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• W3033735980 abstract "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 and Oncology, Centro Nacional de Biotecnología (CNB/CSIC), Madrid, Spain Search for more papers by this author Raquel Blanco Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB/CSIC), Madrid, Spain Search for more papers by this author Josefina Casas Department of Biological Chemistry, Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain CIBER Liver and Digestive Diseases (CIBER-EDH), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author María E Sáez Centro Andaluz de Estudios Bioinformáticos (CAEBi), Seville, Spain Search for more papers by this author Elena Rodríguez-Bovolenta Department of Cell Biology and Immunology, Centro de Biología Molecular Severo Ochoa (CBMSO/CSIC), Madrid, Spain Search for more papers by this author Itziar de Rojas Alzheimer Research Center, Memory Clinic of the Fundació ACE, Institut Català de Neurociències Aplicades, Barcelona, Spain Search for more papers by this author Carina Drechsler Signaling Research Centers BIOSS and CIBSS, University of Freiburg, Freiburg, Germany Department of Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany Institute for Pharmaceutical Sciences, University of Freiburg, Freiburg, Germany Search for more papers by this author Luis Miguel Real Unit of Infectious Diseases and Microbiology, Hospital Universitario de Valme, Seville, Spain Department of Biochemistry, Molecular Biology and Immunology, School of Medicine, Universidad de Málaga, Málaga, Spain Search for more papers by this author Gemma Fabrias Department of Biological Chemistry, Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain CIBER Liver and Digestive Diseases (CIBER-EDH), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Agustín Ruíz Alzheimer Research Center, Memory Clinic of the Fundació ACE, Institut Català de Neurociències Aplicades, Barcelona, Spain CIBER Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Mario Castro orcid.org/0000-0003-3288-6144 Interdisciplinary Group of Complex Systems, Escuela Técnica Superior de Ingeniería, Universidad Pontificia Comillas, Madrid, Spain Search for more papers by this author Wolfgang WA Schamel orcid.org/0000-0003-4496-3100 Signaling Research Centers BIOSS and CIBSS, University of Freiburg, Freiburg, Germany Department of Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany Centre for Chronic Immunodeficiency (CCI), University of Freiburg, Freiburg, Germany Search for more papers by this author Balbino Alarcón orcid.org/0000-0001-7820-1070 Department of Cell Biology and Immunology, Centro de Biología Molecular Severo Ochoa (CBMSO/CSIC), Madrid, Spain Search for more papers by this author Hisse M van Santen orcid.org/0000-0003-0769-4511 Department of Cell Biology and Immunology, Centro de Biología Molecular Severo Ochoa (CBMSO/CSIC), Madrid, Spain Search for more papers by this author Santos Mañes Corresponding Author [email protected] orcid.org/0000-0001-8023-957X Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB/CSIC), Madrid, Spain Search for more papers by this author Ana Martín-Leal Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB/CSIC), Madrid, Spain Search for more papers by this author Raquel Blanco Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB/CSIC), Madrid, Spain Search for more papers by this author Josefina Casas Department of Biological Chemistry, Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain CIBER Liver and Digestive Diseases (CIBER-EDH), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author María E Sáez Centro Andaluz de Estudios Bioinformáticos (CAEBi), Seville, Spain Search for more papers by this author Elena Rodríguez-Bovolenta Department of Cell Biology and Immunology, Centro de Biología Molecular Severo Ochoa (CBMSO/CSIC), Madrid, Spain Search for more papers by this author Itziar de Rojas Alzheimer Research Center, Memory Clinic of the Fundació ACE, Institut Català de Neurociències Aplicades, Barcelona, Spain Search for more papers by this author Carina Drechsler Signaling Research Centers BIOSS