Impaired chemokine-induced migration during T-cell development in the absence of Jak 3

Abstract

The arrival of bone marrow T-cell progenitors to the thymus, and the directed migration of thymocytes, are thought to be regulated by the expression of chemokines and their receptors. Recent data has shown that the Jak/Stat signalling pathway is involved in chemokine receptor signalling. We have investigated the role of Jak 3 in chemokine-mediated signalling in the thymus using Jak 3(-/-) mice. These mice show defects in T-cell development, as well as in peripheral T-cell function, resulting in a hypoplastic thymus and an altered T-cell homeostasis. Here we demonstrate, for the first time, that bone marrow progenitors and thymocytes from Jak 3(-/-) mice have decreased chemotactic responses to CXCL12 and CCL25. We also show that Jak 3 is involved in signalling through CCR9 and CXCR4, and that specific inhibition of Jak 3 in wild-type progenitors and thymocytes decreases their chemotactic responses towards CCL25 and CXCL12. Finally, quantitative reverse transcription-polymerase chain reaction analysis showed that thymocytes from Jak 3(-/-) mice express similar levels of CXCR4 and CCR9 compared to wild-type mice. Altogether, deficient CCL25- and CXCL12-induced migration could result in a homing defect of T-cell progenitors to the thymus, as well as in a deficient thymocyte migration through the thymic stroma. Our results strongly suggest that the absence of Jak 3 affects T-cell development, not only through an impaired interleukin-7 receptor (IL-7R)-mediated signalling, but also through impaired chemokine-mediated responses, which are crucial for thymocyte migration and differentiation.

Figure 1

Jak 3-deficient thymocytes have impaired chemotactic responses towards CXCL12 and CCL25. Total thymocytes were isolated from 4–10-week-old Jak 3^–/– or Jak^+/– mice and allowed to migrate, in a chemotaxis assay, in the presence of 0–1000 ng/ml chemokines, as described in the Materials and methods. (a) Chemotaxis responses of thymocytes from Jak 3-deficient thymocytes and their heterozygous littermates, to chemokines CXCL12 and CCL25. Left panel, cells were stained with calcein-AM prior to the chemotaxis assay, and migrated cells were quantified as described in the Materials and methods. Results are from one representative experiment (n = 5) and expressed as counts/mm² (× 10⁴). Right panel, number of thymocytes that migrated through the membrane were counted by microscopy in a high-power field (hpf). (b) Chemotaxis assays towards CCL4, CCL5 and CCL11. The cells that migrated through the membrane were counted by microscopy in a hpf. The data are from representative experiments (n = 5) showing the mean ± standard error (SE) of eight different fields counted under microscopy in a hpf of replicate wells. Statistical analysis was performed using the unpaired two-tailed Student's t-test. Asterisks indicate a P-value of < 0·05.

Figure 2

Jak 3^–/– bone marrow progenitors show diminished migration towards CXCL12 and CCL25. Data represent chemotaxis assays of bone marrow progenitors isolated from 4–10-week-old Jak 3^–/– mice or age-matched wild-type mice. Bone marrow progenitors were isolated from the femurs of Jak 3^–/– and Jak 3^+/+ mice and allowed to migrate in a chemotaxis assay in the presence of 0–1000 ng/ml of chemokines. Left panel, data showing chemotactic responses to CCL25. Right panel, data representing chemotactic responses to CXCL12. The data shown are from representative experiments (of a total of five) showing the mean ± standard error (SE). The cells that migrated through the membrane were counted in eight different fields under microscopy in a high-power field (hpf) of replicate wells. Statistical analysis was performed using the unpaired two-tailed Student's t-test. Asterisks indicate a P-value of < 0·05.

Figure 3

Haematopoietic bone marrow progenitors respond to CXCL12 and CCL25. Total bone marrow cells from wild-type and Jak 3^–/– mice were stained with Sca-1- and c-kit-specific antibodies to characterize the percentages of haematopoietic bone marrow progenitors in these mice. A representative experiment (out of five) is shown in (a). Total bone marrow cells from C57/BL6 mice were subjected to chemotaxis assays using a transwell technique (b). As shown, a significant percentage of the migrated cells towards CXCL12 and CCL25 (10 ng/ml of chemokine), were positive for Sca-1 and c-kit. A representative experiment (of a total of three) is shown.

Figure 4

Pharmacological inhibition of Jak 3 abrogates the chemotactic response of normal thymocytes to CCL25 and CXCL12. Total thymocytes (a) or bone marrow cells (b) from adult mice were preincubated with inhibitor WHI-P131, or pertussis toxin (PTX), and allowed to migrate in a chemotaxis assay in the presence of 0–1000 ng/ml of the specific chemokine. Cells were stained with calcein prior to performing the chemotaxis assay. Migration was calculated as counts of fluorescence/mm², measured using an FX Imager, as described in the Materials and methods. Representative experiments are shown (n=3). The response to CCL25 (left) and CXCL12 (centre), was greatly decreased in WHI-P131-treated thymocytes, while a lower inhibition was observed in the chemotactic response towards CCL11 (right). The G_αi inhibitor, PTX, completely blocked chemotaxis towards all chemokines tested. Bars represent the mean of triplicate wells ± standard error of the mean (SEM).

Figure 5

CCL25 and CXCL12 induce tyrosine phosphorylation of Jak 3. A representative experiment of n = 5 is shown. Thymocytes (30 × 10⁶) from 4–10-week-old C57/BL6 mice were stimulated with CXCL12 or CCL25 for 15 and 60 seconds, and Jak 3 phosphorylation was analysed after immunoprecipitation and blotting with the phosphotyrosine antibody, 4G10 (top panel). As a positive control, thymocytes were stimulated with recombinant interleukin-7 (IL-7) (100 ng/ml) After stripping, the membrane was reprobed with Jak 3 polyclonal antisera (888) (bottom panel). Densitometric analysis was performed on phosphotyrosine and Jak 3 blots, and the phosphorylation level of Jak 3 was calculated as the ratio of the densitometric intensities of the anti-phosphotyrosine signal to the anti-Jak 3 signal of the respective bands. The data were normalized to the values obtained with thymocytes treated in media alone.

Figure 6

Expression of CCR9 and CXCR4 in Jak 3^–/– thymocytes. Real time reverse transcription–polymerase chain reaction (RT–PCR) was performed on cDNA obtained from total RNA (DNase treated) isolated from 6-week-old Jak 3^–/– and C57/BL6 mice (pools of six mice). Murine CCR9-, CXCR4- and HPRT-specific primers were used in the PCR and amplified using an ABI PRISM 7000 Sequence Detection System. Data show a representative experiment (of a total of six) performed in triplicate. Data (mean ± standard deviation) are plotted as relative chemokine receptor (CKR) mRNA, calculated by normalizing the values obtained with the CCR9 and CXCR4 primers with their respective HPRT controls, as described in the Materials and methods. Analysis of significance was performed using the program Qgene, as previously described.