Chaperone-mediated autophagy regulates T cell responses through targeted degradation of negative regulators of T cell activation

Abstract

Chaperone-mediated autophagy (CMA) targets soluble proteins for lysosomal degradation. Here we found that CMA was activated in T cells in response to engagement of the T cell antigen receptor (TCR), which induced expression of the CMA-related lysosomal receptor LAMP-2A. In activated T cells, CMA targeted the ubiquitin ligase Itch and the calcineurin inhibitor RCAN1 for degradation to maintain activation-induced responses. Consequently, deletion of the gene encoding LAMP-2A in T cells caused deficient in vivo responses to immunization or infection with Listeria monocytogenes. Impaired CMA activity also occurred in T cells with age, which negatively affected their function. Restoration of LAMP-2A in T cells from old mice resulted in enhancement of activation-induced responses. Our findings define a role for CMA in regulating T cell activation through the targeted degradation of negative regulators of T cell activation.

Figure 1. TCR engagement activates CMA

(a-b) Immunoblot of LAMP-2A in total cell lysates (representative of 5 experiments) and quantification of Lamp2a gene expression by qPCR (relative to resting cells) in naïve CD4⁺ T cells and T_H1 and T_H2 cells following activation with anti-CD3 and anti-CD28 antibodies. (c) LAMP-2A (green) expression determined by immunofluorescence in resting and activated T_H1 cells. Nuclei were stained with DAPI (blue). Original magnification 630x. Quantification of number of lysosomes (mean+s.e.m.) performed in 5 fields (20-30cells) from each of three different experiments (n=3; t test; *p=0.013). (d) Electron microscopy images of resting and 24 hours activated T_H1 cells. Fields show representative cells and higher-magnification fields show individual lysosomes (black arrows denote lysosomes). Morphometric analysis performed in 8 micrographies (original magnification ×12,000), corresponding to cells from two different experiments. Values are mean+s.e.m.. (n=2; t test; *p =0.037; **p=0.034). (e) KFERQ–PA–mCherry1 distribution in T_H1 cells transiently transfected with a CMA reporter vector, photoactivated and stimulated with anti-CD3+anti-CD28 in the presence or absence of 3-Methyladenine (3-MA). Images show representative cells (with arrows indicating mCherry positive puncta; original magnification of 630x). Quantification shown in graph (mean+s.e.m. of 3 different experiments with >50 cells counted per experiment) (n=3; ANOVA with Tukey post-test; *p<0.05). (f) Hsc70 detected by immunoblot in cytosolic (Cyto) and lysosomal fractions from resting (Lys-R) and stimulated (Lys-A) T_H1 cells. LAMP-1 and LAMP-2A are used as controls. Blot is representative of 6 different experiments. (g) Immunoblot of LAMP-2A in resting T_H1 cells and cells activated in the presence or absence of N-acetylcysteine (NAc) (representative of two different experiments). (h) Lamp2a gene expression quantified by qPCR in T_H1 cells and cells activated in the presence or absence of NAc (values, fold increase relative to resting cells, are mean+s.e.m.; n= 4; t test; *p=0.007). (i-j) Relative luciferase activity (to resting cells) in T_H1 cells transiently transfected with a vector containing a luciferase reporter under the control of the Lamp2 proximal promoter and stimulated with (i) plate-bound anti-CD3 and anti-CD28 or (j) 50 μM paraquat (Pq) or 1 mM H₂O₂ for for 16 hours. Results are mean+s.e.m. from (i) 4 or (j) 3 different experiments. (k) Immunoblot showing LAMP-2A protein in total cell lysates (representative blot from 2 similar experiments) of resting and activated (24 hours) T_H1 cells transfected with a control non-targeting siRNA (Scr) or siRNAs specific for Duox1 (Dx) or Uqcrfs1 (Uq). (l) Immunoblot showing LAMP-2A in lysates from T_H1 cells resting or activated in the presence or absence of cyclosporine A (CsA) (representative of 5 different experiments). (m) Chromatin immunoprecipitation showing NFAT1 recruitment to the Lamp2 gene promoter (% of input precipitated using anti-NFAT1 antibodies) in resting and activated T_H1 cells. Binding to Adad1 (Ctrl), is used as negative control, while binding to the Il2 promoter is used as positive control. Graph shows mean+s.e.m. from 4 independent experiments.

