IL-7Rα on CD4+ T cells is required for their survival and the pathogenesis of experimental autoimmune encephalomyelitis

Abstract

Background: The IL-7 receptor alpha (IL-7Rα) binds both IL-7 and thymic stromal lymphopoietin (TSLP). IL-7Rα is essential for the development and survival of naive CD4⁺ T cells and their differentiation to effector/memory CD4⁺ T cells. Mice lacking IL-7Rα have severe lymphopenia and are resistant to experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. However, it has been reported that IL-7Rα on peripheral CD4⁺ T cells is disposable for their maintenance and EAE pathogenesis, which does not align with the body of knowledge on the role of IL-7Rα in the biology of CD4⁺ T cells. Given that a definitive study on this important topic is lacking, we revisited it using a novel approach, an inducible knockout of the IL-7Rα gene in CD4⁺ T cells.

Methods: We generated Il7ra^fl/fl/CD4CreER^T2 double transgenic mouse line (henceforth CD4^ΔIl7ra), susceptible to tamoxifen-induced knockout of the IL-7Rα gene in CD4⁺ T cells. CD4^ΔIl7ra mice were immunized with MOG_{35 - 55} for EAE induction and monitored for disease development. The expression of IL-7Rα, CD4⁺ T cell numbers, and MOG_{35 - 55}-specific CD4⁺ T cell response was evaluated in the central nervous system (CNS) and lymphoid tissues by flow cytometry. Additionally, splenocytes of CD4^ΔIl7ra mice were stimulated with MOG_{35 - 55} to assess their proliferative response and cytokine production by T helper cells.

Results: Loss of IL-7Rα from the surface of CD4⁺ T cells in CD4^ΔIl7ra mice was virtually complete several days after tamoxifen treatment. The loss of IL-7Rα in CD4⁺ T cells led to a gradual and substantial decrease in their numbers in both non-immunized and immunized CD4^ΔIl7ra mice, followed by slow repopulation up to the initial numbers. CD4^ΔIl7ra mice did not develop EAE. We found a decrease in the total numbers of TNF-, IFN-γ-, IL-17 A-, and GM-CSF-producing CD4⁺ T cells and regulatory T cells in the spleens and CNS of immunized CD4^ΔIl7ra mice. Tracking MOG_{35 - 55}-specific CD4⁺ T cells revealed a significant reduction in their numbers in CD4^ΔIl7ra mice and decreased proliferation and cytokine production in response to MOG_{35 - 55}.

Conclusion: Our study demonstrates that IL-7Rα on peripheral CD4⁺ T cells is essential for their maintenance, immune response, and EAE pathogenesis.

Keywords: CD4+ T cell; Experimental autoimmune encephalomyelitis; IL-7 receptor alpha; Multiple sclerosis.

Fig. 1

Tamoxifen-induced loss of IL-7Rα on CD4⁺ T cells in CD4^ΔIl7ra mice. Adult (3–5 months old) male and female CD4^ΔIl7ra and control CD4CreER^T2 mice were treated once with 250 mg/kg of tamoxifen via oral gavage. Test and control mice sacrificed at each time point were matched by sex and age. Mice were sacrificed every 24 h for 6 days and evaluated by flow cytometry for IL-7Rα on total, naïve (CD62L⁺CD44^lo), and effector/memory (CD62L⁻CD44^hi) CD4⁺ T cells and total CD8⁺ T cells from the blood, spleen, LNs, and BM. (A) Flow cytometry histograms showing IL-7Rα staining on total CD4⁺ T cells after tamoxifen treatment. Data for a single representative mouse at each time point is shown. (B) Proportions (3–4 mice/group/time point) of IL-7Rα⁺CD4⁺ T cells and total CD8⁺ T cells. Data are presented as mean ± SEM. The unpaired t-test was used for sample comparisons. *p ≤ 0.05