and CIBSS, University of Freiburg, Freiburg, Germany Department of Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany Institute for Pharmaceutical Sciences, University of Freiburg, Freiburg, Germany Search for more papers by this author Luis Miguel Real Unit of Infectious Diseases and Microbiology, Hospital Universitario de Valme, Seville, Spain Department of Biochemistry, Molecular Biology and Immunology, School of Medicine, Universidad de Málaga, Málaga, Spain Search for more papers by this author Gemma Fabrias Department of Biological Chemistry, Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain CIBER Liver and Digestive Diseases (CIBER-EDH), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Agustín Ruíz Alzheimer Research Center, Memory Clinic of the Fundació ACE, Institut Català de Neurociències Aplicades, Barcelona, Spain CIBER Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Mario Castro orcid.org/0000-0003-3288-6144 Interdisciplinary Group of Complex Systems, Escuela Técnica Superior de Ingeniería, Universidad Pontificia Comillas, Madrid, Spain Search for more papers by this author Wolfgang WA Schamel orcid.org/0000-0003-4496-3100 Signaling Research Centers BIOSS and CIBSS, University of Freiburg, Freiburg, Germany Department of Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany Centre for Chronic Immunodeficiency (CCI), University of Freiburg, Freiburg, Germany Search for more papers by this author Balbino Alarcón orcid.org/0000-0001-7820-1070 Department of Cell Biology and Immunology, Centro de Biología Molecular Severo Ochoa (CBMSO/CSIC), Madrid, Spain Search for more papers by this author Hisse M van Santen orcid.org/0000-0003-0769-4511 Department of Cell Biology and Immunology, Centro de Biología Molecular Severo Ochoa (CBMSO/CSIC), Madrid, Spain Search for more papers by this author Santos Mañes Corresponding Author [email protected] orcid.org/0000-0001-8023-957X Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB/CSIC), Madrid, Spain Search for more papers by this author Author Information Ana Martín-Leal1,‡, Raquel Blanco1,‡, Josefina Casas2,3, María E Sáez4, Elena Rodríguez-Bovolenta5, Itziar Rojas6, Carina Drechsler7,8,9, Luis Miguel Real10,11, Gemma Fabrias2,3, Agustín Ruíz6,12, Mario Castro13, Wolfgang WA Schamel7,8,14, Balbino Alarcón5, Hisse M Santen5 and Santos Mañes *,1 1Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB/CSIC), Madrid, Spain 2Department of Biological Chemistry, Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain 3CIBER Liver and Digestive Diseases (CIBER-EDH), Instituto de Salud Carlos III, Madrid, Spain 4Centro Andaluz de Estudios Bioinformáticos (CAEBi), Seville, Spain 5Department of Cell Biology and Immunology, Centro de Biología Molecular Severo Ochoa (CBMSO/CSIC), Madrid, Spain 6Alzheimer Research Center, Memory Clinic of the Fundació ACE, Institut Català de Neurociències Aplicades, Barcelona, Spain 7Signaling Research Centers BIOSS and CIBSS, University of Freiburg, Freiburg, Germany 8Department of Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany 9Institute for Pharmaceutical Sciences, University of Freiburg, Freiburg, Germany 10Unit of Infectious Diseases and Microbiology, Hospital Universitario de Valme, Seville, Spain 11Department of Biochemistry, Molecular Biology and Immunology, School of Medicine, Universidad de Málaga, Málaga, Spain 12CIBER Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain 13Interdisciplinary Group of Complex Systems, Escuela Técnica Superior de Ingeniería, Universidad Pontificia Comillas, Madrid, Spain 14Centre for Chronic Immunodeficiency (CCI), University of Freiburg, Freiburg, Germany ‡These authors contributed equally to this work. **Corresponding author. Tel: +34 91 585 4840; Fax: +34 91 372 0493; E-mail: [email protected] EMBO J (2020)39:e104749https://doi.org/10.15252/embj.2020104749 See also: C Matti & DF Legler (August 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract CCR5 is not only a coreceptor for HIV-1 infection in CD4+ T cells, but also contributes to their functional fitness. Here, we show that by limiting transcription of