Expression of the ether-a-gò-gò-related gene 1 channel during B and T lymphocyte development: role in BCR and TCR signaling

Abstract

The functional relevance of K⁺ and Ca²⁺ ion channels in the "Store Operated Calcium Entry" (SOCE) during B and T lymphocyte activation is well proven. However, their role in the process of T- and B- cell development and selection is still poorly defined. In this scenario, our aim was to characterize the expression of the ether à-go-go-related gene 1 (ERG1) and K_V1.3 K⁺ channels during the early stages of mouse lymphopoiesis and analyze how they affect Ca²⁺signaling, or other signaling pathways, known to mediate selection and differentiation processes of lymphoid clones. We provide here evidence that the mouse (m)ERG1 is expressed in primary lymphoid organs, bone marrow (BM), and thymus of C57BL/6 and SV129 mice. This expression is particularly evident in the BM during the developmental stages of B cells, before the positive selection (large and small PreB). mERG1 is also expressed in all thymic subsets of both strains, when lymphocyte positive and negative selection occurs. Partially overlapping results were obtained for K_V1.3 expression. mERG1 and KV1.3 were expressed at significantly higher levels in B-cell precursors of mice developing an experimental autoimmune encephalomyelitis (EAE). The pharmacological blockage of ERG1 channels with E4031 produced a significant reduction in intracellular Ca²⁺ after lymphocyte stimulation in the CD4⁺ and double-positive T-cell precursors' subsets. This suggests that ERG1 might contribute to maintaining the electrochemical gradient responsible for driving Ca²⁺ entry, during T-cell receptor signaling which sustains lymphocyte selection checkpoints. Such role mirrors that performed by the shaker-type K_V1.3 potassium channel during the activation process of mature lymphocytes. No effects on Ca²⁺ signaling were observed either in B-cell precursors after blocking K_V1.3 with PSORA-4. In the BM, the pharmacological blockage of ERG1 channels produced an increase in ERK phosphorylation, suggesting an effect of ERG1 in regulating B-lymphocyte precursor clones' proliferation and checkpoint escape. Overall, our results suggest a novel physiological function of ERG1 in the processes of differentiation and selection of lymphoid precursors, paving the way to further studies aimed at defining the expression and role of ERG1 channels in immune-based pathologies in addition to that during lymphocyte neoplastic transformation.

Keywords: B and T lymphocytes development4; BCR and TCR signalling2; Ca 2+ signalling1; ERG1 K + channel3; ERK phosphorylation; Kv1.3 channel6; SOCE Ca 2+ overload5.

Figure 1

ERG1 expression in hemopoietic organs of C57BL/6 and SV129 mice. Representative histograms showing anti-ERG1 (AF488) fluorescence analyzed by flow cytometry of cells purified from different lymphoid organs of C57BL/6 (A) and of SV129 representative animals (B). Control—histograms show the fluorescence of ERG1 or Kv1.3 unstained control. Bar graphs showing the mean fluorescence intensity (MFI) of cells purified from different lymphoid organs of C57BL/6 (n = 10) (C) and of SV129 (n = 3) strain (D). Error bars indicate the standard deviation among mice of both sexes, 3 months of age. Statistical analysis was performed by two-tailed T-test (0.001 < p ≤ 0.01 **, p ≤ 0.001 ***).

Figure 2

ERG1 expression in different lymphoid populations. Representative histograms showing anti-ERG1 (AF488) fluorescence analyzed by flow cytometry in the different lymphoid precursors’ subpopulations purified from different lymphoid organs of C57BL/6 (A) and of SV129 strain (B). Control—histograms show the fluorescence of ERG1 or Kv1.3 unstained control. Bar graphs representing ERG1 expression levels in different lymphoid organs (bars marked with a line pattern) and subsets (bars with no pattern, gated as described in Supplementary Figure 2 ) of C57BL/6 (n = 10 (C) and SV129 (n = 3 (D) animals of 3 months of both sexes, normalized on the ERG1 MFI of the PBMC. Histograms represent the mean the fluorescence analyzed in the different animals, and error bars indicate the standard deviation. Statistical analysis was performed by two-tailed T-test (0.01 < p ≤ 0.05 *, 0.001 < p ≤ 0.01 **, p ≤ 0.001 ***).

Figure 3

Kv1.3 expression in hemopoietic organs of SV129 mice. Representative histograms showing anti-Kv1.3 (AF488) fluorescence analyzed by flow cytometry of cells purified from different lymphoid organs of C57BL/6 (A) and of SV129 representative animals (B). Control—histograms show the fluorescence of ERG1 or Kv1.3 unstained control. Bar graphs showing the mean fluorescence intensity (MFI) of cells purified from different lymphoid organs of C57BL/6 (n = 10) (C) and of SV129 (n = 3) strain (D). Error bars indicate the standard deviation among mice of both sexes, 3 months aged. Statistical analysis was performed by two-tailed T-test (0.01 < p ≤ 0.05 *, 0.001 < p ≤ 0.01 **, p ≤ 0.001 ***).

