. J. Biol. Chem. 275, 3603–3609 (2000). 3. Monks, C.R., Kupfer, H., Tamir, I., Barlow, A. & Kupfer, A. Selective modulation 35. Bi, K. et al. Antigen-induced translocation of PKC-θ to membrane rafts is required of protein kinase C-θ during T-cell activation. Nature 385, 83–86 (1997). for T cell activation. Nat. Immunol. 2, 556–563 (2001). 4. Monks, C.R., Freiberg, B.A., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional segre 36. Huang, J. et al. CD28 plays a critical role in the segregation of PKC θ within the gation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998). immunologic synapse. Proc. Natl. Acad. Sci. USA 99, 9369–9373 (2002). 5. Pfeifhofer, C. et al. Protein kinase C θ affects Ca2+ mobilization and NFAT cell 37. Acuto, O. & Michel, F. CD28-mediated co-stimulation: a quantitative support for activation in primary mouse T cells. J. Exp. Med. 197, 1525–1535 (2003). TCR signalling. Nat. Rev. Immunol. 3, 939–951 (2003). 6. Sun, Z. et al. PKC-θ is required for TCR-induced NF-κB activation in mature but 38. Rudd, C.E., Taylor, A. & Schneider, H. CD28 and CTLA-4 coreceptor expression not immature T lymphocytes. Nature 404, 402–407 (2000). and signal transduction. Immunol. Rev. 229, 12–26 (2009). 7. Hayashi, K. & Altman, A. Protein kinase C θ (PKCθ): a key player in T cell life and 39. Sanchez-Lockhart, M., Graf, B. & Miller, J. Signals and sequences that control death. Pharmacol. Res. 55, 537–544 (2007). CD28 localization to the central region of the immunological synapse. J. Immunol. 181, 8. Coudronniere, N., Villalba, M., Englund, N. & Altman, A. NF-κB activation induced 7639–7648 (2008). by T cell receptor/CD28 costimulation is mediated by protein kinase C-θ. Proc. 40. Villalba, M. et al. A novel functional interaction between Vav and PKCθ is required Natl. Acad. Sci. USA 97, 3394–3399 (2000). for TCR-induced T cell activation. Immunity 12, 151–160 (2000). 9. Baier-Bitterlich, G. et al. Protein kinase C-θ isoenzyme selective stimulation of the 41. D’Ambrosio, D., Cantrell, D.A., Frati, L., Santoni, A. & Testi, R. Involvement of transcription factor complex AP-1 in T lymphocytes. Mol. Cell. Biol. 16, 1842–1850 p21ras in T cell CD69 expression. Eur. J. Immunol. 24, 616–620 (1994). (1996). 42. Spitaler, M., Emslie, E., Wood, C.D. & Cantrell, D. Diacylglycerol and protein kinase 10. Altman, A. et al. Positive feedback regulation of PLCγ1/Ca2+ signaling by PKCθ in D localization during T lymphocyte activation. Immunity 24, 535–546 (2006). restimulated T cells via a Tec kinase-dependent pathway. Eur. J. Immunol. 34, 43. Stahelin, R.V. et al. Mechanism of diacylglycerol-induced membrane targeting and 2001–2011 (2004). activation of protein kinase Cδ. J. Biol. Chem. 279, 29501–29512 (2004). 11. Lin, X., O’Mahony, A., Geleziunas, R. & Greene, W.C. Protein kinase C θ participates 44. Carrasco, S. & Merida, I. Diacylglycerol-dependent binding recruits PKCθ and in NF-κB/Rel activation induced by CD3/CD28 costimulation through selective RasGRP1 C1 domains to specific subcellular localizations in living T lymphocytes. activation of IκB β (IKKβ). Mol. Cell. Biol. 20, 2933–2940 (2000). Mol. Biol. Cell 15, 2932–2942 (2004). 12. Manicassamy, S., Sadim, M., Ye, R.D. & Sun, Z. Differential roles of PKC-θ in the 45. Thuille, N. et al. Critical role of novel Thr-219 autophosphorylation for the cellular regulation of intracellular calcium concentration in primary T cells. J. Mol. Biol. 355, function of PKCθ in T lymphocytes. EMBO J. 24, 3869–3880 (2005). 347–359 (2006). 46. Colon-Gonzalez, F. & Kazanietz, M.G. C1 domains exposed: from diacylglycerol binding 13. Marsland, B.J., Soos, T.J., Spath, G., Littman, D.R. & Kopf, M. Protein kinase C to protein-protein interactions. Biochim. Biophys. Acta 1761, 827–837 (2006). θ is critical for the development of in vivo T helper Th2 cell but not Th1 cell 47. Boschelli, D.H. Small molecule inhibitors of PKCθ as potential antiinflammatory responses. J. Exp. Med. 200, 181–189 (2004). therapeutics. Curr. Top. Med. Chem. 9, 640–654 (2009).