specific ceramide synthases, CCR5 signaling reduces ceramide levels and thereby increases T-cell antigen receptor (TCR) nanoclustering in antigen-experienced mouse and human CD4+ T cells. This activity is CCR5-specific and independent of CCR5 co-stimulatory activity. CCR5-deficient mice showed reduced production of high-affinity class-switched antibodies, but only after antigen rechallenge, which implies an impaired memory CD4+ T-cell response. This study identifies a CCR5 function in the generation of CD4+ T-cell memory responses and establishes an antigen-independent mechanism that regulates TCR nanoclustering by altering specific lipid species. Synopsis CCR5 deficiency and the ccr5Δ32 polymorphism endow antigen-experienced CD4+ T cells with a ceramide-rich lipid environment, which restricts TCR nanoclustering. This reduces antigenic sensitivity and impairs CD4+ T:B cell cooperation for humoral responses after antigen re-encounter. CCR5 provides antigen-independent signals that regulate TCR nanoclustering in antigen-experienced CD4+ T cells. CCR5-induced TCR nanoclustering is independent of the CCR5 costimulatory activity on CD4+ T cells. CCR5 signals restrain transcription of ceramide synthase 2; this maintains control of the de novo Cer biosynthetic pathway and reduces ceramide levels. CCR5 maximizes restimulation of memory CD4+ T cells and production of high affinity class-switched antibodies after antigen re-encounter. Introduction The C-C motif chemokine receptor 5 (CCR5) is a seven-transmembrane G protein-coupled receptor (GPCR) expressed on the surface of several innate and adaptive immune cell subtypes, including effector and memory CD4+ T lymphocytes (Gonzalez-Martin et al, 2012). CCR5 acts also a necessary coreceptor for infection by HIV-1. An HIV-resistant population served to identify a 32-bp deletion within the CCR5 coding region (ccr5Δ32), which yields a non-functional receptor (Blanpain et al, 2002). Since ccr5Δ32 homozygous individuals are seemingly healthy, a radical body of thought considers that CCR5 is dispensable for immune cell function. Experimental and epidemiological evidence nonetheless indicates that CCR5 has an important role in innate and acquired immune responses. CCR5 and its ligands C-C motif ligand 3 (CCL3; also termed macrophage inflammatory protein [MIP]-1α), CCL4 (MIP-1β), CCL5 (regulated upon activation, normal T cell expressed and secreted [RANTES]), and CCL3L1 have been associated with exacerbation of chronic inflammatory and autoimmune diseases. Despite varying information due probably to ethnicity effects (Lee et al, 2013; Schauren et al, 2013), further complicated in admixed populations (Toson et al, 2017), epidemiological studies support the ccr5Δ32 allele as a marker for good prognosis for these overreactive immune diseases (Vangelista & Vento, 2017). In contrast, ccr5Δ32 homozygotes are prone to fatal infections by several pathogens such as influenza, West Nile, and tick-borne encephalitis viruses (Lim & Murphy, 2011; Falcon et al, 2015; Ellwanger & Chies, 2019). The mechanisms by which the ccr5Δ32 polymorphism affects all these pathologies have usually been linked to the capacity of CCR5 to regulate leukocyte trafficking. For example, CCR5 deficiency reduces recruitment of influenza-specific memory CD8+ T cells and accelerates macrophage accumulation in lung airways during virus rechallenge (Dawson et al, 2000; Kohlmeier et al, 2008); this could lead to acute severe pneumonitis, a fatal flu complication. CCR5 nonetheless has migration-independent functions that maximize T-cell activation by affecting immunological synapse (IS) formation (Molon et al, 2005; Floto et al, 2006; Franciszkiewicz et al, 2009) as well as T-cell transcription programs associated with cytokine production (Lillard et al, 2001; Camargo et al, 2009). CCR5 and its ligands are also critical for cell-mediated immunity to tumors and pathogens, including HIV-1 (Dolan et al, 2007; Ugurel et al, 2008; González-Martín et al, 2011; Bedognetti et al, 2013). Whereas the role of CCR5 in T-cell priming is well established, its involvement in memory responses