ADAP and SKAP 55 deficiency suppresses PD ‐1 expression in CD 8 + cytotoxic T lymphocytes for enhanced anti‐tumor immunotherapy

• DOI: 10.15252/emmm.201404578
• Publication Date: 2015-04-08T04:31:19Z
• Authors: Chunyang Li; Weiyun Li; Jun Xiao; Shaozhuo Jiao; Fei Teng; Shengjie Xue; Chi Zhang; Chun Sheng; Qibin Leng; Christopher E Rudd; Bin Wei; Hongyan Wang
• Keywords: Cancer Immunotherapy

Research Article7 April 2015Open Access Source Data ADAP and SKAP55 deficiency suppresses PD-1 expression in CD8+ cytotoxic T lymphocytes for enhanced anti-tumor immunotherapy Chunyang Li Key Laboratory of Systems Biology, Institute Biochemistry Cell Shanghai Institutes Biological Sciences, Chinese Academy Shanghai, China Search more papers by this author Weiyun Jun Xiao Normal University, Shaozhuo Jiao Fei Teng Shengjie Xue Chi Zhang Chun Sheng Qibin Leng Pasteur Christopher E Rudd Cambridge Medical Research, Cambridge, UK Bin Wei Corresponding Author State Virology, Wuhan Wuhan, Hongyan Wang Information Li1, Xiao1,2, Jiao1, Teng1, Xue1, Zhang1, Sheng2, Leng3, Rudd4, 5 1 1Key 2Shanghai 3Institute 4Cambridge 5State *Corresponding author. Tel: [phone]; E-mail: [email protected] 21-54921086; EMBO Mol Med (2015)7:754-769https://doi.org/10.15252/emmm.201404578 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision process including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract negatively regulates (CTL) cytotoxicity immunity. However, it is not fully understood how on CTL regulated during immunotherapy. In study, we have identified that ADAP-SKAP55 signaling module reduced Fyn-, Ca2+-, NFATc1-dependent manner. DC vaccine-based tumor prevention therapeutic models, knockout or showed heightened protection from formation metastases mice effector cells. Interestingly, CTLA-4 levels percentages infiltrating CD4+Foxp3+ Tregs remained unchanged. Furthermore, adoptive transfer SKAP55-deficient ADAP-deficient CTLs significantly blocked growth increased Pretreatment wild-type with NFATc1 inhibitor CsA could also downregulate enhance efficacy. Together, propose targeting unrecognized ADAP-SKAP55-NFATc1-PD-1 pathway might increase efficacy Synopsis can via transcription factor CTLs. Targeting enhances against cells vitro vivo. regulate Ca2+- Deficiency reduces expression. SKAP55- improve DC-based vaccine therapy. Adoptive blocks recipient mice. Introduction Cytotoxic (CTLs) play an important role responses. Following recognition antigens professional dendritic (DCs), naive undergo extensive expansion, acquire function, differentiate into tumor-specific kill Although vaccination pulsed has been shown prolong survival time cancer patients, majority had only marginal clinical activity (Bhardwaj, 2007). It critical dissect key proteins determine antigen-induced The inhibitory receptors Programmed Death Receptor (PD-1) T-lymphocyte Antigen 4 (CTLA-4, known as CD152) are inducible expressed activated response various kinds such antigen presented DCs context, ligand PD-L1 wide variety tumors, blockade promotes (Curiel et al, 2003). demonstrated PD-1-deficient TCR transgenic rejection vivo (Blank 2004). Therapies PD-1, PD-L1, therefore promising strategies CTL-mediated (Gravitz, 2013; Gubin 2014; Herbst Powles Tumeh 2014). Despite this, delineated prevent T-cell activation mediated protein-tyrosine phosphorylation cascade Lck, Fyn, ZAP-70 their immune cell-specific adaptor proteins. These lack definable catalytic activities, but instead, possess binding domains sites multimeric complexes. adaptors (src kinase-associated protein 55 kDa; termed SKAP1/src protein-1) (adhesion degranulation promoting protein; Fyb/Fyn SLAP-130/SLP-76-associated 130 kDa) located at killing synapses between Previous studies predominantly cells, constitutively bind together (Liu 1998; Marie-Cardine 1998). Further, stabilize level (Huang 2005; interacts SLP-76 (Src homology 2 (SH2) domain-containing leukocyte 76 kDa), binds RapL (regulator cell adhesion polarization enriched lymphoid tissues) RIAM (Rap1-GTP-interacting molecule) generate 'inside-out' integrin (Griffiths 2001; Peterson 2003, 2004, 2007; Menasche Raab 2010). Since integrin-mediated synapse facilitates lyse (Schmits 1996; Franciszkiewicz 2013), expected cytotoxicity. Surprisingly, study shows increases therapy specifically without affecting tumor-infiltrating regulatory (Tregs). We suggest axis novel mechanism response. Results decreases positively Integrin plays 2013). asked whether Wild-type (KO) were crossed OT-I mice, naïve isolated incubated 10 nM OVA257-264 peptide-pulsed splenocytes OVA257-264-specific To examine cytotoxicity, KO OVA257-264-pulsed EL-4 which derived lymphoma C57Bl6 used targets. As expected, was recruited LFA-1 (Fig 1A). During