Background
CD4
+ CD25
+regulatory T cells (Treg) are of central importance for the maintenance of peripheral tolerance and the regulation of cellular immune responses. Tregs is divided into two groups based on their origin and phenotypic characteristics: naturally occurring Tregs (nTregs) from the thymus and induced Tregs (iTregs) from the periphery [
1‐
4]. Foxp3 is an important marker [
5] and a key transcription factor that regulates the differentiation and function of Tregs. The homeostasis of Treg cells is important for sustaining their function and maintaining the immune balance. Appropriate Treg homeostasis at the periphery plays a crucial role in the maintenance of self-tolerance. Disturbances in this balance are frequently associated with autoimmune diseases. Unlike the homeostasis of naive conventional T cells, Treg homeostasis at the periphery is a much more dynamic process, and its underlying molecular mechanisms has been one of the important topics in this field.
The NF-κB signaling pathways, including the canonical and non-canonical NF-κB signaling pathways, play an important role in the development and maintenance of the peripheral Treg population [
6,
7]. RelB is an important transcription factor of non-canonical pathway of NF-κB family that regulates diverse immune and inflammatory responses [
8‐
10]. Previous studies showed that germline deletion of
RelB caused perturbation in the T cell repertoire, which suggests that RelB is required for T cell development [
11‐
13]. However, the role of RelB in the development of Foxp3
+ regulatory T cells (Tregs) remains controversial. A study reported that the percentage of Foxp3
+ Tregs was increased in
RelB deficient mice, but the absolute number of CD4
+Foxp3
+ Tregs is comparable to that of
RelB+/− mice [
14]. By contrast, other studies reported normal T cell development in
RelB−/− mice [
15,
16]. These differences may occur since Treg development is influenced by stromal cells of lymphoid origin, and RelB is involved in the regulation of stromal cells [
17‐
22]. Additionally, Foxp3
+ Tregs from
RelB deficient mice up-regulated certain activation markers and effector molecules on the cell surface [
20]. Furthermore, the intrinsic role of RelB signaling in regulating the homeostasis and competitive fitness of Tregs was also identified [
23]. However, the inhibitory function of RelB on effector T cells is not different from the role of Tregs in wild-type mice [
20]. Currently, the role of RelB in the generation and suppressive activities of Foxp3
+ Tregs is still not clear.
Our study used chimeric mouse models with bone marrow cells from wild-type (WT) or RelB deficient mice to study the role of RelB in regulating the proliferation and function of Tregs and their subsets. We also investigated the possible mechanisms of RelB on the proliferation of Tregs to demonstrate the role of RelB in the homeostatic proliferation and function of Treg.
Discussion
In this study, we demonstrated that
RelB deficiency exerts a profound effect on the proliferation of Foxp3
+ Tregs, and markedly expands the Treg pool in the periphery. RelB, a component of the NF-κB complex of transcription factors, is a critical regulator of differentiation among medullary thymic epithelial cells and hematopoietic cells [
15,
18,
19]. Besides decreased thymic cellularity,
RelB−/− mice had the following abnormal phenotypes: multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegaly due to extramedullary haematopoiesis, and a decreased population of thymic dendritic cells.
RelB deficiency decreases the population of Tregs in the thymus but increases the thymus Treg populations of bone marrow chimeric mice. These findings indicate that RelB may play a critical role in regulating Treg cell development and homeostasis by influencing the function and status of non-hematopoietic cells.
RelB deficiency did not influence the proliferation and survival of Tregs directly in our experiments nor other reports [
20]. RelB may control the development and proliferation of Tregs through extrinsic cell populations [
11,
12,
27‐
31]. Dendritic cells (DC) and Foxp3
− T cells were confirmed to regulate the expansion of Tregs through RelB-dependent secretion of cytokines secretion [
32,
33]. The effect of the NF-κB pathway on T cell activation is largely driven through the activation of DC. Thus, the development of Tregs in RelB
−/− mice was influenced by exogenic factors. Additionally, mice deficient in the non-canonical NF-κB component gene NF-κB 2 (p100), which inhibits RelB activation and participates in RelB nuclear activity, showed normal thymic development and suppressive function of Tregs. However, they had higher populations of peripheral “effector-phenotype” Tregs (eTregs) [
23,
34,
35]. This demonstrates that with p100 inhibition of RelB possibly maintaining the suppressive functions of Treg. Therefore, RelB may be a negative regulator but not the master regulator of Treg development.