Fig. 2

IL-7Rα gene knockout in CD4⁺ T cells abrogates EAE. Mice were treated with two doses of tamoxifen for two consecutive days, and IL-7Rα loss on blood CD4⁺ T cell was confirmed 5 days later. Mice were then immunized for EAE induction. (A) EAE clinical course in tamoxifen-pretreated CD4^ΔIl7ra and CD4CreER^T2 control mice. (B) Numbers of CD45^hi cells isolated from the CNS at EAE peak in control mice. (C) The expression of IL-7Rα on CD4⁺ and CD8⁺ T cells from the spleen, LNs, and CNS at the preclinical phase (8 d.p.i.), clinical onset (12 d.p.i.), and EAE peak (18 d.p.i.) in control mice. Results are expressed as the mean ± SEM with n ≥ 5 per group from 3 independent experiments. Data were analyzed by Student’s t-test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 3

IL-7Rα gene knockout in CD4⁺ T cells reduces their numbers in immunized CD4^ΔIl7ra mice. Numbers of mononuclear cells (A) isolated from the CNS, LNs, and spleen of CD4^ΔIl7ra and CD4CreER^T2 control mice at preclinical (8 d.p.i.), onset (12 d.p.i.), and peak (18 d.p.i.) of EAE in control mice. The frequencies and absolute numbers of total (A), naïve (B), and effector/memory (C) CD4⁺ T cells are also shown. Results are expressed as the mean ± SEM with n ≥ 5 per group from 2 independent experiments. Data were analyzed by Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Fewer cytokine-producing Th cells are present in immunized CD4^ΔIl7ra mice. Numbers of cytokine-producing CD4⁺ T cells from the spleen, LNs, and CNS of CD4^ΔIl7ra and the control mice during EAE. Results are expressed as the mean ± SEM with n ≥ 4 per group from 2 independent experiments. Data were analyzed by Student; s t-test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 5

Increased proportions of Treg cells among CD4⁺ T cells of CD4^ΔIl7ra mice. (A) Representative flow cytometry plots of CD4⁺Foxp3⁺ cells in gated CD4⁺ T cells at EAE peak in CD4^ΔIl7ra and control mice. (B) A total number of CD4⁺Foxp3⁺ T cells in the CNS, LNs, and spleen of CD4^ΔIl7ra mice compared with control mice at different time points during EAE. Results are expressed as the mean ± SEM with n ≥ 4 per group from 3 independent experiments and were analyzed by Student’s t-test; *P < 0.05; **P < 0.01; ****P < 0.0001

Fig. 6

IL-7Rα gene knockout in CD4⁺ T cells abrogates MOG-specific response. Spleen, LNs, and CNS cells from MOG_{35 − 55}-immunized CD4^ΔIl7ra and control mice were harvested and stained with I-Ab MOG_{35 − 55} MHC tetramer-PE and stained for CD4 and CD45 and analyzed by flow cytometry. (A) The frequencies of MOG_{35 − 55}-specific CD4⁺ T cells in total CD4⁺ T cells and absolute numbers of MOG_{35 − 55}-specific CD4⁺ T cells are shown. Results are expressed as the mean ± SEM with n ≥ 3 per group from 2 independent experiments. (B) Dot plots from the CNS of one representative experiment are shown (percentage of tetramer⁺ cells within the CD45⁺CD4⁺ T cells)

Fig. 7

Diminished MOG_{35 − 55}-specific CD4⁺ T cell response in CD4^ΔIl7ra mice. For cytokine measurement and proliferation assay, splenocytes were seeded (2.5 × 10⁵ cells per well) in a 96-well plate and activated with MOG_{35 − 55}. Parallel samples were activated with anti-CD3 and anti-CD28 mAbs. (A) After four days, cell numbers were elucidated by XTT assay. The proliferation of MOG_{35 − 55}-stimulated cells is represented as a percentage of the proliferation of a corresponding sample activated with anti-CD3 and anti-CD28 mAbs. (B) Cytokine concentrations were measured by ELISA in the culture supernatants at the onset of EAE in control mice. Results are expressed as the mean ± SEM for triplicate cultures of splenocytes from 2 independent experiments (n = 7) and were analyzed by Student; s t-test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001