Figure 2. Silencing of LAMP-2A expression impairs T cell activation

(a) Immunoblot of LAMP-2A protein in sorted GFP⁺ T cells transduced with a lentivirus expressing GFP and a specific shRNA for Lamp2a (shL2A) or a non-targeting control shRNA (Scr). (b) IL-2 and INF-γ production measured by ELISA in resting and 24 hours stimulated (anti-CD3+anti-CD28) T_H1 cells transduced with a lentivirus expressing shL2A or Scr. Results are mean+s.e.m. from 4 (IL-2) or 5 (IFN-γ) different experiments (t test; *p=0.031; **p=0.038). (c) T cell proliferation measured by BrdU incorporation in resting and 24 hours stimulated T_H1 cells transduced with a lentivirus expressing shL2A or Scr. Results are mean+s.e.m. from 4 different experiments (t test *p=0.013). (d) Quantification of Annexin V⁺ cells (mean+s.e.m.) in resting and stimulated T_H1 cells transduced with a lentivirus expressing shL2A or Scr (n=3).

Figure 3. Degradation of Rcan-1 and Itch by CMA in activated T cells

(a) Immunoblot showing enrichment of Itch and Rcan-1 in lysosomal fractions isolated from activated (24 hours antti-CD3+anti-CD28) T_H1 cells compared to lysosomes obtained from resting cells. LAMP-1 is used as control. (b) Itch and Rcan-1 detected by immunoblot in lysosomal fractions from T_H1 cells activated for 24 hours where lysosomal proteases had been inhibited or not for the last 3 hours with NH₄Cl and leupeptin (Lys.Inhib.). LAMP-1 is used as control. (c) Immunoblot showing total cellular Itch and Rcan-1 protein in resting T_H1 cells and in cells activated (24 hours) in the presence or absence of N-acetylcysteine (N-Ac). β-actin is used as control. (d) Immunoblot of cell lysates from T_H1 cells transfected with plasmids expressing GFP or Myc tagged wild type or KFERQ-mutants (GFP-mu-Itch and Myc-mu-Rcan-1) Itch or Rcan-1, and activated for 24 hours with anti-CD3+anti-CD28. Efficiency of transfection is controlled in all cells with a co-transfected GFP expression plasmid. Blots are representative of 4 (a and b), 2 (c) and 3 (d) different experiments. (e-f) Cell proliferation and IL-2 production measured in T_H1 cells transfected with plasmids expressing GFP (Ctrl), wild type Itch, mu-Itch, Rcan-1, mu-Rcan-1 and activated with anti-CD3+anti-CD28 for 24 (IL-2 production) or 48 (proliferation) hours Results are mean+s.e.m. from three different experiments (t test; (e) *p=0.019; **p=0.0034; (f) *p=0.046; **p=0.044).

