CD200R activation on naïve T cells by B cells induces suppressive activity of T cells via IL-24

Abstract

CD200 is an anti-inflammatory protein that facilitates signal transduction through its receptor, CD200R, in cells, resulting in immune response suppression. This includes reducing M1-like macrophages, enhancing M2-like macrophages, inhibiting NK cell cytotoxicity, and downregulating CTL responses. Activation of CD200R has been found to modulate dendritic cells, leading to the induction or enhancement of Treg cells expressing Foxp3. However, the precise mechanisms behind this process are still unclear. Our previous study demonstrated that B cells in Peyer's patches can induce Treg cells, so-called Treg-of-B (P) cells, through STAT6 phosphorylation. This study aimed to investigate the role of CD200 in Treg-of-B (P) cell generation. To clarify the mechanisms, we used wild-type, STAT6 deficient, and IL-24 deficient T cells to generate Treg-of-B (P) cells, and antagonist antibodies (anti-CD200 and anti-IL-20RB), an agonist anti-CD200R antibody, CD39 inhibitors (ARL67156 and POM-1), a STAT6 inhibitor (AS1517499), and soluble IL-20RB were also applied. Our findings revealed that Peyer's patch B cells expressed CD200 to activate the CD200R on T cells and initiate the process of Treg-of-B (P) cells generation. CD200 and CD200R interaction triggers the phosphorylation of STAT6, which regulated the expression of CD200R, CD39, and IL-24 in T cells. CD39 regulated the expression of IL-24, which sustained the expression of CD223 and IL-10 and maintained the cell viability. In summary, the generation of Treg-of-B (P) cells by Peyer's patch B cells was through the CD200R-STAT6-CD39-IL-24 axis pathway.

Keywords: B cells; CD200; IL-24; Peyer’s patch; Treg cell.

Fig. 1

Peyer’s patch B cells provided CD200 to phosphorylate STAT6 in T cells, which in turn stimulate T cell expressing CD200R. (A, B) Flow cytometry analysis of CD200 expression by Peyer’s patch B cells, phosphorylated STAT6 and CD223. Naïve CD4 T cells cultured with Peyer’s patch B cells plus anti-CD3 and anti-CD28 antibodies in presence of anti-CD200 antibody (antagonist, red: isotype, blue: anti-CD200). Naïve CD4 T cells were stimulated with anti-CD3 and anti-CD28 antibodies in presence of anti-CD200R antibody (agonist, orange: isotype, green: anti-CD200R). The amount of IL-4 in culture supernatant was applied for ELISA analysis. (C) FACS analysis of the expression of CD200 and CD200R by Treg-of-B cell with phosphorylated STAT6 (DMSO and WT group) or without phosphorylated STAT6 (AS, AS1517499 and STAT6KO group). (red: DMSO group; orange: wild type group, WT; blue: AS group; green: STAT6KO group). (D) FACS analysis of the expression of CD200 and CD200R by T cells stimulated with anti-CD3 and anti-CD28 antibody with or without Peyer’s patch B cells for three days (green: T only group; red: T cultured with B, T + B, group). Data are representative of three to four different experiments. Results are expressed as the mean ± SEM. *p < 0.05, **p < 0.01 compared with isotype group, wild type group, DMSO group or T + B group

Fig. 2

CD200-CD200R interaction participated in Treg-of-B (P) cell generation. To investigate whether CD200-CD200R interaction affects the generation of Treg-of-B (P) cells or act as the suppressive molecule, the agonist anti-CD200R antibody and antagonist anti-CD200 antibody were applied during Treg-of-B (P) cell generation or suppressive function assay. The functionality of Treg-of-B (P) cells was assessed based on their ability to inhibit the proliferation of responder T cells. Briefly, anti-CD200 (labeled as P_CD200) or anti-CD200R (labeled as P_CD200R) antibodies were administrated in T cell cocultured with Peyer’s patch B cells (Treg-of-B induction). Isotype antibody was used as control group (labeled as P_iso). After three-day coculture, the three different groups of Treg-of-B (P) cells were harvested and cultured with CD4 + CD25- responder T cells (A). To investigate the role of CD200 in suppressive ability, Treg-of-B (P) cells were harvested and then cultured with responder T cells in presence of anti-CD200 (labeled as αCD200) or anti-CD200R (labeled as αCD200R) antibodies (B). To measure the responder T cell proliferative response, 1 µCi of 3 H-thymidine was added to the culture for the last 16 h. Thymidine uptake was determined using a β-counter. Data are representative of three different experiments. Results are expressed as the mean ± SEM. *p < 0.05, **p < 0.01 compared with isotype group