Furthermore, our study showed the increased Tregs in RelB−/− bone marrow chimeric mice were primarily nTregs rather than iTregs. These results suggest that RelB may control the homeostasis of nTregs but not iTregs. However, a small proportion of RelB−/− CD4+ T cells were induced to be Foxp3+ Tregs compared with WT CD4+ T cells. This indicates the role of RelB in induced Foxp3+ Treg. Further studies are undoubtedly required to explore the mechanism of RelB on the regulation of nTregs and iTregs.
Additionally, RelB may regulate the transcription of genes involved in the generation of Tregs [
20]. Tregs in
RelB−/− mice upregulated certain activation markers and effector molecules on the cell surface, including CTLA-4, KLRG1, and TIGIT [
20]. Our research and other studies [
14,
20,
33] have found that
RelB−/− Tregs showed similar suppressive activities as their WT counterparts. The deletion of RelB did not influence the proliferation of Tregs in vitro, but increased the proliferation of Tregs in vivo. These data suggest that the role of RelB in the function and proliferation of FoxP3
+ Tregs. Therefore, the molecular mechanismof RelB on Tregs remain to be studied in the future.
Furthermore, we found that with IL-2 stimulation, the levels of pSTAT5 in Tregs from
RelB−/− mice were not higher than those from WT Treg. RelB deficiency by itself does not affect the Treg functions. Previous reports had shown that the expansion of Foxp3
+ Tregs in
RelB−/− mice were mediated primarily by the hyperactivation of FoxP3
− T effector cells that spontaneously produce increased levels of IL-2, a growth factor for Foxp3
+ Tregs [
18,
19,
36‐
39]. IL-2 is an activator of STAT5 signaling [
40]. Upon IL-2 stimulation, RelB seems to regulate Treg proliferation independently of the STAT5 pathway.
RelB−/− Treg cells may have a weaker response to IL-2 than WT Treg cells. Whether IL-2 promotes the proliferation of
RelB−/− Tregs through other factors or pathways requires further investigation.
Methods
Animals
RelB−/− mice and CD45.1 mice (6 weeks of age, female) were gifts from Dr. Y. X. Fu (University of Texas Southwestern Medical Center, Dallas, TX, USA). WT C57BL/6 mice (6 weeks of age, female) were purchased from Vital River Laboratory Animal Technology (Beijing, China). All mice were housed under specific pathogen-free conditions in the laboratory animal room of Institute of Biophysics, Chinese Academy of Sciences. All animals were housed with a 12 h light/dark cycle on ventilated racks with corncob bedding. The cage temperature was maintained from 68 to 76 degrees Fahrenheit. Five animals were housed in each cage. Animals were fed and given water every day. All procedures were performed in compliance with guidelines for the care and use of laboratory animals and were approved by the ethics committee of the Institute of Biophysics, Chinese Academy of Sciences (Beijing, China) and Shandong Academy of Medical Sciences (Shandong, China).
Bone marrow chimeric construction
Six-week-old CD45.1 mice were irradiated with a 10 Gy dose of Co60. The next day, WT or RelB−/− (CD45.2) mice were euthanized and bone marrow cells were extracted from the thigh bone to form a single-cell suspension. Donor cells (5 × 106) were intravenously transferred to recipients. Mice were continuously fed sulfamethoxazole and trimethoprim (Bactrim) for 4 weeks starting 1 day before irradiation. Five mice per group were used for experiments. Six to 8 weeks later, mice were euthanized by CO2 inhalation followed by cervical dislocation for thymocyte and splenocyte analysis.
