Loss of B7-H1 expression by recipient parenchymal cells leads to expansion of infiltrating donor CD8+ T cells and persistence of graft-versus-host disease

Abstract

Previous experimental studies have shown that acute graft-versus-host disease (GVHD) is associated with two waves of donor CD8(+) T cell expansion. In the current studies, we used in vivo bioluminescent imaging, in vivo BrdU labeling, and three different experimental GVHD systems to show that B7-H1 expression by recipient parenchymal cells controls the second wave of alloreactive donor CD8(+) T cell expansion and the associated second phase of GVHD. Loss of B7-H1 expression by parenchymal cells during the course of GVHD was associated with persistent proliferation of donor CD8(+) T cells in GVHD target tissues and continued tissue injury, whereas persistent expression of B7-H1 expression by parenchymal cells led to reduced proliferation of donor CD8(+) T cells in GVHD target tissues and resolution of GVHD. These studies demonstrate that parenchymal cell expression of B7-H1 is required for tolerizing infiltrating T cells and preventing the persistence of GVHD. Our results suggest that therapies designed to preserve or restore expression of B7-H1 expression by parenchymal tissues in the recipient could prevent or ameliorate GVHD in humans.

Fig. 1. Loss of B7-H1 expression by parenchymal tissues in TBI-conditioned recipients was associated with chronic inflammation

WT BALB/c recipients were pretreated with PBS or anti-CD3 mAb (5 μg/g) seven days before transplantation. On day 0, 8 hours after 800 cGy TBI, CD8⁺ T cells (20×10⁶) from luc⁺ C57BL/6 donors and BM cells (50×10⁶) from WT C57BL/6 donors were injected i.v. into recipients. Recipients were monitored for clinical GVHD 1-2 times a week. In vivo donor CD8⁺ T cell expansion was visualized with in vivo BLI every 5 days for up to 40 days after transplantation. (A) A representative series of in vivo BLI pictures depicting donor CD8⁺ T expansion is shown for 1 of 8 recipients in each group. (B) Kinetic curves of whole body emission of photons per second (mean ± SE, N=8). (C) Clinical GVHD scores (mean ± SE, N=8). (D) On days 0, 10, 20, 30, and 40 after transplantation, hepatocytes from the recipients with or without anti-CD3 pretreatment were isolated and stained with anti-B7-H1or isotype control. One representative FACS pattern is shown of three replicate experiments. Hepatocytes from D40 were also used for real-time PCR of B7-H1 mRNA. Relative expression levels of B7-H1 mRNA is shown (mean ± SE, N=4). (E) On day 30 after transplantation, recipients with or without anti-CD3 pretreatment were given 150 μg recombinant murine IFN-γ by i.v. injection. 48 hours later, hepatocytes from the recipients were stained with anti-B7-H1 or isotype control. One representative FACS pattern is shown of three replicate experiments. (F-G) On day 30 after transplantation, the infiltrating mononuclear cells (0.2×10⁶/well) from liver tissues were stimulated with PMA (10ng/ml) and ionomycin (200ng/ml) for 72 hours, and then the culture supernatants were measured with ELISA. (F) left panel: IFN-γ concentration (mean ± SE, N=4); right panel: relative expression levels of IFN-γR mRNA. (G) IL-6, IL-2, TNF-α concentration (mean ± SE, N=4).

Fig. 2. Induction of hepatocyte expression of B7-H1 via hydrodynamic injection of B7-H1 cDNA inhibited tissue infiltrating donor CD8⁺ T cell expansion

