NAC1 confines virus-specific memory formation of CD4+ T cells through the ROCK1-mediated pathway

Abstract

Nucleus accumbens-associated protein 1 (NAC1), a transcriptional cofactor, has been found to play important roles in regulating regulatory T cells, CD8⁺ T cells, and antitumor immunity, but little is known about its effects on T-cell memory. In this study, we found that NAC1 expression restricts memory formation of CD4⁺ T cells during viral infection. Analysis of CD4⁺ T cells from wild-type (WT) and NAC1-deficient (^-/- ) mice showed that NAC1 is essential for T-cell metabolism, including glycolysis and oxidative phosphorylation, and supports CD4⁺ T-cell survival in vitro. We further demonstrated that a deficiency of NAC1 downregulates glycolysis and correlates with the AMPK-mTOR pathway and causes autophagy defective in CD4⁺ T cells. Loss of NAC1 reduced the expression of ROCK1 and the phosphorylation and stabilization of BECLIN1. However, a forced expression of ROCK1 in NAC1^-/- CD4⁺ T cells restored autophagy and the activity of the AMPK-mTOR pathway. In animal experiments, adoptively transferred NAC1^-/- CD4⁺ T cells or NAC1^-/- mice challenged with VACV showed enhanced formation of VACV-specific CD4⁺ memory T cells compared to adoptively transferred WT CD4⁺ T cells or WT mice. This memory T-cell formation enhancement was abrogated by forcing expression of ROCK1. Our study reveals a novel role for NAC1 as a suppressor of CD4⁺ T-cell memory formation and suggests that targeting NAC1 could be a new approach to promoting memory CD4⁺ T-cell development, which is critical for an effective immune response against pathogens.

Keywords: CD4+ T cells; NAC1; ROCK1; cellular metabolism; memory formation.

Figure 1.. Loss of NAC1 alters the survival, function, and metabolism of CD4⁺ T cells.

(A&B) T cells from pooled LNs and spleen of WT and NAC1^−/− mice were analyzed by flow cytometry for cell frequency. The data shown are representative of five mice per group of three independent experiments. *, p < 0.05. (C-H) Purified CD4⁺ T cells were isolated from WT and NAC1^−/− mice and stimulated with coated anti-CD3 Ab and soluble anti-CD28 Ab. The plotting data shown are representative of three identical experiments. (C) The cell recovery was examined with trypan blue exclusion from day 0 to day 8. The frequency of T cells on day 0 was assigned a value of 100%. *, p < 0.05; **, p < 0.01. Repeats n= 4. (D) T cells were stimulated, and intracellular cytokines (IL-2 and IFN-γ) were analyzed through flow cytometry. Repeats n=3. (E&F) 2X10⁵ stimulated CD4⁺ T cells were seeded into Seahorse XF96 culture plate. The Extracellular Acidification Rate (ECAR) was monitored with Rotenone and Antimycin A (ROT/AA), 2-deoxy-glucose (2-DG) drugs injection at different time points in the figure. Sample number (n=4), **, p <0.01. (G&H) 2X10⁵ stimulated CD4⁺ T cells were seeded into Seahorse XF96 culture plate. The Oxygen Consumption Rate (OCR) was measured with Oligomycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), Rotenone and Antimycin A (ROT/AA) drugs injections at different time points in the figure. Sample number (n=4), **, p <0.01.

Figure 2.. NAC1 regulates glycolysis of CD4⁺ T cells through the AMPK-mTOR pathway.

CD4⁺ T cells were isolated from the pooled LNs and spleen of WT and NAC1^−/− mice and activated with coated anti-CD3 Ab and soluble anti-CD28 Ab. Proteins were extracted from the cells on different days. (A) p-mTOR/ mTOR activity was checked by immunoblots on day 3. The blot images are representative of three identical experiments. *, p < 0.05. (B) The activated T cells were treated with DMSO, dactolisib (PI3K inhibitor), MK-2206 2HCl (AKT inhibitor), OSU-03012 (PDK1 inhibitor) according to the concentration indicated in the figures. p-S6 protein was analyzed by flow cytometry using intracellular staining on day 0. Mean Fluorescence intensity (MFI) was calculated. Repeats n=3. (C) PI3K and AKT expression were tested by immunoblots. Repeats n=3. (D) PDK1 expressions were verified on day 0, day 3, and day 6. Repeats n=3. (E-F) AMPK activity was checked on day 3 and day 6. The ratio of AMPK/ p-AMPK is calculated by ImageJ. The blot images are representative of three identical experiments. **, p < 0.01.

Figure 3.. Effect of NAC1 expression on autophagy in CD4⁺ T cells.

CD4⁺ T cells were isolated from the pooled LNs and spleen of WT or NAC1^−/− mice and stimulated with coated anti-CD3 Ab and soluble anti-CD28 Ab. (A) Cells were cultured with either DMSO (Control) or bafilomycin for 2 hours before protein extraction on day 3 and day 6. Autophagic adaptor proteins, including both P-62 and LC3, were analyzed by immunoblot. The blot images are representative of three identical experiments. (B-D) Purified CD4⁺ T cells were cultured for 3 days. Repeats n=3. (B&C) Confocal microscope analysis on P-62 expression. Cells were stained with CD4 marker (Red), nucleus (Blue), and P-62 (Green). The scale bar is 20 μm. The green puncta for 3 cells were accumulated together for comparison. ***, p < 0.001. An unpaired student T-test was used for quantification. (D) P-62 MFI was analyzed by flow cytometry. (E-G) Autophagosome formation was checked by transmission electron microscope (TEM). The scale bar is 2 µm. Both autophagosome numbers and area were calculated and quantified based on more than 10 micrographics. Blue arrows or red arrows were used to indicate the autophagosome in WT or NAC1^−/− CD4⁺ T cells. *, p < 0.05; **, p < 0.01.

