CCR 5 deficiency impairs CD 4 + T‐cell memory responses and antigenic sensitivity through increased ceramide synthesis

• DOI: 10.15252/embj.2020104749
• Publication Date: 2020-06-11T15:57:22Z
• Authors: Ana Martín‐Leal; Raquel Blanco; Josefina Casas; María E Sáez; Elena Rodríguez‐B...; Itziar de Rojas; Carina Drechsler; Luis Miguel Real; Gemma Fabrias; Agustín Ruíz; Mario Castro; Wolfgang WA Schamel; Balbino Alarcón; Hisse M van Santen; Santos Mañes
• Keywords: CD4-Positive T-Lymphocytes; Humoral responses; Receptors, CCR5; 610; Ceramides; Membrane phase; ccr5[delta]32; Mice; 03 medical and health sciences; Animals; Humans; T-cell receptor; Humoral response; Antigens; Mice, Knockout; 0303 health sciences; membrane phase; Articles; 3. Good health; HEK293 Cells; HIV ‐1 infection; sphingolipid; CCR5; Immunologic Memory; humoral response; Ceramide synthesis

Article11 June 2020Open Access Source DataTransparent process CCR5 deficiency impairs CD4+ T-cell memory responses and antigenic sensitivity through increased ceramide synthesis Ana Martín-Leal Department of Immunology Oncology, Centro Nacional de Biotecnología (CNB/CSIC), Madrid, Spain Search for more papers by this author Raquel Blanco Josefina Casas Biological Chemistry, Institute Advanced Chemistry Catalonia (IQAC-CSIC), Barcelona, CIBER Liver Digestive Diseases (CIBER-EDH), Instituto Salud Carlos III, María E Sáez Andaluz Estudios Bioinformáticos (CAEBi), Seville, Elena Rodríguez-Bovolenta Cell Biology Immunology, Biología Molecular Severo Ochoa (CBMSO/CSIC), Itziar Rojas Alzheimer Research Center, Memory Clinic the Fundació ACE, Institut Català Neurociències Aplicades, Carina Drechsler Signaling Centers BIOSS CIBSS, University Freiburg, Germany Faculty Biology, Pharmaceutical Sciences, Luis Miguel Real Unit Infectious Microbiology, Hospital Universitario Valme, Biochemistry, School Medicine, Universidad Málaga, Gemma Fabrias Agustín Ruíz Enfermedades Neurodegenerativas (CIBERNED), Mario Castro orcid.org/0000-0003-3288-6144 Interdisciplinary Group Complex Systems, Escuela Técnica Superior Ingeniería, Pontificia Comillas, Wolfgang WA Schamel orcid.org/0000-0003-4496-3100 Centre Chronic Immunodeficiency (CCI), Balbino Alarcón orcid.org/0000-0001-7820-1070 Hisse M van Santen orcid.org/0000-0003-0769-4511 Santos Mañes Corresponding Author [email protected] orcid.org/0000-0001-8023-957X Information Martín-Leal1,‡, Blanco1,‡, Casas2,3, Sáez4, Rodríguez-Bovolenta5, Rojas6, Drechsler7,8,9, Real10,11, Fabrias2,3, Ruíz6,12, Castro13, Schamel7,8,14, Alarcón5, Santen5 *,1 1Department 2Department 3CIBER 4Centro 5Department 6Alzheimer 7Signaling 8Department 9Institute 10Unit 11Department 12CIBER 13Interdisciplinary 14Centre ‡These authors contributed equally to work. **Corresponding author. Tel: [phone]; Fax: [phone]; E-mail: EMBO J (2020)39:e104749https://doi.org/10.15252/embj.2020104749 See also: C Matti & DF Legler (August 2020) PDFDownload PDF article text main figures. Peer ReviewDownload a summary editorial decision including letters, reviewer comments feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures Info Abstract is not only coreceptor HIV-1 infection in T cells, but also contributes their functional fitness. Here, we show that limiting transcription specific synthases, signaling reduces levels thereby increases antigen receptor (TCR) nanoclustering antigen-experienced mouse human cells. This activity CCR5-specific independent co-stimulatory activity. CCR5-deficient mice showed reduced production high-affinity class-switched antibodies, after rechallenge, which implies an impaired response. study identifies function generation establishes antigen-independent mechanism regulates TCR altering lipid species. Synopsis ccr5Δ32 polymorphism endow cells with ceramide-rich environment, restricts nanoclustering. T:B cell cooperation humoral re-encounter. provides signals regulate CCR5-induced costimulatory on restrain synthase 2; maintains control novo Cer biosynthetic