The secreted form of p28 subunit of interleukin (IL)-27 inhibits biological functions of IL-27 and suppresses anti-allogeneic immune responses

Abstract

Interleukin-27 (IL-27) is a new IL-12-related heterodimeric cytokine comprising a novel p28 molecule and the Epstein-Barr-virus-induced gene 3 (EBI3) molecules. It augments initiation of T helper type 1-mediated immunity by enhancing the proliferation and cytokine production of T cells. In this study, we examined whether a secreted form of IL-27 subunits would inhibit IL-27-mediated immunological responses. COS-7 cells transduced with the mouse (m) p28 gene secreted a monomeric mp28 protein; however, those transduced with the mEBI3 gene did not detect a mEBI3 protein in the culture supernatants. The secreted mp28 prevented the IL-27-mediated signal transduction and activator of transcription 1 phosphorylation and subsequently inhibited the IL-27-mediated intercellular adhesion molecule-1 induction and interferon-gamma production in CD4(+) T cells. We generated mp28-expressing murine carcinoma Colon 26 cells and inoculated a mixture of the mp28- and mIL-27-expressing Colon 26 cells into syngeneic BALB/c mice. Simultaneous production of mp28 and mIL-27 from Colon 26 cells suppressed IL-27-mediated anti-tumour effects in the mice. We examined the p28-mediated immune suppression by inoculating mp28-expressing myoblasts into allogeneic mice. Forced production of mp28 suppressed the allogeneic cytotoxic T-lymphocyte induction and subsequently retarded the graft rejection. Furthermore, production of both mp28 and mp40, which inhibits the functions of IL-12 and IL-23, prolonged the graft survival longer than the grafts expressing either mp28 or mp40. We propose that p28 can be a regulatory subunit for IL-27-mediated cellular immune responses and a possible therapeutic agent to suppress unfavourable immune responses.

Figure 1

Murine p28 but not mEBI3 was secreted as a monomeric protein. (a) COS-7 cells were transfected with mp28-myc or mEBI3-myc DNA. The cell lysates and supernatants were separated with sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) under reduced or non-reduced conditions. The myc-tagged protein was examined with Western blot analysis. (b) COS-7 cells were transfected with mp28-myc, mEBI3-haemagglutinin (HA) or both DNAs. The culture supernatants were separated by SDS–PAGE under reduced conditions. The myc- or HA-tagged protein was examined with Western blot analyses.

Figure 2

Murine (m) p28 inhibited interleukin-27 (IL-27)-dependent biological responses. (a) Naïve CD4⁺ T cells were stimulated with recombinant mIL-27 in the presence of the supernatants from the mp28-transfected or parental COS-7 cells for 24 hr and then restimulated with IL-12 for 24 hr. Interferon-γ (IFN-γ) in the cell-free supernatants was measured and the data represent mean ± standard deviation (SD) of triplicate samples. Asterisk indicates the statistical significance (P< 0·01 with Student’s t-test). (b) Naïve CD4⁺ T cells were stimulated with plate-coated anti-CD3 and anti-CD28 monoclonal antibodies and were cultured with or without IL-27 in the presence of the supernatants from the mp28-transfected or parental Chinese hamster ovary cells for 16 hr. The cells were analysed for their surface expression of intercellular adhesion molecule 1 (ICAM-1) with flow cytometry. The number in each panel showed the mean fluorescence intensity of ICAM-1 expression.

Figure 3

Murine (m) p28 inhibited interleukin-27 (IL-27)-mediated signal transducer and activator of transcription 1 (STAT1) activation. Naïve CD4⁺ T cells were stimulated with or without recombinant mIL-27 in the presence of the supernatants from the mp28-transfected (p28 sup.) or parental COS-7 cells (Control sup.) for 15 min. The cell lysates were subjected to Western blot analyses with anti-tyrosine-phosphorylated STAT1 (pY-STAT1) and anti-STAT1 (p91) antibodies. STAT1 phosphorylation levels were calculated as described in the Materials and methods and are summarized in the graph under the pictures using arbitrary units that were standardized with control supernatants as 1. Asterisks indicate statistical significance (P< 0·01). N.D.: not detected.

Figure 4

Murine (m) p28-producing tumour cells prevented interleukin-27 (IL-27)-dependent anti-tumour effects in vivo. (a) Northern blot analysis of the mp28 and IL-27 gene expressions in Colon 26, Colon 26/p28 and Colon 26/IL-27 cells with the p28-specific probe (upper panel). Ribosomal RNAs were shown as a loading control (lower panel). PA317/p28 is a positive control for the p28 gene. (b) Flow cytometric analysis of major histocompatibility complex (MHC) class I molecules on Colon 26, Colon 26/IL-27 and Colon 26/p28 cells. The cells were stained with anti-H-2K^d, anti-H-2D^d and anti-H-2L^d (thick line), or control monoclonal antibodies (thin line). (c) Tumour growth of the IL-27- and p28-producing tumour cells. The mixtures of Colon 26/IL-27 cells with either Colon 26 (open circles) or Colon 26/p28 (closed circles) (total 1 × 10⁶ cells), or the equal cell number of Colon 26 cells (closed squares) were inoculated subcutaneously into syngeneic mice. Data represent mean ± SD (n = 3).

Figure 5

Production of murine (m) p28 and mp40 prolonged allograft survival in allogeneic host animals. (a) Reverse transcription–polymerase chain reaction analysis of the gene expression of mp28, mp40 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, as a control) in C2Z, C2Zp28 or C2Zp40 cells. (b) Expression of H-2K^k and H-2D^k on C2Z, C2Zp28 and C2Zp40 cells. They were stained with anti-H-2K^k and anti-H-2D^k (thick line), or control antibodies (thin line). (c) The β-galactosidase activities in C2Z, C2Zp28 and C2Zp40 cells. Data indicate mean ± SD of triplicate experiments. (d) C2Z, C2Zp28 or C2Zp40 cells (total 1 × 10⁶ cells), or mixture of C2Zp28 and C2Zp40 cells (5 × 10⁵ C2Zp28 and 5 × 10⁵ C2Zp40) were inoculated into the quadriceps of naïve C57BL/6 mice (n = 4) and killed 21 days after inoculation. The formaldehyde-fixed tissues were stained with X-gal. (e) The percentage of β-galactosidase-positive areas of each tissue was measured. Data indicated mean ± SD. Asterisks indicate statistical significances when compared with the C2Z-inoculated mice. (*P< 0·05 with Student’s t-test and **P< 0·05 with Welch’s t-test).

Figure 6

Murine p28 suppressed allogeneic cytotoxic T lymphocyte (CTL) induction. (a) Seven days after myoblast inoculation, the splenocytes of C57BL/6 mice that were inoculated with C2Z (open circles), C2Zp28 (closed circles) or C2Zp40 cells (open squares) were cultured for 5 days with mitomycin C-treated C2Z cells. Cytolytic activities against C2Z and EL-4 cells were measured at various effector : target (E : T)ratios. Data represent mean ± SD of triplicate samples. Asterisks indicate statistical significances when compared with the C2Z-inoculated mice (P< 0·05 with Student’s t-test). (b) Splenocytes of naïve C57BL/6 mice were cultured with mitomycin C-treated C2Z (open circles) or C2Zp28 (closed circles) cells. Cytolytic activities against C2Z and EL-4 cells were measured at various E : T ratios. Data represent mean ± SD of triplicate samples. Asterisks indicate statistical significances when compared with the C2Z-stimulated splenocytes (P< 0·05 with Student’s t-test).