aDVANCE ONLINE PUBLICATION nature immunology ONLINE METHODS at 37 °C followed by two additional retroviral infections at daily intervals. Antibodies and reagents. Monoclonal antibodies (mAbs) to mouse CD3 After the final infection, cells were washed and cultured in RPMI medium (145-2C11), CD28 (37.51), IL-4 (11B11), interferon-γ (XMG1.2) and the p40 containing 10% (vol/vol) FBS and recombinant IL-2 (200 U/ml) for another subunit of IL-12–IL-23 (C17.8) were from BioLegend, as were fluorophore- 3 d before restimulation with mAb to CD3 plus mAb to CD28. conjugated anti-IL-4 (11B11), anti-interferon-γ (XMG1.2), anti-17A (TC11- 18H10.1), anti-CD69 (H1.2F3) and anti-CD25 (PC61). Anti-PKC-θ was from Immunoprecipitation and immunoblot analysis. Retrovirally transduced BD Transduction Laboratories (27/PKC) or Cell Signaling Technology (2059). CD4+ T cells were stimulated for 5 min with mAb to CD3 plus mAb to CD28. Antibody specific for the C terminus of PKC-θ that is cross-reactive with Cell lysis in 1% (wt/vol) digitonin lysis buffer, immunoprecipitation and PKC-δ (C-18) and polyclonal hamster anti-CD28 (PV.1) were from Santa Cruz immunoblot analysis were done as described27. Biotechnology. Anti-talin (8d4) was from Sigma. Cell tracker blue (CMAC), Alexa Fluor 647–conjugated anti-mouse immunoglobulin (A-21236) and Luciferase reporter assay. Lucifer reporter genes for RE/AP or NFAT have Alexa Fluor 555–conjugated anti-rabbit immunoglobulin (A-21425) were been described8,40. MCC-specific hybridoma T cells were transfected through from Molecular Probes. Recombinant mouse IL-3, IL-4, IL-6, IL-12, stem cell the use of Ingenio electroporation reagent (Mirus Bio). Transfected cells were factor and transforming growth factor-β were from PeproTech. Digitonin was stimulated for 6 h with APCs consisting of DCEK fibroblasts stably expressing from EMD Chemicals. OVA peptide (amino acids 323–339) and MCC peptide I-Ek and B7-1, which were prepulsed with MCC peptide (10 µg/ml). Cells were (amino acids 88–103) were from Genescript. then lysed and luciferase activity was quantified and normalized to the activity of the cotransfected β-Gal reporter gene. Plasmids. Retroviral plasmids encoding full-length human PKC-θ and PKC-δ were generated by PCR amplification and were subcloned into the retrovi- Immunofluorescence microscopy. Full-length human PKC-θ and its ral vector pMIG48. PKC-θ–∆V3 (deletion of amino acids 282–379), PKC-θ+ derivatives were cloned as GFP fusion proteins in the retroviral vector pMX. δV3 (replacement of PKC-θ V3 with amino acids 282–358 of PKC-δ) and Retroviral supernatants were used for infection of preactivated CD4+ T cells. PKC-δ+θPR (insertion of amino acids 328–336 of human PKC-θ between Transduced cells were allowed to ‘rest’ for an additional 3 d. On the day of the amino acids Ile312 and Tyr313 of PKC-δ) were constructed by overlap- the experiment, APCs were prepared from wild-type B6 splenocyte samples ping PCR. PKC-θ point mutants were generated with a QuikChange II Site- that were depleted of CD4+ T cells with beads coated with anti-CD4 (L3T4). Directed Mutagenesis Kit (Stratagene). The PKC-θ V3 expression vector Those APCs were stained with CellTracker Blue (CMAC; 20 µM) and then was constructed by in-frame subcloning of sequence encoding amino acids pulsed for 30 min at 37 °C with 5 µM OVA peptide. Those APCs were allowed 282–379 of human PKC-θ into a pMIG vector containing sequence encoding to conjugate for 20 min at 37 °C before fixation with 4% (wt/vol) parafor- an N-terminal Myc tag. V3-4PA and V3-∆PR were generated by site-directed maldehyde and permeabilization with PBS supplemented with 1% (wt/vol) mutagenesis and overlapping PCR, respectively, of pMIG-V3. Vectors encod- BSA and 0.1% (vol/vol) Triton X-100. Cells were immunostained for 1 h at ing PKC-enhanced GFP fusion proteins were generated by PCR and subcloned 25 °C with the appropriate antibodies in PBS supplemented with 1% (wt/vol) into the retroviral vector pMX26. The fluorescent tag was attached to the BSA and 0.3% (wt/vol) saponin. Immunofluorescene images were captured C terminus of each PKC by a polyglycine linker (LESGGGGSGGGG). with a Marianas digital fluorescence-microscopy system (Intelligent Imaging Innovations) as described49. Mice and primary cell cultures. B6 mice were