Skint8, a Novel B7 Family-Related Molecule, Negatively Regulates T Cell Responses

Abstract

Immune responses are tightly controlled by T cell costimulatory and coinhibitory molecules. In this study, we identify Skint8 as a new member of the T cell coinhibitory group, whose extracellular domains share significant homology with existing B7 family members. Skint8 mRNA is expressed in resting and activated B cells, monocytes, and CD4 T cells. The Skint8 putative receptor is expressed on activated CD4 and CD8 T cells, B cells, monocytes and dendritic cells. Recombinant Skint8-IgG Fc fusion protein inhibits T cell proliferation, activation, and cytokine production in vitro. In vivo administration of Skint8-IgG Fc reduces T cell activation and alleviates experimental autoimmune encephalomyelitis in mice. The findings broaden our understanding of the regulation of immune responses and may have implications for treating immune-related diseases.

Figure 1.

The expression pattern of Skint8 mRNA in tissues and immune cells. (A) RNA was isolated from indicated tissues of C57BL/6 mice. The expression of Skint8 mRNA was determined by RT-PCR. GAPDH was used as a loading control. (B) CD4⁺ and CD8⁺ T cells, B220⁺ B cells, CD11b⁺ monocytes, F4/80⁺ macrophages, and CD11c⁺ DCs were magnetically isolated from splenocytes. (C) To activate T cells, purified CD4⁺ or CD8⁺ T cells were incubated with anti-CD3 (1 μg/ml) and anti-CD28 (0.5 μg/ml) antibodies for 3 days. To activate B cells, monocytes, macrophages, or DCs, the purified immune cells were incubated with LPS (10 μg/ml) for 3 days. RNA was isolated from the (B) resting and (C) activated immune cells. The expression of Skint8 mRNA was determined by RT-PCR. (D) The expression of Skint8 mRNA was determined by qRT-PCR. Each column was normalized with GAPDH. *P<0.05 compared with resting cells. The data are representative of 3 independent experiments.

Figure 2.

The expression pattern of the Skint8 putative receptor. (A, B) Spleen cells were harvested from C57BL/6 mice. To obtain activated T cells, the spleen cells were incubated with anti-CD3 (1 μg/ml) and anti-CD28 (0.5 μg/ml) antibodies for 3 days. To obtain activated B cells, monocytes and DCs, the spleen cells were incubated with LPS (10 μg/ml) for 3 days. The freshly harvested and activated immune cells were stained with biotinylated Skint8-Ig or control Ig, followed by streptavidin-PE, as well as anti-CD4, CD8, B220, CD11b, or CD11c antibodies to identify the immune cells. (A) Representative flow cytometric profiles and (B) Statistical analysis showing the binding of Skint8-Ig or control Ig to freshly harvested and activated immune cells. (B) Data are presented as relative fluorescence intensity (RFI) for cell binding of Skint8-Ig versus control Ig. (C) HEK-293 cells were transfected with an expression vector containing the murine PD-1, CD28, BTLA, CTLA-4, or ICOS gene and screened for cells stably expressing each gene. The transfected cells were stained with antibody against the respective PD-1, CD28, BTLA, CTLA-4, or ICOS protein (open histograms) or isotype antibody (shaded histograms). Representative flow cytometric profiles showing the expression of each receptor. (D) PD-1, CD28, BTLA, CTLA-4, or ICOS gene-transfected HEK-293 cells were stained with biotinylated Skint8-Ig or control Ig, followed by streptavidin-PE. Representative flow cytometric profiles showing the binding of Skint8-Ig (open histograms) or control Ig protein (shaded histograms) to the transfected cells. The data are representative of 3 independent experiments.

Figure 3.

