Ndrg3 is a critical regulator of peripheral T cell maturation and homeostasis

Abstract

To provide protection, anticipatory T cell-dependent immunity is reliant on the generation and maintenance of a naïve T cell repertoire, which is sufficiently diverse to ensure recognition of newly encountered antigens. Therefore, under steady-state conditions, a given individual needs to maintain a large pool of naïve T cells, ready to respond to potential threats. Here, we demonstrate that N-myc downstream-regulated gene 3 (Ndrg3) is essential for naïve T cell stability. Mice with T cell-specific Ndrg3 loss are lymphopenic, with reduced numbers of conventional T cells and natural killer T cells. We show that in the absence of Ndrg3, naïve CD8+ T cells exhibit high rates of both proliferation and apoptosis, phenotypes that could be partially rescued by transgenic expression of a high-avidity T cell receptor. Furthermore, Ndrg3-deficient cells were refractory to interleukin-4, resulting in reduced Eomes induction, and a decreased virtual memory population. Our study therefore identifies Ndrg3 as an unexpected, pleiotropic regulator of T cell homeostasis.

Fig. 1.. Ndrg3 is indispensable for peripheral T cells.

Total thymocyte numbers from Ndrg3^ctrl (Ndrg3^+/fl;Lck-cre+) and Ndrg3^TKO (Ndrg3^fl/fl;Lck-cre+) mice are shown in (A). Proportions and cell numbers of DN, DP, CD4SP, and CD8SP thymocytes are depicted in (B). Total splenocyte numbers from Ndrg3^ctrl and Ndrg3^TKO are shown in (C). Proportions (D) and cell numbers (E) of TCRβ+CD4+ and TCRβ+CD8+ cells in the spleen. Representative gating of naïve (CD62L+CD44^low), CM (CD62L+CD44^high), and EM (CD62L−CD44^high) subsets in TCRβ+CD4+ (F) and TCRβ+CD8+ (G) splenocytes. Proportions and cell numbers of naïve (H), EM (I), and CM (J) TCRβ+CD4+ and TCRβ+CD8+ splenocytes are quantified. Scatter dot plots are presented as the means ± SD with each symbol representing an individual mouse. Data collected from ≥2 experiments. Comparison between groups was calculated with unpaired t tests. **P < 0.01, ****P < 0.0001, and nonsignificant (ns) data indicate P > 0.05.

Fig. 2.. Ndrg3 deficiency impairs T cell homeostasis and results in increased apoptosis.

Representative histograms of EGFP expression from Rag2-EGFP+Ndrg3^ctrl (Ndrg3^+/fl;Lck-cre+;Rag2-EGFP) and Ndrg3^TKO (Ndrg3^fl/fl;Lck-cre+;Rag2-EGFP) mice in CD8SP thymocytes and TCRβ+CD8+ splenocytes are shown in (A) and (B), respectively. Proportions and cell numbers of RTE and mature naïve (MN) T cells are quantified in (C). Proportions of annexin V+ cells among CD8SP and RTE are presented in (D). Annexin V+ proportions in CD8SP thymocytes and CD44^low and CD44^high TCRβ+CD8+ splenocytes from Ndrg3^ctrl and Ndrg3^TKO mice are depicted in (E). Cell cycle analysis of TCRβ+CD8+CD62L+CD44^low cells is shown in (F). G₀ (Ki-67^negDAPI^low), G₁ (Ki-67^posDAPI^low), S (Ki-67^posDAPI^intermidiate), and G₂/M (Ki-67^posDAPI^high). Representative gating of Ki-67+ cells in TCRβ+CD8+CD62L+CD44^low population is shown and quantified in (G). Flow cytometry histograms and quantifications of CD5 and CD127 expression in TCRβ+CD8+CD62L+CD44^low cells are shown in (H) and (I), respectively. Naïve CD8 (CD8+CD62L+CD44^low) T cells (8 × 10⁴) from Ndrg3^ctrl and Ndrg3^TKO mice were labeled with cell proliferation dye (CPD) and stimulated with anti-CD3– and anti-CD28–coated beads for 48 hours at 37°C. Depicted in (J) are flow cytometry histograms illustrating division peaks (left panel) and quantifications of dividing cell proportions (middle panel) and numbers (right panel) after 48 hours of culture. Representative gating and proportions of CD69+ cells in proliferating naïve T cells are presented in (K). Scatter dot plots are presented as the means ± SD with each symbol representing an individual mouse. Data were collected from ≥2 experiments. Comparison between groups was calculated using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns data indicate P > 0.05.

Fig. 3.. High-affinity P14 TCR partially rescues naïve T cell populations in Ndrg3^TKO mice.