Figure 4. Reduced T cell responses in CMA-deficient mice

(a) Immunoblot of LAMP-2A in CD4⁺ T cells, liver, kidneys, thymus and lung of L2A-cKO (KO) mice or wild type littermates (WT). (b) Lamp2a, b and c mRNA expression (mean+s.e.m, expressed as relative to WT values) measured by qPCR in T cells from WT or L2A-cKO mice (n=3). (c) Macroautophagy flux (LC3 flux) measured in resting CD4⁺ T cells from WT and KO mice or cells activated with anti-CD3 and anti-CD218 for 24 hours (Act). (d) Representative image (from 6 different mice) of spleens from WT and L2A-cKO mice. Distribution of peripheral CD4⁺ and CD8⁺ T cells was determined by FACS. (e-f) Cell proliferation and IL-2 production measured in CD4⁺ T cells isolated from spleens of WT and KO mice following stimulation for 48 hours. Results are mean+s.e.m. of 4 (e) 6 (f) independent experiments (t test, (e) *p=0.0062; (f) *p=0.006). g. Immunoblot of Itch and Rcan-1 proteins in total lysates from activated (24 hours) CD4⁺ T cells of 3 different KO mice compared to WT littlermate controls. * Non-specific band. (h) Expression of Lamp2a mRNA assessed by qPCR in samples obtained from resting or activated for 24 hours CD4⁺ T cells isolated form WT or KO mice Data (mean+s.e.m. from 3 different experiments) is presented as fold induction of mRNA expression following activation. (i-j) Recall responses to OVA_323-339 peptide (cell proliferation and IL-2 production) in draining lymph nodes isolated from OVA_323-339-immunized KO mice and WT littermates. Results are mean+s.e.m. from 4 different experiments (t test; (i) *p=0.0054; (j) *p=0.0052). (k-l) Recall responses (cell proliferation and cytokine production) to Listeria in CD4⁺ T cells isolated from naïve or previously infected WT or KO mice. Graphs show mean+s.e.m. from 6 mice analyzed in two independent experiments (t test; (k) *p=0.032; (l) *p=0.02). (m) Listeria CFU in spleen and liver measured in previously immunized WT and KO mice after reinfection with a high dose of Listeria. Results show mean+s.e.m. from data obtained in triplicate from three different mice (t test; *p=0.008; **p=0.001).

Figure 5. Deficient regulation of LAMP-2A expression in aged T cells

(a) LAMP-2A protein detected by immunoblot in CD4⁺ T cells from 4-month and 22-month old mice that were left resting or stimulated with anti-CD3+anti-CD28 for 24 hours. Image representative of 3 different experiments. (b) Immunoblot showing Rcan-1 and Itch in CD4⁺ T cells from 4 and 22 month old mice stimulated for 24 hours. A quantification of blots from 3 different mice is shown (% of the protein content in 4 month old mice. mean+s.e.m.; t test; *p=0.044). (c-d) Immunoblot of LAMP-2A in total cell lysates prepared form resting and activated (24 hours) naive and memory CD4⁺ T cells (c). Similar experiments using naïve and memory CD4⁺ T cells from 4 month and 22 month old mice (d). A representative immunoblot and the quantification from blots of 3 different young and 3 old mice are shown (mean+s.e.m.; t test; *p=0.009). (e) EMSA showing NFAT binding using nuclear extracts from CD4⁺ T cells isolated from 3, 12 or 22 month old mice and activated for 4 hours. (N:NFAT; FP: free probe) (f) ROS production measured by FACS using CellRox-green in CD4⁺ T cells isolated from 4 or 22 month old mice and activated for 12 hours. Data (mean+s.e.m. from 3 different mice) is presented as mean fluorescence intensity (MFI). (g-i) Immunoblot for LAMP-2A (g), proliferation (h) and IL-2 production (i) measured in T_H1 cells from 4 or 22 month old mice transduced with a retrovirus expressing human LAMP-2A and GFP (RV-Lamp2a) or just GFP (RV-GFP). Infected cells were sorted by GFP expression, cultured for 2 weeks and then stimulated for 24 to 48 hours. Results are mean+s.e.m. from 4 (h) and 5 (i) different experiments (ANOVA with Tukey post-test; *p<0.05). (j) Immunoblot showing LAMP-2A in total cell lysates obtained from resting and activated CD4⁺ T cells from two human donors (A and B). (k) Lamp2a mRNA expression measured by real time PCR in naïve and memory CD4⁺ T cells obtained from 3 young (26±2 years old) and 6 old (72±6 years old) human donors and activated for 24 hours. 2^−ΔCt values (mean+s.e.m.) were calculated using actin as a reference gene (Mann-Whitney; *p=0.0033; **p=0.047).