Fig. 3

CD200-CD200R interaction between T cells helps T cells survive. (A) T cells cultured with Peyer’s patch B cells (labeled as T + B) could express CD200. (B, C) FACS analysis of the expression of phosphorylated STAT6, CD223 and CD200R. To determine the role of CD200 expressed by T cells, Peyer’s patch B cells were depleted after culturing with T cells one day. The remaining T cells were cultured for another two days (labeled as T-B). The culture supernatant was harvested for determination of the amount of IL-4 by ELISA (D). (E) To confirm that CD200-CD200R interaction between neighboring T cells was important for Treg-of-B (P) cell suppressive ability, anti-CD200 antibody was applied in the T-B groups (labeled as T-B + αCD200). These Treg-of-B (P) cells were harvested for suppression function test (left). Phosphorylated STAT6, the amounts of IL-4 and cell viability were determined by FACS analysis and ELISA, respectively (right). Data are representative of three to four different experiments. Results are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, compared with T-B group or responder T cell only group (labeled as -)

Fig. 4

CD39, which is regulated by CD200-CD200R interaction, participated in Treg-of-B (P) cell generation. (A, B) FACS analysis of CD39 expression by wild type T cells (labeled as T + B), STAT6 knock T cells (labeled as T_ko+B), T cells with STAT6 inhibitor AS (labeled as T + B + AS) or T cells with anti-CD200 antibody (labeled as T + B + αCD200), cultured with Peyer’s patch B cells for two days. (C, D) FACS analysis of CD223 and phosphorylated STAT6 by T cells cultured with Peyer’s patch B cells with (labeled as T + B + 39inh) or without (labeled as T + B) CD39 inhibitor for three days. Treg-of-B (P) cells were harvested and separated to two parts. One part for FACS analysis. The second part for detection of IL-10. After restimulation of Treg-of-B (P) cells, supernatant was harvested for ELISA. (E) To investigate the role of CD39 in Treg-of-B (P) cell suppressive ability, CD39 inhibitor was applied in the process of Treg-of-B (P) cell suppression function test (left). In addition, the role of CD39 in Treg-of-B (P) cell generation was evaluated. CD39 inhibitor was added in T cell cultured with Peyer’s patch B cells (right). Data are representative of three to four different experiments. Results are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, compared with T + B group or responder T cell only group (labeled as -). #p < 0.05, compared with Treg-of-B (P) DMSO group (labeled as Treg/B(D))

Fig. 5

IL-24, which is regulated by CD200-CD200R interaction, participated in Treg-of-B (P) cell generation. (A, B) The expression of IL-24 was analyzed by quantitative PCR (QPCR) and ELISA. Wild type T cells or STAT6 knockout T cells were cultured with Peyer’s patch B cells (labeled as Treg/B, T(ko)reg/B, respectively). T cells only were applied as control group (labeled as T). To determine the role of CD200 in regulating IL-24, T cells were cultured with Peyer’s patch B cells in presence of anti-CD200 antibody (labeled as Treg/B + aCD200). After three days, T cells and Treg-of-B (P) cells were harvested for QPCR or restimulation for ELISA. (C) To determine the role of IL-24 in Treg-of-B (P) cell suppressive ability, wild type and IL-24 knockout T cells were used in Treg-of-B (P) cell generation (labeled as T(wt)reg/B and T(ko)reg/B, respectively). Wild type Treg-of-B (P) cells were harvested and applied for suppressive function test in presence of different concentration of soluble IL-20RB (0, 100, 1000 and 2000 pg/ml), or anti-IL20RB antibody. Responder T cells plus recombinant IL-24 (50 ng/ml) was used as control group. (D) FACS analysis of the expression of IL-20RB on T cells, Treg-of-B (P) cells and responder T cells. After three days generation, T cells and Treg-of-B (P) cells were harvested and cocultured with responder T cells. To distinguish responder T cells and T cells or Treg-of-B (P) cells, responder T cells were labeled with CFSE before coculturing. After two days coculture, four cell subsets were analyzed by FACS. Four cell subsets: Treg-of-B (P) cell (red color), T cell (dark gray), responder T cell (cultured with Treg-of-B (P) cell, green color), responder T cell (cultured with T cell, purple color). (E) To analyze whether IL-24 affect Treg-of-B (P) cell suppressive ability, analysis of the expression of CD223, the amount of IL-4 and cell viability were performed. Treg-of-B (P) cells were restimulated with or without anti-IL-20RB for two days. Cells were applied for analyzing CD223 expression and cell viability. Culture supernatant was harvested for analyzing IL-10 level. (F) (left) T cells were cultured with Peyer’s patch B cells with or without CD39 inhibitor (labeled as Treg/B and Treg/B_(CD39inh), respectively). After three days, different Treg-of-B (P) cell groups were applied for restimulation and culture supernatant was harvested for analysis of IL-24 production. (right) Treg-of-B (P) cells were restimulation with or without anti-IL-20RB. After two days, cells were harvested for analysis of CD39 expression. Results are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 compared with T group, Treg/B group, Treg/B + αCD200 group or responder T cell only group (labeled as -). ^#p < 0.05, ^##p < 0.01, compared with T(wt)reg/B group, T(ko)reg/B group or sIL-20RB (0) group

Fig. 6

Peyer’s patch B cells induce Treg cells with CD200R-STAT6-CD39-IL-24 axis pathway. Peyer’s patch B cells provided the first signal, CD200, to activate T cells, with the following CD39 expression. CD39 regulate the increased expression of IL-24 that could sustain the CD223 and IL-10 production, and upregulate Treg-of-B (P) cell viability