Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire

Abstract

The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Nck deletion results in peripheral T-cell lymphopenia. (A) Total lymph node (LN) cells were markedly decreased in Nck.T−/− mice (n = 16, triangles), in comparison with Nck.T+/+ controls (n = 11, circles; P < 0.0001). The number of Nck-deficient CD4+ and CD8+ T cells was also significantly reduced compared with that of Nck.T+/+ mice. (B) The expression of CD44 was increased on gated CD4+ and CD8+ T cells from Nck.T−/− mice (line) compared with Nck.T+/+ controls (shaded histograms).

Fig. 2.

Nck-deficient T cells are hyporesponsive to TCR-mediated stimulation. (A) Proliferation of CFSE-labeled T cells from Nck.T−/− (n = 9, Lower) and Nck.T+/+ (n = 7, Upper) mice was assessed upon in vitro stimulation with low (1 μg/mL) or high (10 μg/mL) doses of anti-CD3ε ± CD28. Nck-deficient T-cell responses were reduced compared with those of Nck+/+ T cells. (B) However, upon stimulation with PMA and a calcium ionophore, T cells from Nck.T−/− (n = 4, filled bar) and Nck+/+ (n = 5, open bar) mice exhibited similar levels of proliferation, as assessed by [3H]TdR incorporation. (C) Nck.T−/− (n = 6) T cells failed to respond to stimulation with low (1 μg/mL, P < 0.0001, Left) and high (10 μg/mL, P = 0.003, Right) doses of anti-CD3ε compared with Nck.T+/+ controls (n = 6). At low doses of anti-CD3ε, low doses of anti-CD28 failed to rescue Nck-deficient T-cell proliferation (1–2.5 μg/mL, P < 0.005). At high doses of anti-CD3ε, Nck-deficient T-cell proliferation was restored by lower doses of anti-CD28 (1 μg/mL, P < 0.001; anti-CD28 > 2.5 μg/mL, P = n.s.). (D) T-cell cultures (αCD3ε/αCD28, 1 μg/mL, 60 h) from Nck.T−/− (n = 6, filled bar) mice exhibited a 6- to 18-fold reduction in the content of IL-2 compared with Nck.T+/+ (n = 6, open bar) T-cell cultures (P < 0.0001), as assessed by ELISA. Data are representative of three independent experiments. (E) Nck2 expression was assessed by semiquantitative RT-PCR on resting (T0) and stimulated (T72h) T cells from Nck.T+/+ (n = 2, Left) and Nck.T−/− (n = 3, Right) mice. T cells from Nck.T−/− mice lacked Nck2 expression before or after stimulation with high doses of anti-CD3/CD28.

Fig. 3.

Nck is required for low-avidity T cells. (A) The percentage of splenic CD8+ T cells expressing the transgenic TCRβ (TCRβT, Vβ8.1/2) and TCRα (TCRαT, T3.70) chains was reduced in RAG2−/−.HY-TCR.Nck.T−/− females (n = 5, Lower) in comparison with RAG2−/−.HY-TCR.Nck.T+/+ females (n = 5; Upper). (B) In RAG2+/+.HY-TCR.Nck.T−/− females (Lower), an overall decrease in splenic CD8+ T cells (Left) was associated to a drastic reduction in T cells expressing both TCRαT and TCRβT (Right) compared with Nck.T+/+ controls (Upper). (C) The percentage of TCRαT βT+ cells was assessed in on gated CD8 single positive thymocytes (Upper) and CD8+ splenic T cells (Lower) from RAG2+/+.HY-TCR.Nck.T−/− females (n = 9, triangles) and RAG2+/+.HY-TCR.Nck.T+/+ controls (n = 9, circles). Nck deletion did not affect the percentage of TCRαT βT+ in mature CD8+ thymocytes (P = 0.24) but resulted in a strong reduction of splenic TCRαT βT+CD8+ T cells (P < 0.0001). Lines represent the arithmetic mean. (D) In the spleen, the percentage of CD8+ T cells was comparable in Nck.T−/− (n = 9) and Nck.T+/+ (n = 9) P14-TCR mice (Left). Gated CD8+ T cells expressed similar levels of the transgenic TCRα (Vα2) and TCRβ (Vβ8.1/2) chains in the presence/absence of Nck (Right). All data are representative of at least three independent experiments.

Fig. 4.

Differential requirement for Nck of low- and high-avidity T-cell responses. (A) CFSE-labeled HY-TCR T cells from RAG−/−. HY-TCR.NckT−/− (n = 6) and Nck.T+/+ (n = 7) females were stimulated with anti-CD3ε (10 μg/mL, Right) or with D1 dendritic cells loaded with peptides of different affinity—namely, the hyperagonist C2A, the agonist HY, the weak agonist K1A, and the “null” peptide gp33. In the CFSE/FSC dot plots, the gates define actively proliferating cells (FSChighCFSEint), viable nondividing cells (FSCintCFSEhigh), and apoptotic/dead cells (FSClow). Nck-deficient T cells exhibited a lower proliferative activity than Nck.T+/+ T cells in response to the HY and K1A peptides (A and B) (HY: P = 0.002; K1A: P = 0.048). In contrast, Nck.T+/+ and Nck.T−/− T cells mounted comparable responses to the C2A hyperagonist. The response of Nck-deficient HY-TCR T cells (n = 5) to optimal CD3 stimulation was impaired, when compared with that of control T cells (A) (n = 7, P = 0.004). Data are representative of three separate experiments, collectively depicted in B. (C) CFSE-labeled P14-TCR T cells from Nck.T−/− and Nck.T+/+ mice were stimulated with dendritic cells loaded with different doses of the agonist peptide gp33. The percentage of actively proliferating (FSChigh) Nck.T−/− (n = 9) and Nck.T+/+ (n = 9) P14-TCR+ T cells was comparable. Proliferation of Nck-deficient (n = 5) P14-TCR T cells was impaired in response to suboptimal doses of anti-CD3ε (P = 0.06). Cumulative results from five (gp33) or three (anti-CD3ε) independent experiments are shown.

Fig. 5.

Altered signaling in Nck-deficient thymocytes and mature T cells. (A) Peripheral T cells from Nck.T+/+ (n = 6, Upper) and Nck.T−/− (n = 6, Lower) mice were stained with the CD3ε.PRS-specific APA1/1 mAb, under resting conditions (0 min; shaded histogram) and 10 min (solid line) or 30 min (broken line) after CD3ε engagement (10 μg/mL, Right). Isotype controls under resting (dotted line) and stimulating (not shown) conditions had superimposable profiles. (B) Nck-deficient CD4+ and CD8+ T cells (n = 3, dotted line) exhibited lower intracellular calcium flux than Nck.T+/+ T cells (n = 3, solid line) upon CD3ε cross-linking. Data are representative of three separate experiments. (C) Reduced levels of ERK phosphorylation (Left) were detected upon CD3ε stimulation of CD8+ and CD4+ (not shown) peripheral T cells from Nck.T−/− mice (n = 6) compared with Nck.T+/+ (n = 6) T cells. In contrast, no differences were identified in the kinetics and levels of p38 phosphorylation (Right) upon CD3ε stimulation in CD4+ (data not shown) and CD8+ peripheral T cells from Nck.T−/− mice (n = 3) compared with Nck.T+/+ (n = 3) T cells. Dotted lines represent isotype controls.