The effects of Skint8-Ig protein on T cell proliferation and activation in vitro. (A) T cells were purified from spleen cells of C57BL/6 mice by magnetic separation. The cells were cultured on plates pre-coated with (A) anti-CD3 antibody (1 μg/ml) and graded doses of Skint8-Ig (1840, 3728, 7456 ng/ml) or equimolar amounts of control Ig (990, 1980, 3960 ng/ml) protein, or (B) anti-CD3 (1 μg/ml) and anti-CD28 (0.5 μg/ml) antibodies in the presence of Skint8-Ig or equimolar amount of control Ig for 3 days. [³H] thymidine (1 μCi/well) was added to the cultures 12 hours before harvest. T cell proliferation was measured by [³H] thymidine incorporation. (C-E) Spleen cells were labelled with CFSE and cultured in anti-CD3 antibody precoated 96-well plates in the presence of indicated doses of Skint8-Ig or control Ig for 3 days. The cells were analyzed for CFSE levels by CD4⁺ and CD8⁺ T cells. (C) Representative flow cytometric analysis of CFSE distribution of CD4⁺ and CD8⁺ T cells, and (D, E) statistical analysis of T cell proliferation. (F, G) Purified CD4⁺ or CD8⁺ T cells were cultured with anti-CD3 antibody and graded doses of Skint8-Ig or equimolar amounts of control Ig protein for 3 days. T cell proliferation was measured by [³H] thymidine incorporation as in (A). (H-L) Spleen cells were cultured with (H, I) anti-CD3 antibody or (J-L) anti-CD3 and anti-CD28 antibodies in the presence of Skint8-Ig or control Ig. The cells were analyzed for the expression of CD69 24 hours later. (H, J) Representative flow cytometric profiles, and (I, K, L) statistical analyses of the percentages of CD69⁺ cells in CD4⁺ or CD8⁺ T cells. The data are expressed as mean ± SD and representative of 3 independent experiments. * P<0.05 compared with control Ig.

Figure 4.

The effects of Skint8-Ig protein on cytokine production from T cells in vitro. Purified murine T cells were cultured with plate-bound (A-C) anti-CD3 antibody (1 μg/ml) in the presence of Skint8-Ig (3728 ng/ml) or equimolar amount of control Ig (1980 ng/ml), or (D, E) anti-CD3 (1 μg/ml) and anti-CD28 (0.5 μg/ml) antibodies in the presence of Skint8-Ig (6 μg/ml) or equimolar amount of control Ig for 3 days. (A) The levels of IFNγ and TNFα (pg/ml) in the supernatant were measured by ELISA kits. (B-E) The T cells were stimulated with phorbol myristate acetate and ionomycin 4 hours before harvesting, and then stained with antibodies against CD4, IFNγ and TNFα. The percentages of the cytokine positive cells in CD4⁺ T cells were determined by flow cytometry. (B, D) Representative flow cytometric profiles, and (C, E) statistical analyses of the percentages of cytokines in CD4⁺ T cells. The data are expressed as mean ± SD and representative of 3 independent experiments. * P<0.05 compared with control Ig.

Figure 5.

Skint8-Ig attenuates EAE in mice. C57BL/6 mice were immunized with 200 μg MOG_35–55 emulsified in CFA and 500 ng of purified Bordetella pertussis toxin. The mice were injected i.p. with 25 μg Skint8-Ig or control Ig protein 3 times per week from day 0. EAE development was monitored. (A) Mean clinical scores, (B-F) Thirty five days after immunization, spleens were harvested and analyzed for (B, C) CD69 expression by CD4 T cells, and (D, E) CD44^loCD62L^hi naïve and CD44^hiCD62L^lo effector memory CD4 T cells. (F-S) In separate experiments, groups of EAE mice were sacrificed at (F-I, N) prime time (day 10) or (J-M, O, P-S) peak time (day 22) of EAE. (F, G, J, K) The percentage of CD4⁺ T cells in the spleen, and (H, I, L, M) the percentage of CD4⁺CD25⁺Foxp3⁺ Tregs in CD4⁺ T cells were examined. (N, O) The splenic CD4⁺ T cells were stimulated with MOG (20 μg/ml) in vitro for 3 days; the supernatant was analyzed for the production of the indicated cytokines by ELISA. (P-S) The CNS infiltrating (P, Q) CD4⁺ T cells and (R, S) CD4⁺CD25⁺Foxp3⁺ Tregs were analyzed. The data are pooled from 2 independent experiments (n=8 for Skint8-Ig or control Ig groups each). * P<0.05 compared with control Ig-treated mice.