B7-H1 expression on non-B and non-T cells promotes distinct effects on T- and B-cell responses in autoimmune arthritis

Abstract

The immune system has developed several regulatory mechanisms to maintain homeostasis of adaptive immune responses. T-cell programmed death (PD)-1 recognition of B7-H1 (PD-L1) expressed on APC and non-lymphoid tissue regulates T-cell activation. We show that B7-H1(-/-) mice exhibit exacerbated proteoglycan (PG)-induced arthritis and increased Th-1 CD4(+) T-cell responses. Unexpectedly, the PG-specific antibody response in B7-H1(-/-) mice was diminished. A reduction in the number of peanut agglutinin(+) GC coincided with a decrease in CD19(+) GL-7(+) CD95(+) GC B cells that was a result of increased caspase-induced apoptosis. The percent of CD38(+) CD138(+) emerging plasma cells was decreased. B7-H1(-/-) mice exhibited an increased frequency of CD4(+) PD-1(hi) CXCR5(hi) ICOS(hi) CD62L(lo) T follicular helper cells that displayed a hyperactive phenotype with increased expression of mRNA transcripts for Bcl6, IL-21, and the apoptosis-inducer molecule FasL. In cell transfer of B7-H1(-/-) cells into SCID mice, non-B and non-T cells were sufficient to normalize the antibody response, T-cell hyperactivity, and the development of PG-induced arthritis. These findings indicate that B7-H1 on non-B and non-T cells signals through PD-1 on T effector cells to prevent excessive activation and reduce autoimmune arthritis. Furthermore, these findings demonstrate a novel role for B7-H1 expression in promoting B-cell survival by regulating the activation of T follicular helper cell.

Figure 1. Exacerbation of PGIA and autoreactive CD4⁺ T cell responses in B7-H1^−/− mice

WT BALB/c (n=10–15) and PD-L1^−/− (n=10–15) mice were monitored for arthritis severity (A) and incidence (B) following three i.p. immunizations of human PG in DDA at 3-week intervals. Hindlimbs were harvested from WT (C & E) or B7-H1^−/− (D & F) mice 6 and 10 weeks after the initial PG immunization. Sections were stained with H&E. Magnifications of 4× (C & D) and 10× (E & F) were used for histological scores (G) were averaged from both hind joints from 3–6 mice per group and are representative of two independent experiments. Proliferation in response to PG (10μg/ml) restimulation (H) of CD4⁺ cells collected at 10 weeks was measured by [³H]-thymidine incorporation during the last 24 hours of 5 day cultures. Flow cytometry was used to determine the percentage of CD25⁺ FoxP3⁺ Tregs (I) and expression level of CD62L (J) of splenic CD4⁺ T cells. IFN-γ (K & L), IL-4 (M & N) and IL-17 (O & P) were measured by ELISA (K, M & O) of supernatants from 4 day cultures or by ELISPOT assays (L, N & P). Values are means ± SEM and are representative of three independent experiments. *, p< 0.05, Mann-Whitney U.

Figure 2. Autoantibody titers are diminished in B7-H1^−/− mice

WT (n=5–10) and B7-H1^−/− (n=5–10) mice were immunized i.p. with PG-DDA 3 times at three week intervals as indicated by arrows. Serum samples collected from at weeks 1, 4, 7 and 10 after the initial PG-DDA immunization were measured for the presence of autoantibody. Titers of both human-PG-specific IgG1 (A) and IgG2a (B) and mouse-PG-specific IgG1 (C) and IgG2a (D) antibodies were measured by ELISA. Values are means ± SEM and are representative of four independent experiments. *, p< 0.05, Mann-Whitney U.

Figure 3. Germinal center B cells and plasma cells are decreased in B7-H1^−/− mice

Spleens from WT and B7-H1^−/− mice (n=3–4) were isolated 1 week after the second PG-DDA immunization. Germinal center B cells were assessed by PNA staining of frozen sections (A) and by dual staining of CD19⁺ cells with GL-7 and CD95 (B). Plasma cells were also identified using flow cytometry by CD38 and CD138 double positive cells (C). Spleens from naïve WT or B7-H1^−/− mice were assessed for T1, T2, marginal zone (MZ) and follicular (FO) B cell subsets (D) or for the proliferative capacity of total splenic B cells to response to LPS (0.4 or 2 μg/ml) or anti-CD40 (3 μg/ml) + anti-IgM (3 μg/ml) (E). Values are means ± SEM and are representative of two independent experiments. *, p< 0.05, Mann-Whitney U.

Figure 4. B7-H1 negatively regulates T_FH cells

Spleens from WT (n=4–5) or B7-H1^−/− (n=4) mice were harvested 10 days following the second PG-DDA immunization. Absolute number (A), percent CaspGLOW⁺ (B & C) and ICOS mean fluorescence intensity (MFI) (D) of splenic T_FH cells (CD3⁺CD4⁺PD-1^hiCXCR5^hiICOS^hiCD62L^lo) were assessed by flow cytometry. mRNA was isolated from sorted naïve, T_FH and non-T_FH cells from WT or B7-H1^−/− and assessed for Bcl-6 (E), IL-21 (F) and FasL (G) transcripts by qPCR. GL-7⁺CD95⁺ GC B cell death was assessed by CaspGLOW and flow cytometry (H & I). Values are means ± SEM and are representative of two independent experiments. *, p< 0.05, Mann-Whitney U.

Figure 5. Stimulatory B7-H1 signals are not provided by B or T cells

Isolated splenic B cells (2×10⁷) from WT or B7-H1^−/− mice were coinjected with B-depleted (2×10⁷) splenic cells from WT or B7-H1^−/− intravenously into SCID mice recipients. One week following cell transfer, mice began PGIA immunization regime. SCID mice groups were reconstituted as follows: WT B-depleted cells + WT B cells (T^WT B^WT) (n=5); WT B-depleted cells + B7-H1^−/− B cells (T^WT B^B7-H1−/−) (n=5); B7-H1^−/− B-depleted cells + B7-H1^−/− B cells (T^B7-H1−/− B^B7-H1−/−) (n=5). Spleens were collected 1 week following the second PG-DDA immunization and analyzed by flow cytometry for GC B cells as GL-7⁺ CD95⁺ of CD19⁺ (A) along with PCs as CD38⁺ CD138⁺ of total cells (B). Autoantibodies titers (C–E) were evaluated by ELISA from serum samples collected at 8 weeks. Values are means ± SEM and are representative at least two independent experiments. *, p< 0.05, Mann-Whitney U.

Figure 6. B cell B7-H1 regulates T cell activation in vitro, but non-B and T cell tissue expression of B7-H1 can regulate excessive PGIA in vivo

Purified splenic. naïve or LPS pre-activated WT or B7-H1^−/− B cells were cocultured with purified CD4⁺ T cells from PG immunized WT mouse spleens for 5 days with or without PG (10μg/ml) restimulation. Proliferation (A) was measured through [³H]-thymidine in the final 24 hours. SCID mice were reconstituted with splenic B and T cells from WT and B7-H1^−/− mice as described previously. Following cell transfer and PG-DDA immunizations mice were monitored for PGIA severity (B) and incidence (C). Spleens were harvested at week 8. Proliferation (D) of splenic CD4+ T cells in responses to PG (10μg/ml) restimulation was measured by ³H-thymidine incorporation for the final 24 hours of 5 day cultures. Values are means ± SEM and are representative of three independent experiments. *, p< 0.05, Mann-Whitney U.