housed, maintained under specific pathogen–free conditions and manipulated according to a protocol Bone marrow chimeras. Bone marrow chimeras of irradiated Rag1−/− mice approved by the La Jolla Institute for Allergy and Immunology Animal Care were produced as described48. Bone marrow cells were flushed from the femurs Committee. Prkcq−/− OT-II mice were generated by intercrossing of OT-II and tibias of Prkcq−/− mice. Lineage-negative bone marrow cells were selected mice and Prkcq−/− mice (a gift from D. Littman). CD4+ T cells were isolated by with a Lineage Cell Depletion column (Miltenyi Biotec) and were cultured for positive selection with beads coated with mAb to CD4 (L3T4; Miltenyi Biotec) 24 h in DMEM (Mediatech) containing 10% (vol/vol) FBS, 10 ng/ml of IL-3, and were cultured in RPMI-1640 medium (Mediatech) supplemented with 20 ng/ml of IL-6 and 50 ng/ml of stem cell factor. Cells were infected with 10% (vol/vol) heat-inactivated FBS, 2 mM glutamine, 1 mM sodium pyruvate, retrovirus for 3 consecutive days. GFP+ cells were sorted, and 2 × 105 cells 1 mM MEM nonessential amino acids and 100 U/ml each of penicillin G were injected intravenously into irradiated Rag1−/− mice, followed by analysis and streptomycin (Life Technologies). Differentiation into TH1, TH2 or TH17 6–8 weeks after the transfer. effector cells was achieved as described49,50. Naive CD4+ T cells were preactivated for 48 h with plate-bound mAb to CD3 (5 µg/ml) and soluble mAb to Adoptive transfer and OVA-induced airway inflammation. CD4+ T cells CD28 (2.5 µg/ml) in the presence of polarizing conditions (TH1: 100 U/ml from OT-II B6 mice were cultured with plate-bound anti-CD3 (5 µg/ml), of IL-2, 20 U/ml of IL-12 and 10 µg/ml of IL-4 mAb; TH2: 100 U/ml of IL-2, soluble anti-CD28 (2.5 µg/ml) and IL-2 (100 U/ml) under TH1- or TH2- 200 U/ml of IL-4, 10 µg/ml each of mAb to IL-12 and mAb to interferon-γ; polarizing conditions, as described above. GFP+ cells were sorted, and 6 TH17: 5 ng/ml of TGFβ, 20 ng/ml of IL-6, 10 µg/ml each of mAb to IL-4 and 1 × 10 cells were injected intravenously into naive wild-type B6 mice. Then mAb to interferon-γ). Two rounds of retroviral transduction were performed 1 d later, mice received aerosolized OVA (5 mg/ml in 20 ml PBS) for 30 min before cells were allowed to ‘rest’ for 3 d in the presence of 100 U/ml of IL-2. once a day for 3 consecutive days by ultrasonic nebulization. Mice were On day 7, cells were restimulated for 8 h with plate-bound mAb to CD3 plus killed 24 h after the final challenge and lung inflammation was assessed. mAb to CD28 (10 µg/ml each) without additional cytokines, and intracellular Bronchoalveolar lavage fluid was collected and cytokines were measured cytokines were stained in the presence of GolgiStop (BD Biosciences). as described14.

Retroviral transduction. Platinum-E packaging cells were plated in a six-well Statistical analysis. One-way analysis of variance with post-hoc Bonfferoni’s plates in 2 ml RPMI medium plus 10% (vol/vol) FBS. After 24 h, cells were corrections was used for statistical analyses, with P values of less than 0.05 transfected with retroviral plasmid DNA (5 µg) through the use of TransIT- being considered statistically significant. LT1 transfection reagent (Mirus Bio). After overnight incubation, the medium 48. Holst, J. et al. Generation of T-cell receptor retrogenic mice. Nat. Protoc. 1, 406–417 was replaced and cultures were maintained for at least another 24 h. Retroviral (2006). supernatants were then collected and filtered, supplemented with 5 µg/ml of 49. Becart, S. et al. Tyrosine-phosphorylation-dependent translocation of the SLAT polybrene and 200 U/ml of recombinant IL-2 and then used to infect CD4+ protein to the immunological synapse is required for NFAT transcription factor activation. Immunity 29, 704–719 (2008). T cells that had been preactivated with plate-bound anti-CD3 (5 µg/ml), solu- 50. Canonigo-Balancio, A.J., Fos, C., Prod’homme, T., Becart, S. & Altman, A. SLAT/ ble anti-CD28 (5 µg/ml) and recombinant IL-2 (200 U/ml). Plates were centri- Def6 plays a critical role in the development of Th17 cell-mediated experimental fuged for 1 h at 800g and were incubated for at least 4 h at 32 °C and overnight autoimmune encephalomyelitis. J. Immunol. 183, 7259–7267 (2009).

doi:10.1038/ni.2120 nature immunology