priming phase, observed IL-2 production (Supplementary Fig S1A) did affect (i.e. CD11a) S1B). Figure 1. decrease A. conjugated 30 min, fixed, stained anti-SKAP55 (red), anti-LFA-1 (green) Hoechst (blue). B, C. WT SKAP55−/− generated Tg then EL4 targets h assess different Effector:Target ratios (B; mean triplicates ± SD); surface mRNA (C). Graphs representative least three independent experiments. D. (3 × 106) injected C57BL/6 followed injection non-pulsed (CFSEhi) (CFSElo) (5 measure (mean SD, n = 3 per group). Representative data E. pretreated anti-PD-1 antibody IgG control, incubation ability SD). F. transfected plasmids expressing SKAP55-GFP EGFP, treated (left panel) assay SD) (right panel). information: Statistical significance determined unpaired two-tailed Student's t-test. Download figure PowerPoint greater compared controls effector-to-target 1B). absence decreased both 1C). resting basal significant difference. Next, method i.v. mice; CFSElow) CFSEhigh) target 1:1 ratio Non-pulsed lysed internal controls. Compared controls, killed vivo, flow cytometry 26.9% vs. 41.5% alive CFSElow-target left after CTLs) 1D). Importantly, addition restored similar 1E). over-transfected GFP-SKAP55 S1C), GFP-SKAP55+ assay. Overexpression GFP+ control 1F). By using genetic overexpression strategy, unexpectedly inhibits reduce Because reported 2005), next checked regulating function CTL. After peptide, changing S2A B). Similar SKAP55, accumulated 2A). S2C) 2B), while 2C). agreement levels, E:T 2D). Significantly, treatment ADAP−/− 2E). 2. CFSE-labeled anti-ADAP (red). B. GFP, stimulated detect C, unpulsed (C), (D; μg/ml Naïve WT, SKAP55−/−, ADAP−/−, SKAP55−/−ADAP−/− splenocytes, plate-bound anti-CD3/CD28 12 48 check effect ADAP/SKAP55 double (DKO), crossbred get DKO anti-CD3/CD28, those 2F). Also, anti-CD3/CD28-stimulated degree S2D). Taken together, dependent NFATc1, Ca2+, Fyn factors (nuclear c1, NFAT2) (Agnellini Oestreich 2008), c-Fos (Xiao 2012) Blimp-1 (Shin 2009) cooperated According previous findings, highly dephosphorylated moves faster SDS–PAGE gels than phosphorylated inactive form. noticed peptide stimulation elevated total CTLs, suppressed ADAP- 3A). immunostaining confirmed nuclear localized peptide-stimulated well 3B, quantified Supplementary S3A). addition, S3B). measured interaction promoter electrophoretic mobility shift (EMSA) biotin-labeled NFAT-binding DNA probes (N1). stimulation, amount probe/protein complexes enhancement inhibited 3C S3C). unlabeled competitor probe (Comp. N1) mutant contains same sequence except carrying mutated site mutN1) Comp. N1 abrogated probe-NFAT complex, mutN1 failed achieve effect. indicates specificity probe, described (Oestreich 2008). 3. PD1 A–C. expression, entry immunoblotting (A) (B). Alternatively, extracts these containing EMSA. included DMSO μM CsA, PP2 75 2APB, ADAP, overexpressed pGL3-NFAT Luciferase reporter plasmid Jurkat Cells anti-CD3 anti-CD28 presence 2APB 6 h, measuring luciferase readings available online figure. [emmm201404578-sup-0002-SDataFig3.pdf] report suggested much lesser S3D), Blimp1 S3E). cyclosporine A (CsA) block translocation (Flanagan 1991), (Su Navarro-Antolin 2000). our S3F). This following dominant reduction (even under condition expression). 3D). dissected intracellular effectors NFATc1-driven others previously tyrosine kinase phosphorylates (Marie-Cardine 1997, Liu physically activates SHP-2 (Tang 1999), recruits transduces negative signals mainly (Yokosuka 2012). performed immunoprecipitation anti-Fyn, pulled down endogenous vice versa. anti-SHP-2 co-precipitated S3G). (or ADAP) 293 observe direct interaction. (Filby anti-CD3-stimulated Fyn-deficient severely diminished dephosphorylation NFAT calcium flux (Sugie 2004), induce (Macian, 2005). examined specific (2-aminoethoxydiphenyl borate) respectively, wild-type, 3E). overexpressing NFAT-luciferase plasmid, tandem repeats site. readings, 3F). deletion SH3 domain (termed SKAP55-ΔSH3), disrupts (Duke-Cohan 2006), abolish S3H). reports upregulate exposure high hinders generation functional memory (de Goër de Herve consistent observation production, tumors vaccine-induced Wild-type, immunized twice DCs, s.c. E.G7 based endogenously synthesizes presents peptide. repressed skin S4A). confirm phenotype EL4) neither cell-line nor tumor-type specific, vaccine-mediated model physiological conditions. melanoma B16F10 lysates challenged lungs 26 days later 4A, Multiple formed strongly right 4. day −14 −7 prepulsed lysates. On 0, inoculated Number lung counted challenge (n ≥ mice). At 0 (B, before injection) (C, injection), CD44hiCD8+ CD44hiCD4+ Foxp3+CD4+ analyzed D, granzyme B samples prepared lung-infiltrated lungs, MLN, spleen Mann–Whitney U-test. re