Figure 4.. NAC1 regulates autophagy of CD4⁺ T cells through ROCK1-mediated phosphorylation of BECLIN1.

(A) The BECLIN1 and PIK3C3 expression on day 0, day 3, and day 6. T cells were activated, and proteins were extracted from the activated cells on different days. The ratio of BECLIN1/ β-ACTIN was calculated by ImageJ. The blot images are representative of three identical experiments. *, p < 0.05; **, p < 0.01; ns, p > 0.05. (B) T cells mRNA was extracted from activated T cells on day 0. cDNA was used for Autophagy Pathway RNA Microarray. mRNA expression ratio of NAC1^−/− / WT had displayed among 84 autophagy pathway-related genes. Repeats n=3. (C) Beclin1 mRNA expression was confirmed on day 0 and day 3 by qPCR. ns, p > 0.05. Sample number = 4. (D) T cell proteins were extracted from either WT or NAC1^−/− CD4⁺ T cells on day 3 for immunoprecipitation with either anti-BECLIN1 or anti-IgG antibodies. The eluted fractions were detected with anti-p-Threonine and anti-BECLIN1 immunoblots. Repeats n=3. (E) The ROCK1 protein expression was detected by immunoblot. Repeats n=3. *, p < 0.05 (F) The Rock1 mRNA expression was quantified by qPCR on day 0 and day 3. Sample number = 4. **, p < 0.01; ns, p > 0.05.

Figure 5.. NAC1^−/− mice sustain higher survival of VACV-specific CD4⁺ T cells and enhance formation of the VACV-specific memory CD4⁺ T cells.

WT or NAC1^−/− mice were infected with the vaccinia virus (VACV) at 2X10⁶ PFU. The VACV-infected mice were sacrificed at 1,2,3,4,5 weeks to get the pooled LNs and spleen. Then the tissue was smashed and filtered with 40 μm cell strainers. The red blood cells were removed by RBC lysis buffer. The total cell number for each mouse was calculated by cell counter. Multiple cell surface markers (CD4, CD44, CD69, CD197) and VACV tetramer staining were applied before flow cytometry analysis. The representative data are three identical experiments. Mice number for each group (n=5–7). (A) Representative flow cytometry plotting for those VACV-specific CD4⁺ T cells during 1 to 5 weeks. (B) VACV-specific CD4⁺ T cell number change in 35 days. (C) VACV-specific CD4⁺ T cell frequency survival in 35 days. (D) Representative flow cytometry for VACV-specific memory CD4⁺ T cells and memory subsets analysis on day 35. (E) VACV-specific memory CD4⁺ T cell number on day 35. (F) VACV-specific memory CD4⁺ T cell frequency on day 35. (G) VACV-specific resident-memory CD4⁺ T cell (CD44^highCD69^highCD197^low) frequency on day 35. *, p < 0.05; **, p < 0.01; ns, p > 0.05.

Figure 6.. Adoptive transferred NAC1^−/− CD4⁺ T cells generate a higher number of memory T cells.

Either WT or NAC1^−/− mice (Thy1.2⁺) were sacrificed as the donor mice. Then the naive CD4⁺ T cells were purified from pooled LNs and spleen. The WT or NAC1^−/− CD4⁺ T cells were cultured for 3 days and 1X10⁶ cells were adoptively transferred into randomly chosen recipient mice (Thy1.1⁺) without irradiation treatment. The same day after ACT, mice were challenged with 2X10⁶ PFU VACV. After 35 days, the pooled LNs and spleen were collected for flow cytometry analysis. Cells were stained with Thy1.2, CD44, CD69, and CD197 surface markers and VACV tetramer staining. The representative data are three identical experiments. Mice number for each group (n=5). (A) The diagram for the experiment design. (B) Representative flow cytometry for the donor VACV-specific memory T cells and memory subsets on day 35. (C) Donor VACV-specific memory T cell number. (D) Donor VACV-specific memory T cell frequency. (E) Donor VACV-specific resident-memory T cell frequency. *, p < 0.05; ns, p > 0.05. (F&G) VACV-specific memory T cell exhaustion marker PD-1, Tim-3 expression. Repeats n=3. (H&I) VACV-specific memory T cell functional cytokines, IL-2 and IFN-γ secretion. Repeats n=3.

Figure 7.. Effect of overexpression of ROCK1 on memory T cell formation in NAC1^−/− CD4⁺ T cells.

The naive WT or NAC1^−/− CD4⁺ T cells were purified from pooled LNs and spleen. T cells were transduced with either empty p-MIG or p-MIG-ROCK1 retrovirus vector. Then the GFP⁺ cells were sorted out for experiments. (A) BECLIN1 expression was examined by immunoblot. Repeats n=3. (B&C) p-AMPK expression was verified by flow cytometry after intracellular staining. The MFI of p-AMPK was calculated and compared. The plotting data shown are representative of three identical experiments (n=3). *, p < 0.05; **, p < 0.01; ns, p > 0.05. (D-F) 2X10⁵ ROCK1 overexpressed WT or NAC1^−/− CD4⁺ T cells (donor Thy1.2⁺) were adoptively transferred into randomly chosen irradiated treatment recipient mice (Thy1.1⁺). Then the mice were challenged with 2X10⁶ PFU VACV. The representative data are three identical experiments. Mouse number for each group (n=5). (D) Representative flow cytometry plotting for the donor VACV-specific memory T cells and memory subsets on day 35. (E) Donor VACV-specific memory T cell number. (F) Donor VACV-specific memory T cell frequency. (G) Proposed model of CD4⁺ T cell memory formation regulated by NAC1.