has not been addressed in depth. Only a single report suggested CCR5 involvement in CD4+ T-cell promotion of memory CD8+ T-cell generation through a migration-dependent process (Castellino et al, 2006). It remains unknown whether CCR5 endows memory T cells with additional properties. One such property is the elevated sensitivity of effector and memory (“antigen-experienced”) CD4+ and CD8+ T cells to their cognate antigen compared to naïve cells (Kimachi et al, 1997; Kersh et al, 2003; Huang et al, 2013). This sensitivity gradient (memory ≫ effector > naïve) in CD8+ T cells is linked to increased valency of preformed T-cell antigen receptor (TCR) oligomers at the cell surface, termed TCR nanoclusters (Kumar et al, 2011). This antigen-independent TCR nanoclustering (Schamel et al, 2005, 2006; Lillemeier et al, 2010; Sherman et al, 2011; Schamel & Alarcon, 2013) enhances antigenic sensitivity by increasing avidity to multimeric peptide-major histocompatibility complexes (Kumar et al, 2011; Molnar et al, 2012) and by allowing cooperativity between TCR molecules (Martínez-Martín et al, 2009; Martín-Blanco et al, 2018). TCRβ subunit interaction with cholesterol (Chol) and the presence of sphingomyelins (SM) are both essential for TCR nanoclustering (Molnar et al, 2012; Beck-Garcia et al, 2015). Replacement of Chol by Chol sulfate impedes TCR nanocluster formation and reduces CD4+CD8+ thymocyte sensitivity to weak antigenic peptides (Wang et al, 2016). Whether antigen-experienced CD4+ T-cell sensitivity is linked to TCR nanoscopic organization and the homeostatic factors that regulate TCR nanoclustering remains unexplored. Given its co-stimulatory role in CD4+ T cells, we speculated that CCR5 signals would affect the antigenic sensitivity of CD4+ memory T cells. To test this hypothesis, we analyzed the function of in vivo-generated memory CD4+ T cells in wild-type (WT) and CCR5−/− mice, and the effect of CCR5 deficiency on CD4 T-cell help in the T-dependent humoral response. We found that CCR5 is necessary for the establishment of a functional CD4 memory response through a mechanism independent of its co-stimulatory role for the TCR signal. We show that CCR5 deficiency does not affect memory CD4 T-cell generation, but reduces their sensitivity to antigen. Our data demonstrate an unreported CCR5 regulatory role in memory CD4+ T-cell function by inhibiting the synthesis of ceramides, which are identified here as negative membrane regulators of TCR nanoscopic organization. Results CCR5 deficiency impairs the CD4+ T-cell memory response To determine the role of CCR5 in CD4+ memory T-cell generation and/or function, we adoptively transferred congenic CD45.1 mice with lymph node/spleen cell suspensions from OT-II WT or CCR5−/− mice (CD45.2) and subsequently infected them with OVA-encoding vaccinia virus; 5 weeks post-immunization, we analyzed spleen CD45.2+ donor cells from OT-II mice. CCR5 expression on OT-II cells affected neither the total number of memory CD4+ T cells (Fig 1A and B) nor the percentage of CD4+ TEM (CD44hi; CD62L−; Fig 1C) or TCM (CD44hi; CD62L+; Fig 1D) cells generated. OT-II WT cells nonetheless had stronger responses to antigenic restimulation than OT-II CCR5−/− memory T cells, as determined by the percentage of interferon (IFN)γ-producing cells after ex vivo stimulation with OVA323–339 (Fig 1E). Figure 1. CCR5 deficiency impairs CD4+ T-cell memory responses A. Representative plots of splenocytes from CD45.1 mice adoptively transferred with CD45.2 OT-II WT or CCR5−/− lymph node cell suspensions, 5 weeks after infection with rVACV-OVA virus. The gating strategy used to identify the memory CD4+ T-cell subtypes is shown (n = 5). B. Absolute number of OT-II cells recovered in spleens of mice as in A (n = 5). C, D. Percentage of CD4+ TEM (C) and TCM (D) in the OT-II WT and CCR5−/− populations (n = 5). E. IFNγ-producing OT-II WT and CCR5−/− memory cells isolated from mice as in (A) and restimulated ex vivo with OVA323–339 (1 μM) (n = 4). F. Immunization scheme for NIP-OVA and NIP-KLH in WT and CCR5−/− mice. G–I. Representative plots (G) and quantification of the