Flow cytometry and antibodies
Mouse splenocytes and thymocytes were prepared from pooled thymus or spleen. Thymic and splenic cells were pre-incubated with Fc-block before staining with other antibodies. The mouse antibodies used included anti-CD4 (RM4–5, eBioscience); anti-CD8 (53–6.7, eBioscience); anti-CD45.1 (A20, BioLegend), anti-CD45.2 (104, eBioscience); anti-CD25 (PC61.5, eBioscience); anti-CD69 (H1.2F3, eBioscience); anti-CD24 (M1/69, eBioscience); anti-Helios (22F6, eBioscience) before flow cytometry analysis. For intracellular staining of Ki-67 (B56, BD), pSTAT5 (47/Stat5(pY694), BD) and Foxp3 (NRRF-30, eBioscience), cells were fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation / Permeabilization Solution Kit (554,714, BD) and stained according to the manufacturer’s protocols. The CD4
+ T cells from the thymus and spleen of WT or
RelB−/− mice were analyzed by flow cytometry and gated as shown in the Supplementary Figure
1. The samples were analyzed using a BD LSRFortessa flow cytometer and FlowJo software (Tree Star Inc). All single-cell suspensions from the tissues were stained with Abs diluted in PBS containing 2% FCS for 30 min on ice.
Adoptive cell-transfer experiments
The WT and RelB−/− splenic CD4+ T cells were labeled with 5.0 μM 5- (and 6-) CFSE (Molecular Probes, Inc. Eugene, OR, USA) for 15 min at 37 °C. Then the cells were washed with PBS twice before re-suspending in PBS. For adoptive transfer, the CD4+ T cells (CD45.1+CD4+) derived from WT or (CD45.2+CD4+) derived from RelB−/− mice were respectively transferred into CD45.1+ CD45.2+ WT B6 mice intravenously. At 3.5 days after cell transfer, the phenotype of transferred CD45.1+ CD4+ T cells or CD45.2+ CD4+ T cells in the host spleen was assessed by flow cytometry by gating on live CD4+ T cells.
The induction of iTregs from the spleen
Naive CD4+ CD25− T cells from WT or RelB−/− splenocytes were sorted using a BD Aria III flow cytometer (BD Biosciences, CA, USA). The purity of CD4+CD25− T cells was routinely above 90%, and these cells were cultured in 96-well plates with TGF-β (5 ng/mL; Peprotech) and IL-2 (50 U/mL; R&D) stimulation. Three days later, flow cytometry was used to measure the expression of CD4+ Foxp3+ T cell (iTreg).
Isolation and induction of Treg precursor cells
Treg precursors were defined as CD4+CD8−CD25+CD69+CD24+ cells that were isolated from the thymocytes of WT or RelB−/− mice and sorted using a BD Aria III flow cytometer (BD Biosciences, CA, USA). The purity of Treg precursor cells was routinely above 90%. Cells were harvested and stimulated with IL-2 (50 U/mL; R&D) for 3 days to induce the Treg phenotype. Single-cell suspensions were collected, stained, and detected using flow cytometry.
The inhibition of Treg on T cells
WT or RelB−/− CD4+ CD25+ Treg cells were isolated and mixed with CFSE-labeled naive conventional CD4+ T cells (2 × 105) at different ratios (Treg: conventional T = 1:1, 1:2). Anti-CD3 (1 μg/mL) and anti-CD28 (1 μg /mL) antibodies were used to stimulate T cell proliferation. Three days later, T cell proliferation was measured by flow cytometry.
Statistical analysis
Flow cytometry data were analyzed with FlowJo (Tree Star) software. Numerical data were processed in Excel (Microsoft) and plotted in Graphpad Prism (Graphpad Software, Inc). Statistical significance was determined using the nonparametric Mann–Whitney U test. *P < 0.05, **P < 0.01, and ***P < 0.001 unless otherwise indicated.
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