The CD8α-PILRα interaction maintains CD8+ T cell quiescence

Abstract

T cell quiescence is essential for maintaining a broad repertoire against a large pool of diverse antigens from microbes and tumors, but the underlying molecular mechanisms remain largely unknown. We show here that CD8α is critical for the maintenance of CD8⁺ T cells in a physiologically quiescent state in peripheral lymphoid organs. Upon inducible deletion of CD8α, both naïve and memory CD8⁺ T cells spontaneously acquired activation phenotypes and subsequently died without exposure to specific antigens. PILRα was identified as a ligand for CD8α in both mice and humans, and disruption of this interaction was able to break CD8⁺ T cell quiescence. Thus, peripheral T cell pool size is actively maintained by the CD8α-PILRα interaction in the absence of antigen exposure.

Fig. 1. Inducible genetic deletion of Cd8a disrupts the homeostasis of memory and naive CD8⁺ T cells in the periphery.

(A and B) FACS-isolated memory CD44^hiCD8⁺ T cells from Cre^+/+Cd8a^+/+ (Cd8a^+/+) or Cre^+/+Cd8a^loxp/loxp (Cd8a^loxp/loxp) mice were CFSE-labeled and transferred into B6 congenic CD45.1 mice followed by tamoxifen treatment. The percentage and absolute number of CD45.2⁺CFSE⁺ cells in the lymph node (A) and spleen (B) cells were examined 34 days after tamoxifen treatment. (C and D) FACS-isolated naïve CD44^loCD8⁺ T cells from Cre^+/+Cd8a^+/+ (Cd8a^+/+) or Cre^+/+Cd8a^loxp/loxp (Cd8a^loxp/loxp) mice were CFSE-labeled and transferred into B6 congenic CD45.1 mice followed by tamoxifen treatment. The percentage and absolute number of CD45.2⁺CFSE⁺ cells in the lymph node (C) and spleen (D) cells were examined 36 days after tamoxifen treatment. In (A to D), left panel: representative flow cytometry plots of CD45.2⁺CFSE⁺ percentage in the lymph node or spleen of each group 34 (A and B) and 36 (C and D) days after tamoxifen treatment; Right panel: bar graphs including all mice from each group. n=5 mice per group in (A to D). Data shown in (A to D) are representative of at least three independent experiments. Mean ± SD is shown. *P<0.05, **P<0.01, ****P<0.0001 by unpaired Student’s t test.

Fig. 2. CD8α maintains memory CD8⁺ T cell quiescence and CD127 and CD122 expression in the periphery.

The lymph node and spleen cells analyzed here were from the same populations used in Fig. 1, A and B. As described in Fig. 1, A and B, memory CD45.2⁺CFSE⁺ T cells were gated and analyzed for: (A) CD69⁺ percentage, (B) FAS MFI, (C) CD122 MFI, (D) CD127 MFI, and (E) CD5 MFI. See also fig. S5 for representative flow cytometry plots and histograms. MFI: mean fluorescence intensity. Mean ± SD is shown. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by unpaired Student’s t test. n=5 mice per group. Data are representative of three independent experiments.

Fig. 3. Identification of PILRα as a ligand for CD8α in both mice and humans.

(A) Screening for the binding partner of human CD8α-hIg fusion protein against a human transmembrane protein library on the Mirrorball Fluorescence Cytometer. Representative images of 384-well plates (left) and single wells (right) are shown. (B) Flow cytometry analysis of human CD8α-hIg fusion protein binding to mock or human PILRA-transfected 293T cells. (C) Flow cytometry analysis of human PILRα-mIg binding to mock or human CD8A-transfected 293T cells. (D) ELISA of biotin-labeled human CD8α-hIg binding to immobilized flag-hIg or human PILRα-hIg. (E) ELISA of biotin-labeled mouse CD8α-his binding to immobilized flag-mIg or mouse PILRα-mIg. (F) Flow cytometry analysis of mouse CD8α-hIg fusion protein binding to mock or mouse Pilra-transfected 293T cells. (G) Flow cytometry analysis of APC-labeled mouse PILRα-hIg protein binding to mock or mouse Cd8a-transfected 293T cells in the presence of anti-mouse CD8α mAb, 3D9 or anti-mouse PILRα mAb, 9B12. In (D and E), values are mean ± SD, and three replicates are included in each concentration. Representative data of two (D), three (E), and at least four (B, C, F and G) independent experiments are shown.

Fig. 4. Blockade of PILRα–CD8α interactions disrupts CD8⁺ T cell homeostasis and quiescence.

(A) B6 WT mice were administered 200 μg of control or 9B12 antibody intraperitoneally (i.p.) every 3-4 days starting on day 0. The frequency and the absolute number of CD8⁺ T cells among lymph node and spleen cells were examined on day 15. (B and C) Thymectomized B6 WT mice were administered 200 μg of control or 9B12 antibody i.p. every 3 days starting from day 0. CD8⁺ T cell frequency and cell number (B), and CD4⁺ T cell frequency and cell number (C) in the lymph node and spleen were examined on Day 14. (D) One day after 200 μg of control or 9B12 antibody was administered to B6 WT mice i.p., lymph node CD8⁺ T cells were analyzed for CD69 expression by flow cytometry. (E) One day after 200 μg of control or 9B12 antibody was administered to B6 WT mice i.p., CD8⁺ T cells purified from lymph node cells were stimulated by PMA, ionomycin, and brefeldin A and analyzed for IFNγ expression by flow cytometry. Representative data of two (B and C), three (A and E), and eight (D) independent experiments are shown. n=3 mice per group in (B, C, D and E). n=5 mice per group in (A). Mean ± SD is shown. *P<0.05, **P<0.01, ****P<0.0001 by unpaired Student’s t test. ns: not significant.