pathway levels. maximizes restimulation high affinity antibodies Introduction The C-C motif chemokine 5 (CCR5) seven-transmembrane G protein-coupled (GPCR) expressed surface several innate adaptive immune subtypes, effector lymphocytes (Gonzalez-Martin et al, 2012). acts necessary HIV-1. An HIV-resistant population served identify 32-bp deletion within coding region (ccr5Δ32), yields non-functional (Blanpain 2002). Since homozygous individuals are seemingly healthy, radical body thought considers dispensable function. Experimental epidemiological evidence nonetheless indicates has important role acquired responses. its ligands ligand 3 (CCL3; termed macrophage inflammatory protein [MIP]-1α), CCL4 (MIP-1β), CCL5 (regulated upon activation, normal secreted [RANTES]), CCL3L1 have been associated exacerbation chronic autoimmune diseases. Despite varying information due probably ethnicity effects (Lee 2013; Schauren 2013), further complicated admixed populations (Toson 2017), studies support allele as marker good prognosis these overreactive diseases (Vangelista Vento, 2017). In contrast, homozygotes prone fatal infections pathogens such influenza, West Nile, tick-borne encephalitis viruses (Lim Murphy, 2011; Falcon 2015; Ellwanger Chies, 2019). mechanisms affects all pathologies usually linked capacity leukocyte trafficking. For example, recruitment influenza-specific CD8+ accelerates accumulation lung airways during virus rechallenge (Dawson 2000; Kohlmeier 2008); could lead acute severe pneumonitis, flu complication. migration-independent functions maximize activation affecting immunological synapse (IS) formation (Molon 2005; Floto 2006; Franciszkiewicz 2009) well programs cytokine (Lillard 2001; Camargo 2009). critical cell-mediated immunity tumors pathogens, (Dolan 2007; Ugurel 2008; González-Martín Bedognetti 2013). Whereas priming established, involvement addressed depth. Only single report suggested promotion migration-dependent (Castellino 2006). It remains unknown whether endows additional properties. One property elevated ("antigen-experienced") cognate compared naïve (Kimachi 1997; Kersh 2003; Huang gradient (memory ≫ > naïve) valency preformed oligomers at surface, nanoclusters (Kumar 2011). (Schamel 2005, Lillemeier 2010; Sherman Alarcon, 2013) enhances increasing avidity multimeric peptide-major histocompatibility complexes Molnar 2012) allowing cooperativity between molecules (Martínez-Martín 2009; Martín-Blanco 2018). TCRβ subunit interaction cholesterol (Chol) presence sphingomyelins (SM) both essential (Molnar 2012; Beck-Garcia 2015). Replacement Chol sulfate impedes nanocluster CD4+CD8+ thymocyte weak peptides (Wang 2016). Whether nanoscopic organization homeostatic factors unexplored. Given speculated would affect To test hypothesis, analyzed vivo-generated wild-type (WT) CCR5−/− mice, effect CD4 help T-dependent We found establishment response signal. does generation, antigen. Our data demonstrate unreported regulatory inhibiting ceramides, identified here negative membrane regulators organization. Results determine and/or function, adoptively transferred congenic CD45.1 lymph node/spleen suspensions from OT-II WT or (CD45.2) subsequently infected them OVA-encoding vaccinia virus; weeks post-immunization, spleen CD45.2+ donor mice. expression affected neither total number (Fig 1A B) nor percentage TEM (CD44hi; CD62L−; Fig 1C) TCM CD62L+; 1D) generated. had stronger than determined interferon (IFN)γ-producing ex vivo stimulation OVA323–339 1E). Figure 1. A. Representative plots splenocytes CD45.2 node suspensions, rVACV-OVA virus. gating strategy used subtypes shown (n = 5). B. Absolute recovered spleens A C, D. Percentage (C) (D) E. IFNγ-producing isolated (A) restimulated (1 μM) 4). F. Immunization scheme NIP-OVA NIP-KLH G–I. (G) quantification frequency (H) absolute (I) Tfh (CD4+CD44+PD-1+CXCR5+) primary immunization (day 7) 7). J, K. ELISA analysis high- (J) low-affinity (K) isotype-specific anti-NIP sera