WT BALB/c recipients were conditioned with 800cGy TBI and then given CD8⁺ T cells (5 × 10⁶) and BM cells (50 × 10⁶) from WT C57BL/6 donors. 20 days after transplantation, the recipients were given hydrodynamic injection of B7-H1 cDNA or vector control. Thereafter, the recipients were monitored for clinical GVHD. (A) 24 hours after hydrodynamic injection, hepatocytes from recipients treated with B7-H1 plasmid or vector control were stained for anti-B7-H1 or IgG isotype control. A representative FACS pattern is shown from one of three replicate experiments. (B) Clinical GVHD scores (mean ± SE, N=13). (C) Survival curves (N=13). (D) 50 days after transplantation, sorted donor CD8⁺ T cells (0.2 ×10⁶/well) from liver tissues of recipients treated with B7-H1 plasmid or vector control were stimulated with immobilized anti-CD3 for 72 hours, and ³H-TdR incorporation was measured. Mean ± SE of CPM of ³H-TdR incorporation from 4 replicate experiments is shown. (E) 50 days after transplantation, liver tissue was harvested for histopathologic evaluation. The mean ± SE of pathology scores of 6 recipients per group are shown.

Fig. 3. Expression of B7-H1 by recipient tissues prevented the second wave of donor CD8⁺ T cell expansion and the second phase of GVHD

WT BALB/c or B7-H1^−/− recipients were conditioned with anti-CD3-based regimen and then given CD8⁺ T cells (20×10⁶) from luc⁺ C57BL/6 donors and BM cells (50×10⁶) from WT C57BL/6 donors. Recipients were monitored for clinical GVHD 1-2 times a week. Donor CD8⁺ T cell expansion was visualized with in vivo BLI for more than 50 days after transplantation. Additional recipients were used to compare donor T cell yield in lymphoid tissues and GVHD target tissues and to evaluate histopathology. (A) A representative series of in vivo BLI pictures depicting donor CD8⁺ T expansion is shown for 1 of 8 recipients in each group. (B) Kinetic curves of whole body emission of photons per second (mean ± SE, N=8). (C, D) Clinical GVHD score (mean ± SE) and survival curves, N=8. (E-G) Additional recipients were used for enumerating thymocytes, MNC in spleen, and donor T cells in spleen, liver, and skin via staining of H-2K^b, TCRβ, CD4 and CD8, 35 days after transplantation. (E) Yields of CD4⁺CD8⁺ thymocytes (mean ± SE, N=4). (F) Yields of MNCs in spleen (mean ± SE, N=4). (G) Yields of donor CD8⁺ T cells in spleen, liver and skin (mean ± SE, N=4). (H) Histopathology scores (mean ± SE, N=6).

Fig. 4. Expression of B7-H1 by parenchymal tissues prevented the second wave of donor CD8⁺ T cell expansion and the second phase of GVHD in Rag-2^−/− recipients

Rag2^−/− BALB/c mice were given lethal TBI (800 cGy) and then given BM cells (5×10⁶) from B7-H1^−/− Rag2^−/− BALB/c mice to create B7-H1^+/+ chimeras that had B7-H1 expression only in parenchymal cells. Conversely, B7-H1^−/− chimeras with expression of B7-H1 in hematopoietic cells but not in parenchymal cells were established by transferring BM cells from Rag-2^−/− mice into B7-H1^−/−Rag2^−/− mice. The control Rag-2^−/− mice were given TBI and BM cells from Rag-2^−/− donors. 60 days later, the B7-H1^+/+ chimeras and the B7-H1^−/− chimeras were used as transplantation recipients without conditioning. (A-B) B7-H1^−/− chimeras were given CD8⁺ T cells (20 × 10⁶) and BM cells (50 × 10⁶) from C57BL/6 donors.14 days after transplantation, the recipients were given i.p. injections of anti-B7-H1 mAb or control rat IgG (5 μg/g) every other day for 30 days. Recipients were monitored for clinical GVHD and survival. (A) Kinetic curves of clinical score (N=9). (B) Survival curves (N=9). (C-F) B7-H1^−/− chimeras and control Rag-2^−/− mice were given luc⁺CD8⁺ T cells (20 × 10⁶) from luciferase-transgenic C57BL/6 donors and BM cells (50 × 10⁶) from WT C57BL/6 donors. Thereafter, recipients were monitored for clinical GVHD once or twice weekly. Donor CD8⁺ T cell expansion was visualized with in vivo BLI for more than 40 days after transplantation. (C) A representative series of in vivo BLI pictures depicting donor CD8⁺ T expansion is shown for 1 of 8 recipients in each group. (D) Kinetic curves of whole body emission of photons per second (N=8). (E) Clinical GVHD scores (N=8). (F) Survival curves (N=8).