frequency (H) and absolute number (I) of Tfh cells (CD4+CD44+PD-1+CXCR5+) in the spleen after primary immunization (day 7) with NIP-OVA (n = 7). J, K. ELISA analysis of high- (J) and low-affinity (K) isotype-specific anti-NIP antibodies in sera from OVA/OVA- and OVA/KLH-immunized mice (day 15 post-challenge; n = 5 mice/group). Data representative of one experiment of two. Data information: (B–E, H–K), Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired Student's t-test. Download figure Download PowerPoint We also studied T cell-dependent B-cell responses in WT and CCR5−/− mice after immunization with the hapten 4-hydroxy-3-iodo-5-nitrophenylacetyl coupled to ovalbumin (NIP-OVA; Fig 1F). We detected no difference in the percentage or absolute number of T follicular helper (Tfh) cells (CD4+, CD44hi, CXCR5+, PD1+) between WT and CCR5−/− mice at 7 days post-immunization (Fig 1G–I). At day 30, half of the mice were boosted with the same NIP-OVA immunogen (OVA/OVA) and the other half received NIP conjugated with another carrier protein (OVA/KLH); levels of NIP-specific high- and low-affinity immunoglobulins (Ig) were analyzed 15 days later. Comparison of the humoral responses between OVA/OVA- and OVA/KLH-immunized mice would assess the effect of memory CD4+ T cells specific for the first carrier protein on the humoral response to NIP. There were no differences in high/low-affinity NIP-specific IgM production between WT and CCR5−/− mice with either immunization strategy (Fig 1J and K). CCR5 deficiency markedly impaired the generation of high-affinity class-switched anti-NIP antibodies specifically in OVA/OVA-immunized mice (Fig 1J and K). Since class switching was similar in WT and CCR5−/− OVA/KLH-immunized mice, our results suggest that CCR5 deficiency reduces the generation of high-affinity class-switched immunoglobulins due to deficient memory CD4+ T-cell function. The CCR5 effect on antigen-experienced CD4+ T cells is cell-autonomous To test whether the in vivo memory defect associated with CCR5 deficiency was intrinsic to CD4+ T cells, we activated OT-II WT and CCR5−/− spleen T cells with OVA323–339 antigen for 3 days; after antigen removal, we cultured cells with IL-2 or IL-15. OT-II cells that differentiated in exogenous IL-2 expressed CCL3, CCL4, CCL5, and a functional CCR5 receptor, as determined by their ability to flux Ca2+ and migrate after CCL4 stimulation (Appendix Fig S1A–D). Like CD8+ T cells (Richer et al, 2015), OT-II cells cultured with IL-15 showed a memory-like phenotype (Fig EV1); they were smaller than IL-2-cultured cells and retained CD62L with reduced activation marker expression (CD25, CD69, CD44) compared to IL-2-cultured T cells (Fig 2A). Findings were similar in OT-II WT and CCR5−/− cells (Fig 2B), which reinforced the idea that CCR5 is not involved in CD4+ T memory cell differentiation. Restimulation of IL-2- or IL-15-expanded OT-II lymphoblasts with the OVA323–339 peptide nonetheless indicated that CCR5-expressing cells showed strong proliferation and higher IL-2 production at low antigen concentrations than CCR5-deficient cells (Fig 2C–F), indicative of an increased number of cells responding to antigenic stimulation. CCR5 might thus increase the antigenic sensitivity of antigen-experienced CD4+ T cells in a cell-autonomous manner. Click here to expand this figure. Figure EV1. Gating strategy for characterization of IL-2 and IL-15-expanded OT-II lymphoblastRepresentative examples of the gating strategy used to characterize the IL-2- and IL-15-expanded OT-II lymphoblasts using the indicated markers. Download figure Download PowerPoint Figure 2. CCR5 increases the sensitivity of antigen-experienced CD4+ T cells A, B. Representative histograms and quantification of mean fluorescence intensity (MFI; A) or the percentage of cells positive for the indicated memory markers (B) in OT-II WT and CCR5−/− lymphoblasts expanded in IL-2 or IL-15, as specified. Data shown as mean ± SEM (n ≥ 3). The gating strategy is shown in Fig EV1. C–F. IL-2- (C, D) and IL-15-expanded lymphoblasts (E, F) were restimulated with indicated concentrations of