OVA/OVA- OVA/KLH-immunized 15 post-challenge; n mice/group). Data representative one experiment two. information: (B–E, H–K), mean ± SEM. *P < 0.05, **P 0.01, ***P 0.001, two-tailed unpaired Student's t-test. Download figure PowerPoint studied cell-dependent B-cell hapten 4-hydroxy-3-iodo-5-nitrophenylacetyl coupled ovalbumin (NIP-OVA; 1F). detected no difference follicular helper (Tfh) (CD4+, CD44hi, CXCR5+, PD1+) 7 days post-immunization 1G–I). At day 30, half were boosted same immunogen (OVA/OVA) other received NIP conjugated another carrier (OVA/KLH); NIP-specific immunoglobulins (Ig) later. Comparison assess first NIP. There differences high/low-affinity IgM either 1J K). markedly specifically OVA/OVA-immunized class switching was similar our results suggest deficient cell-autonomous defect intrinsic activated days; removal, cultured IL-2 IL-15. differentiated exogenous CCL3, CCL4, CCL5, receptor, ability flux Ca2+ migrate (Appendix S1A–D). Like (Richer 2015), IL-15 memory-like phenotype EV1); they smaller IL-2-cultured retained CD62L (CD25, CD69, CD44) 2A). Findings 2B), reinforced idea involved differentiation. Restimulation IL-2- IL-15-expanded lymphoblasts peptide indicated CCR5-expressing strong proliferation higher low concentrations 2C–F), indicative responding stimulation. might thus increase manner. Click expand figure. EV1. Gating characterization lymphoblastRepresentative examples characterize using markers. 2. A, histograms fluorescence intensity (MFI; A) positive markers (B) expanded IL-15, specified. SEM ≥ 3). C–F. (C, D) (E, F) OVA323–339; (thymidine incorporation into DNA; E) (by ELISA; D, measured 72 h. presented two-way ANOVA t-test (C–F). modulates partially attributed influences organization, electron microscopy (EM) analyze replicas labeling anti-CD3ε antibody 10 nm gold-conjugated A; image lymphoblast EV2). small larger 4 genotypes 3A). there significant size 3B C). per each condition Table S1). As predicted, ≪ IL-15-differentiated S1E), coincided S1F G). These findings reinforce IL-15-induced versus IL-2-induced effector-like link nevertheless lymphoblasts, independently milieu. EV2. Analysis microscopyRepresentative replica stained Some areas enlarged distribution TCR-stained molecules. Scale bar, 50 nm. 3. A–C. EM (A; 6 cells/genotype; WT: 3,427, CCR5−/−: 3,528 particles), (B; WT, 8 15,419 particles; CCR5−/−, 5,410 particles) (C; 27,518 22,696 particles). field top panel shows gold particle anti-CD3ε-labeled cells; bottom, (mean SEM) particles clusters (gray bars) (red). Insets one, two, three, four, four particles, statistical analysis. Posterior IL-2-expanded (E) clustering parameter b (gray) (red); randomly generated distributions receptors blue. value condition. probability chance nearly 0% ROPE. oligomer BN-PAGE anti-CD3ζ immunoblotting 10, lysed buffer containing digitonin Brij-96. ferritin (f1, 440 f2, 880 kDa forms). ratio monomeric lysis quantified densitometry (right; G. Top, images showing Bottom, (WT, gray bars; 14,680 red; 15,374 (A–C, F, G), one-tailed Using Monte Carlo simulation, applied experimental cluster random particles. cases, observed experimentally differed significantly pure proximity S2). define model accounts dynamics (Castro 2014), Bayesian inference method estimates so-called parameter, b. Based model, concluded approaches 3D E). whereas very 3D), clear separation 3E). analyses provide mathematical framework validates EM. oligomerization blue-native gel electrophoresis (BN-PAGE) Swamy Schamel, digitonin, detergent disrupts components, comparable levels, 3F). Brij96, preserves nanoclusters, notable reduction large Two techniques controls purified selection S3). fewer, those counterparts 3G; Appendix S1), promotes endogenously 2011), it interest know defective suboptimal address question, treated antagonist TAK-779 various intervals throughout culture lymphoblasts. addition phase (blockade function) decreased untreated 4A). treatment did alter S4), CCR5-specific. 4. acti