Figure 4

Kv1.3 expression in different lymphoid populations. Representative histograms showing anti-Kv1.3 (AF488) fluorescence analyzed by flow cytometry in the different lymphoid precursors’ subpopulations purified from different lymphoid organs of C57BL/6 (A) and of SV129 strain (B). Control—histograms show the fluorescence of ERG1 or Kv1.3 unstained control. Bar graphs representing Kv1.3 expression levels in different lymphoid organs (bars marked with a line pattern) and subsets (bars with no pattern, gated as described in Supplementary Figure 1 ) of C57BL/6 (n = 10 (C) and SV129 (n = 3 (D) animals of 3 months of both sexes, normalized on the Kv1.3-AF488 MFI of the PBMC. Histograms represent the mean the fluorescence analyzed in the different animals, and error bars indicate the standard deviation. Statistical analysis was performed by two-tailed T-test (0.01 < p ≤ 0.05 *, 0.001 < p ≤ 0.01 **, p ≤ 0.001 ***).

Figure 5

ERG1 and Kv1.3 expression in different lymphoid populations of the experimental autoimmune encephalomyelitis (EAE) disease murine model Histograms representing ERG1 and Kv1.3 expression levels in the BM (A, C) and in the thymus (B, D) of C57BL/6 (n = 10) and C57BL/6 EAE sacrificed at the peak of the disease, 15 days after the EAE induction (n = 3). Histograms represent the mean of the fluorescence analyzed in the different animals, normalized on the expression in PBMC; error bars indicate the standard deviation. Statistical analysis was performed by two-tailed T-test (0.01 < p ≤ 0.05 *, 0.001 < p ≤ 0.01 **, p ≤ 0.001 ***).

Figure 6

Representative plots showing the variation in Ca²⁺ level before and after BCR and TCR stimulation. Plots showing the Fluo-4 fluorescence in lymphocyte precursors purified from the BM and thymus of a 3-month male C57BL/6 mouse. After 1 min of acquisition, lymphocyte’s precursors have been stimulated with a mix of anti-IgM and anti-CD19 for B cells and with anti-CD3 pre-binding followed by cross-linking with goat anti-hamster IgG for T cells to appreciate the Ca²⁺ entry wave. As control, part of the B and T lymphocytes were treated with ionomycin and EGTA. The moment of the stimulation is indicated by the arrow. Tubes have been immediately reinserted in the cytometer to appreciate the SOCE. The Fluo-4 MFI was calculated before (−) and after (+) the stimulation.

Figure 7

Effect of E4031 and Psora-4 on Ca²⁺ basal level and SOCE following BCR and TCR stimulation in the different lymphocytes’ precursor subsets. The Fluo-4 MFI in the different lymphocyte precursors from 7- and 3-month-old C57BL/6 mice of both sexes was calculated over a period of 1 min before (−) and 1.5 min after the stimulation (+). Bars represent the mean Fluo-4 MFI for each subset in the different experimental conditions, normalized for the untreated and unstimulated (unt, −) B220⁺ (A) or TCRβ⁺ population (B). The effect of E4031 and Psora-4 treatment on the population that responded to the stimulation is shown in (C, D) Statistical analysis was performed by two-tailed T-test (0.01 < p ≤ 0.05 *, 0.001 < p ≤ 0.01 **, p ≤ 0.001 ***).

Figure 8

Effects of ERG1 inhibition on MAPK signal pathways Western blot analysis showing total ERK and ERK phosphorylation on residues Thr 202 and Tyr 204 on total lysates from BM (A) and thymus (B) of four C57BL/6 animals of 3 months stimulated and treated overnight with E4031 [30mM]. For each thymus (n = 4) are reported the unstimulated (−), stimulated (+), stimulated, and treated with E4031 and Psora-4 [5 mM] conditions. Histograms show the fold change in the phosphorylation calculated by WB quantification of pERK in each animal, normalized for the total ERK. Error bars represent standard deviation. Statistical analysis was performed by two-tailed T-test (0.01 < p ≤ 0.05 *).

Figure 9

hKCNH2 expression in different mature B and T lymphoid subsets Transcriptomic analysis based on RNA-seq data of 13 types of human immune cells from 91 donors, deposited on the DICE database (https://dice-database.org), showing hKCNH2 transcripts per million (TPM).