Inhibition of interleukin 7 receptor signaling by antigen receptor assembly

Abstract

After the productive rearrangement of immunoglobulin (Ig) heavy chain genes, precursor (pre-)B lymphocytes undergo a limited number of cell divisions in response to interleukin (IL)-7. Here, we present evidence that this phase of IL-7-dependent expansion is constrained by an inhibitory signal initiated by antigen receptor assembly. A line of pre-B cells from normal murine bone marrow that expresses a mu heavy chain with a D-proximal V(H)7183.2 region divides continuously in IL-7. IL-7 responsiveness ceases upon differentiation to the mu(1), kappa(1) stage, despite continuing expression of the IL-7 receptor (IL-7R), suggesting that antigen receptor assembly inhibits IL-7 responsiveness. This is confirmed by introduction of a rearranged lambda light chain gene, which inhibits proliferative signaling through the IL-7R. Inhibition is specific to the IL-7R, because it is overcome by replacement of the IL-7R cytoplasmic domain with corresponding sequences from the closely related IL-2Rbeta chain. Alteration of a single tyrosine residue, Tyr410, in the IL-7R cytoplasmic domain to phenylalanine also prevents the inhibition of proliferation after antigen receptor assembly. Thus, the loss of IL-7 responsiveness after antigen receptor assembly may be mediated through the recruitment of an inhibitory molecule to this residue. Our findings identify a novel mechanism that limits cytokine-dependent proliferation during B lymphopoiesis. This mechanism may be essential for the proper regulation of peripheral B lymphocyte numbers.

Figure 1

(A) Shows that markers characteristic of the pre-B I stage in differentiation are expressed by the pre-B cell line used in these studies. The panels show the results of flow cytometric analyses of 10,000 cells after staining with antibodies against the indicated markers. Forward and side light scatter profiles were used to exclude dead cells. (B) Demonstrates the dose-dependent proliferation of the pre-B cells in response to IL-7. Proliferation quantified by the MTT assay is plotted on the y-axis against cytokine dose on the x-axis. The solid and dotted lines compare the proliferation of a pre-B cell culture tested at an interval of >1 yr, showing that IL-7–dependent proliferation is not lost after prolonged passage in culture. Results are typical of at least three independent experiments.

Figure 1

(A) Shows that markers characteristic of the pre-B I stage in differentiation are expressed by the pre-B cell line used in these studies. The panels show the results of flow cytometric analyses of 10,000 cells after staining with antibodies against the indicated markers. Forward and side light scatter profiles were used to exclude dead cells. (B) Demonstrates the dose-dependent proliferation of the pre-B cells in response to IL-7. Proliferation quantified by the MTT assay is plotted on the y-axis against cytokine dose on the x-axis. The solid and dotted lines compare the proliferation of a pre-B cell culture tested at an interval of >1 yr, showing that IL-7–dependent proliferation is not lost after prolonged passage in culture. Results are typical of at least three independent experiments.

Figure 2

(A) The sequence of the productive IgH rearrangement from the pre-B cell line. (B) The V region sequence, shaded grey in A, is aligned with the sequence of V_H7183.2. The D segment (shaded black) and the J region (unshaded) are also marked in A.

Figure 3

(A) Shows that κ⁺ and κ⁻ pre-B cells stain with equal frequency and intensity for the murine IL-7 (MIL7) receptor. (B and C) The results of experiments with pre-B cells transduced with retrovirally encoded huIL-4R/IL-7R wild-type (WT) or huIL-4R/IL-2Rβ chimeric receptors. The top panel in each case shows that expression of the transduced receptors is equivalent, whereas the bottom panels demonstrate the marked difference in the capacity of the two chimeric receptors to support the outgrowth of κ⁺ cells. Results are typical of at least three independent experiments.

Figure 4

(A) Shows that pre-B cells transduced with huIL-4R/IL-7RY410F and huIL-4R/IL-7RY456F receptors proliferate as well in response to stimulation of the mutant receptors with IL-4 (dotted line) as they do in response to stimulation of their endogenous IL-7R by IL-7 (solid line). Proliferation quantified by the MTT assay is plotted on the y-axis against cytokine dose on the x-axis. (B and C) Compare the phenotype of pre-B cells transduced with retrovirally encoded huIL-4R/IL-7RY410F or huIL-4R/IL-7RY456F mutant chimeric receptors. The top panel in each case shows that expression of the transduced receptors is equivalent, whereas the bottom panels demonstrate the marked difference in the capacity of the two mutant chimeric receptors to support the outgrowth of κ⁺ cells. Results are typical of at least three independent experiments.