Representative gating of polyclonal CD8 (CD8+T3.70−) and HY CD8 (CD8+T3.70+) cells among splenocytes harvested from 6-week-old female Ndrg3^ctrl HY (Ndrg3^+/fl;Lck-cre+;HY) and Ndrg3^TKO HY (Ndrg3^fl/fl;Lck-cre+;HY) mice is presented in (A). Proportions and numbers of CD8 T cells from the spleen are depicted in (B). Representative gating of naïve CD8 (CD8+CD62L+CD44^low) splenocytes from Ndrg3^ctrl HY and Ndrg3^TKO HY mice is shown in (C), and proportions and numbers are quantified in (D). Median fluorescence intensity (MFI) of CD127 in CD8+ splenocytes is presented in (E). Proportions of annexin V+ cells in HY CD8 splenocytes are shown in (F). Representative gating of polyclonal CD8 (CD8+gp33-tetramer−) and P14 CD8 (CD8+gp33-tetramer+) splenocytes from Ndrg3^ctrl P14 (Ndrg3^+/fl;Lck-cre+;P14) and Ndrg3^TKO P14 (Ndrg3^fl/f;Lck-cre+;P14) mice is illustrated in (G). Proportions and cell numbers of polyclonal CD8 and P14-expressing CD8 splenocytes are quantified in (H). Representative flow cytometry gating of naïve CD8 (CD8+CD62L+CD44^low) polyclonal and P14 CD8 cells is shown in (I) and quantified in (J). MFI of CD127 is shown in (K). The percentage of annexin V+ cells in P14 CD8 splenocytes is presented in (L). HY-TCR data were collected from female mice only. All data originate from ≥2 experiments. Scatter dot plots are presented as the means ± SD with each symbol representing an individual mouse. Comparison between groups was calculated with unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, and ns data indicate P > 0.05.

Fig. 4.. Ndrg3-deficient naïve CD8+ T cells expressing a high-avidity TCR fail to respond normally to activation.

Purified 8 × 10⁴ naïve CD8 splenocytes (CD8+CD62L+) from Ndrg3^ctrl P14 and Ndrg3^TKO P14 mice were labeled with CPD and incubated with anti-CD3– and anti-CD28–coated beads at 37°C for 48 hours. Representative flow cytometry histograms illustrating division peaks are shown in (A). Dividing cell proportions (left panel) and numbers (right panel) are quantified in (B). Expression of CD69 in proliferating cells is presented and quantified in (C). Concentrations (pg/ml) of IFN-γ and TNF-α released to cell culture medium by proliferating (stimulated) or unstimulated (media) P14 naïve CD8 T cells are shown in (D). Scatter dot plots are presented as the means ± SD with each symbol representing an individual mouse. Data were collected from three experiments. Comparison between groups was calculated with unpaired t tests. ***P < 0.001.

Fig. 5.. Ndrg3-deficient CD8+ T cells exhibit changes in the expression of the T-box transcription factors, Eomes and Tbet.

CD8SP TCRβ+MHC1^high thymocytes were sorted and submitted for bulk RNA-seq analysis. The top 25 differentially expressed (DE) genes between Ndrg3^TKO and Ndrg3^ctrl are shown in (A). The heatmap shows the row z-score of counts. Four independent biological replicates were analyzed. Flow cytometry analysis of Eomes expression in CD8SP thymocytes from Ndrg3^ctrl and Ndrg3^TKO mice is illustrated in (B). Representative flow cytometry gating and quantification of Eomes (C) and Tbet (D) expression in TCRβ+CD8+ splenocytes. Flow cytometry plots representing the gating strategy for the coexpression of Eomes and Tbet in TCRβ+CD8+ splenocytes (E) and quantification of proportions in total TCRβ+CD8+ (E), TCRβ+CD8+CD44^low (F), and TCRβ+CD8+CD44^high (G). Representative gating strategy and proportions of CD8+ VM (CD8+CD62L+CD44^highCD49d^low) and TM (CD8+CD62L+CD44^highCD49d^high) splenocytes are presented in (H). Scatter dot plots originate from ≥2 experiments and are presented as the means ± SD with each symbol representing an individual mouse. Comparison between groups was calculated with unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns data indicate P > 0.05.

Fig. 6.. Ndrg3 is required for efficient responses to IL-4.

Sort-purified CD8SP thymocytes (2 × 10⁵) from Ndrg3^ctrl and Ndrg3^TKO mice were plated in 200 μl of IMDM and 10% FCS supplemented with IL-4 (20 ng/ml), cultured for 20 hours at 37°C, and subsequently analyzed by flow cytometry for the induction of Eomes and IL-4Rα. Quantification of Eomes and IL-4Rα expression is shown in (A) and (B), respectively. A drawing of IL-4-Akt–dependent Eomes induction in CD8SP thymocytes is shown in (C). The illustration was generated on the basis of previously published experimental evidence (61) and created with BioRender.com. CD8SP thymocytes were sorted and stimulated with IL-4 the same way as in (A) with addition of 25 nM rapamycin or 5 μM Akti. After 20 hours of incubation, proportions of Eomes and IL-4Rα were measured and are shown in (D) and (E), respectively. Proportions of Eomes expression in total CD8SP thymocytes and CD8+ splenocytes from Ndrg3^ctrl P14 and Ndrg3^TKO P14 mice are quantified in (F) and (G), respectively. Representative gating and quantification of thymic NKT cells (CD1d-tetramer+TCRβ+) from Ndrg3^ctrl Ndrg3^TKO mice are depicted in (H). Scatter dot plots originate from ≥2 experiments and are presented as the means ± SD with each symbol representing an individual mouse. Comparison between groups was calculated with unpaired t tests. *P < 0.05.