OVA323–339; cell proliferation (thymidine incorporation into DNA; C, E) and IL-2 production (by ELISA; D, F) were measured after 72 h. Data are presented as mean ± SEM (n = 5). Data information: *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA (B) or two-tailed unpaired Student's t-test (C–F). Download figure Download PowerPoint CCR5 modulates TCR nanoclustering in antigen-experienced CD4+ T cells The high antigenic sensitivity of antigen-experienced CD8+ T cells was partially attributed to increased TCR nanoclustering (Kumar et al, 2011). To determine whether CCR5 deficiency influences TCR organization, we used electron microscopy (EM) to analyze surface replicas of OT-II WT and CCR5−/− naïve cells and lymphoblasts after labeling with anti-CD3ε antibody and 10 nm gold-conjugated protein A; a representative image of a IL-15-expanded WT lymphoblast is shown (Fig EV2). We found no differences in TCR nanoclusters between OT-II WT and CCR5−/− naïve cells, which had a small percentage of TCR nanoclusters larger than 4 TCR in both genotypes (Fig 3A). In contrast, there was a significant increase in TCR nanocluster number and size in WT compared to CCR5−/− lymphoblasts (Fig 3B and C). The number of TCR nanoclusters per cell analyzed in each condition is also indicated (Appendix Table S1). As predicted, there was a gradient in TCR nanoclustering of naïve ≪ IL-2- < IL-15-differentiated OT-II WT cells (Appendix Fig S1E), which coincided with increased antigenic sensitivity of the IL-15-expanded cells (Appendix Fig S1F and G). These findings thus reinforce the IL-15-induced memory-like phenotype versus the IL-2-induced effector-like phenotype and link TCR nanoclustering with increased sensitivity in antigen-experienced CD4+ T cells. The difference in TCR nanoclustering between WT and CCR5−/− cells was nevertheless similar in IL-2- and IL-15-expanded lymphoblasts, which indicates that CCR5 affects TCR nanoclustering in lymphoblasts independently of the cytokine milieu. Click here to expand this figure. Figure EV2. Analysis of TCR molecules by electron microscopyRepresentative cell surface replica of a IL-15-expanded WT lymphoblast stained with anti-CD3ε antibody and gold-conjugated protein A. Some cell areas have been enlarged to show the distribution of the TCR-stained molecules. Scale bar, 50 nm. Download figure Download PowerPoint Figure 3. CCR5 increases TCR nanoclustering in antigen-experienced CD4+ T cells A–C. Analysis of TCR nanoclustering by EM in OT-II WT and CCR5−/− naïve cells (A; n = 6 cells/genotype; WT: 3,427, CCR5−/−: 3,528 particles), and IL-2- (B; WT, n = 8 cells, 15,419 particles; CCR5−/−, n = 6 cells, 5,410 particles) or IL-15-expanded lymphoblasts (C; WT, n = 8 cells, 27,518 particles; CCR5−/−, n = 7 cells, 22,696 particles). A representative small field image at the top of each panel shows gold particle distribution in the cell surface replicas of anti-CD3ε-labeled cells; at bottom, quantification (mean ± SEM) of gold particles in clusters of indicated size in WT (gray bars) and CCR5−/− cells (red). Insets show the distribution of clusters of one, two, three, four, or more than four particles, and statistical analysis. D, E. Posterior distribution in naïve (D) and IL-2-expanded lymphoblasts (E) of the clustering parameter b for WT (gray) and CCR5−/− cells (red); randomly generated distributions of receptors are shown in blue. The mean value of the b parameter is indicated for each condition. The probability of a chance distribution similar to that determined in cells is nearly 0% by the ROPE. F. Comparison of TCR oligomer size using BN-PAGE and anti-CD3ζ immunoblotting in day 10, IL-2-expanded WT and CCR5−/− OT-II lymphoblasts lysed in buffer containing digitonin or Brij-96. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms). The ratio of TCR nanoclusters to monomeric TCR in each lysis condition was quantified by densitometry (right; n = 5). G. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of CD4+ T cells isolated from OVA/OVA-immunized WT and CCR5−/− mice. Bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size (WT, gray bars; n = 5 cells, 14,680 particles; CCR5−/−, red; n = 7 cells, 15,374 particles). Insets show the distribution between clusters of one, two, three, four, or more than four particles, and statistical analysis. Data information: (A–C, F, G), Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, one-tailed unpaired Student's t-test. Scale bar, 50 nm (A–C, G). Download figure Download PowerPoint Using a Monte Carlo simulation, we applied data from surface replicas of naïve and IL-2-expanded OT-II lymphoblasts to determine whether the experimental frequency of cluster size was due to random distribution of gold particles. In all cases, the cluster distributions observed experimentally differed significantly from pure random proximity between clusters (Appendix Fig S2). To define the differences between OT-II WT and CCR5−/− cells, we used a model that accounts for receptor clustering dynamics (Castro et al, 2014), a Bayesian inference method that estimates the so-called clustering parameter, b. Based on this model, we concluded that the probability of a chance nanocluster distribution similar to that observed for naïve and activated OT-II WT and CCR5−/− cells approaches 0% (Fig 3D and E). Posterior distribution analysis also showed that whereas the clustering parameter was very similar between naïve OT-II WT and CCR5−/− cells (Fig 3D), there was clear separation in lymphoblasts (Fig 3E). These analyses provide a mathematical framework that validates the TCR nanoclustering differences between WT and CCR5−/− cells, as determined by EM. The differences in TCR oligomerization between OT-II WT and CCR5−/− lymphoblasts were also studied using blue-native gel electrophoresis (BN-PAGE) (Schamel et al, 2005; Swamy & Schamel, 2009). Cell lysis with digitonin, a detergent that disrupts TCR nanoclusters into their monomeric components, showed that WT and CCR5−/− lymphoblasts expressed comparable TCR levels, as detected with anti-CD3ζ antibodies (Fig 3F). Cell lysis with Brij96, which preserves TCR nanoclusters, showed a notable reduction in large TCR complexes in CCR5−/− compared to WT lymphoblasts (Fig 3F). Two independent techniques thus support a CCR5 role in TCR nanoscopic organization in antigen-experienced CD4+ T cells. To determine whether CCR5 controls TCR nanoclustering in in vivo-generated memory T cells, we analyzed TCR distribution in surface replicas of CD4+ memory T cells purified by negative selection from OVA/OVA-immunized WT and CCR5−/− mice (Appendix Fig S3). CD4+ memory cells from CCR5−/− mice showed fewer, smaller TCR nanoclusters than those from WT counterparts (Fig 3G; Appendix Table S1), which indicates that CCR5 promotes formation of large TCR nanoclusters in endogenously generated CD4+ memory T cells. CCR5-induced TCR nanoclustering is independent of its co-stimulatory activity Since CCR5 has co-stimulatory functions in CD4+ T-cell priming (Molon et al, 2005; González-Martín et al, 2011), it is of interest to know whether defective TCR clustering in CCR5−/− lymphoblasts is due to suboptimal primary activation of these cells. To address this question, we treated OT-II WT cells with the CCR5 antagonist TAK-779 at various intervals throughout culture and analyzed TCR nanoclusters in IL-2-expanded T lymphoblasts. TAK-779 addition during the priming phase (blockade of CCR5 co-stimulatory function) decreased the percentage of large TCR nanoclusters compared to untreated controls (Fig 4A). TAK-779 treatment did not alter TCR clustering in OT-II CCR5−/− cells (Appendix Fig S4), which indicates that the TAK-779 effect on OT-II cells is CCR5-specific. Figure 4. CCR5-induced TCR nanoclustering is specific and independent of its co-stimulatory activity OT-II WT cells were acti" @default.
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• W3033735980 title "<scp>CCR</scp> 5 deficiency impairs <scp>CD</scp> 4 ⁺ T‐cell memory responses and antigenic sensitivity through increased ceramide synthesis" @default.
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• W3033735980 doi "https://doi.org/10.15252/embj.2020104749" @default.
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