Proline-rich tyrosine kinase-2 is critical for CD8 T-cell short-lived effector fate - PubMed (original) (raw)

Proline-rich tyrosine kinase-2 is critical for CD8 T-cell short-lived effector fate

Sören Beinke et al. Proc Natl Acad Sci U S A. 2010.

Abstract

T-cell interactions with antigen-presenting cells are important for CD8 T-cell effector or memory fate determination. The integrin leukocyte function-associated antigen-1 (LFA-1) mediates T-cell adhesion but the contribution of LFA-1-induced signaling pathways to T-cell responses is poorly understood. Here we demonstrate that proline-rich tyrosine kinase-2 (PYK2) deficiency impairs CD8 T-cell activation by synergistic LFA-1 and T-cell receptor stimulation. Furthermore, PYK2 is essential for LFA-1-mediated CD8 T-cell adhesion and LFA-1 costimulation of CD8 T-cell migration. During lymphocytic choriomeningitis virus infection in vivo, PYK2 deficiency results in a specific loss of short-lived effector CD8 T cells but does not affect memory-precursor CD8 T-cell development. Similarly, lack of LFA-1 primarily impairs the generation of short-lived effector cells. Thus, PYK2 facilitates LFA-1-dependent CD8 T-cell responses and promotes CD8 T-cell short-lived effector fate, suggesting that PYK2 may be an interesting therapeutic target to suppress exacerbated CD8 T-cell responses.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Impaired activation of PYK2-deficient CD8 T cells in response to synergistic TCR and LFA-1 stimulation in vitro. WT or PYK2-deficient T cells were stimulated with plate-bound anti-CD3 antibody at standard concentrations (0.5 μg per well) or at limiting concentrations (0.1 μg per well) in the presence or absence of 0.3 μg per well plate-bound ICAM-1. (A) Proliferation was analyzed by 3H-thymidine uptake at 72 h. Graph shows average signal ± SD: *0.01 < P < 0.05; **0.001 < P < 0.01 (unpaired two-tailed Student's t test). (B) Proliferation was analyzed by CFSE labeling and FACS analysis. (C) IFN-γ and IL-2 expression in CD8 T cells was determined at 40 h by intracellular FACS staining after 5-h Brefeldin A treatment. (A–C) Data are representative of three or more experiments.

Fig. 2.

Fig. 2.

PYK2-deficient CD8 T cells are defective in LFA-1–mediated adhesion, polarity, and transmigration. (A) WT or PYK2-deficient T cells were stimulated as indicated in ICAM-1–coated microwell plates and adherent CD8 T cells were quantified by FACS. (B) WT or PYK2-deficient CD8 T-cell blasts were allowed to adhere to ICAM-1–coated cover slides. F-actin was stained with Alexa488-phalloidin and analyzed by fluorescence microscopy. Data are representative of an average view field from more than three experiments. (C) Migration of WT or PYK2-deficient CD8 T cells in the absence or presence of the indicated chemokines was measured using control or ICAM-1–coated transwell chambers. Migratory activity is presented as a percentage of migrating CD8 T-cell number divided by input CD8 T-cell number. (A and C) Graphs show average cell numbers from three independent experiments ± SEM: *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001 (unpaired two-tailed Student's t test).

Fig. 3.

Fig. 3.

PYK2 deficiency impairs the expansion of CD8 T cells in response to LCMV. (A) WT or PYK2-deficient P14 CD8 T cells were stimulated with dendritic cells and the indicated concentrations of LCMV gp33-41 peptide in vitro. Proliferation was analyzed by 3H-thymidine incorporation. Graph shows average counts ± SD. (B–D) WT (CD45.1+/CD45.2+) and PYK2-deficient (CD45.2+/CD45.2+) P14 CD8 T cells were mixed at a 1:1 ratio and adoptively transferred into host mice (CD45.1+/CD45.1+). The next day, mice were infected with LCMV Armstrong and P14 cells were analyzed at the indicated times. (B) Representative FACS plot of the analysis of congenic markers of P14 CD8 T cells from blood at day 8; numbers shown represent frequencies of the indicated population within CD8 T cells. (C) Graph shows average total P14 CD8 T-cell numbers in blood and spleen from three or more experiments (day 5: n = 10 mice; day 8: n = 20 mice; day 15: n = 6 mice) ± SEM. (D) WT and PYK2-deficient P14 transgenic CD8 T cells that were CFSE-labeled before adoptive transfer were analyzed by FACS after LCMV infection. (A and D) Data are representative of three experiments. (A and C) *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001 (unpaired two-tailed Student's t test).

Fig. 4.

Fig. 4.

PYK2 deficiency results in a loss of short-lived effector cells but does not affect memory precursor cell generation. (A–C) WT and PYK2-deficient P14 CD8 T cells were adoptively transferred and mice were infected with LCMV Armstrong as in Fig. 3. (A) Representative FACS analysis of IL-7Rαlow/KLRG1high short-lived effector versus IL-7Rαhigh/KLRG1low memory precursor cells at day 8 after infection. (B) Graph shows average total cell numbers of short-lived effector cells or memory precursor cells from three or more experiments (day 5: n = 10 mice; day 8: n = 20 mice; day 15: n = 6 mice) ± SEM: **0.001 < P < 0.01; ***P < 0.001 (unpaired two-tailed Student's t test). (C) FACS analysis of BrdU incorporation on day 5 after LCMV infection. Data are representative of three experiments.

Fig. 5.

Fig. 5.

CD11α deficiency primarily affects the generation of short-lived effector cells during LCMV infection. (A–C) WT (CD45.2+/CD45.2+) or CD11α-deficient (CD45.1+/CD45.2+) P14 CD8 T cells were mixed at a 1:1 ratio and adoptively transferred into host mice (CD45.1+/CD45.1+). The next day, chimeric mice were infected with LCMV Armstrong. (A) Representative FACS analysis of congenic markers on blood cells at day 8 after infection. Numbers shown represent frequencies of the indicated population within CD8 T cells. (B) Representative FACS analysis of IL-7Rαlow/KLRG1high short-lived effector versus IL-7Rαhigh/KLRG1low memory precursor cells in the blood at day 8 after infection. (C) Graph shows average total cell numbers from two experiments (day 8: n = 10 mice; day 15: n = 10 mice) ± SEM: *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001 (unpaired two-tailed Student's t test).

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