Fig. 5. Comparison of percentage of CD4⁺CD8⁺ thymocytes and percentage of injected donor CD8⁺ T cells in spleen and GVHD target tissues of recipients with and without GVHD

WT BALB/c recipients were conditioned with 800 cGy TBI or with anti-CD3, vorinostat, and busulfan, and then given CD8⁺ T cells (20×10⁶) from WT (CD45.2) C57BL/6 donors and TCD-BM cells (50×10⁶) from congenic (CD45.1) C57BL/6 donors. 35 days after transplantation, when TBI-conditioned recipients showed severe clinical GVHD, the percentage of CD4⁺CD8⁺ thymocytes and percentage of injected CD8⁺ T cells in spleen and GVHD target tissues were measured by flow cytometry. (A) Thymocytes are shown as CD4 versus CD8 staining. Representative results from 1 of 4 replicate experiments are shown. (B-D) MNC of spleen, liver, and skin were stained for H-2K^b, TCRβ, CD8 and CD45.2. The gating of each subset is indicated by the arrows. Representative results from 1 or 4 replicate experiments are shown.

Fig. 6. Yield of CD4⁺CD8⁺ thymocytes and injected donor CD8⁺ T cells in spleen and GVHD target tissues in recipients with and without GVHD

Based on the percentage of T cell subsets and mononuclear cell numbers from different tissues in Fig. 5, the yield (mean ± SE, N=4) of T cell subsets was calculated. (A) Yield of CD4⁺CD8⁺ thymocytes. (B) Yield of MNC of spleen. (C-D) Yield of total donor T, yield of donor CD8⁺ T, and yield of injected donor CD8⁺ T in spleen, liver, and skin, respectively.

Fig. 7. Comparison of proliferation of the injected donor CD8⁺ T cells in the spleen and GVHD target tissues in recipients with or without GVHD

WT BALB/c recipients were conditioned with 800 cGy TBI or with vorinostat, busulfan and anti-CD3, and then given CD8⁺ T cells (20×10⁶) from WT (CD45.2) C57BL/6 donors and TCD-BM cells (50×10⁶) from congenic (CD45.1) C57BL/6 donors. Beginning on day 35 after transplantation, recipients were given daily i.p. injections of BrdU (50 μg/g bodyweight) for three days, and the percentage of proliferating BrdU⁺ T cells in lymphoid tissue spleen/lymph nodes and GVHD target tissues liver, gut, and skin was measured with flow cytometry on day 38. In addition, hepatocytes were measured for B7-H1 expression. Donor CD8⁺ T cells infiltrating the liver were measured for expression of PD-1 and IL-7Rα. Donor CD8⁺ T cells infiltrating the liver were also sorted for measurement of in vitro proliferation after stimulation with immobilized anti-CD3. (A) Gated H-2K^b+CD45.2⁺CD8⁺ injected donor T cells are shown in BrdU versus CD8. One representative FACS pattern and mean percentage ± SE of BrdU⁺ cells are shown (N= 4). (B) Hepatocytes were isolated and stained for anti-B7-H1 or IgG isotype control. In addition, B7-H1 mRNA levels in hepatocytes were measured by real-time PCR. One representative FACS pattern and relative B7-H1 mRNA expression are shown (mean ± SE, N=4). (C) Liver MNCs from normal BALB/c or recipients were stained for H-2K^b, CD45.2, CD8, PD-1 or IL7Rα. Gated H-2K^b+CD45.2⁺CD8⁺ donor T were shown as a histogram of PD-1 or IL7Rα. One representative FACS pattern and mean percentage ± SE of PD-1⁺ or IL-7Rα⁺ cells are show (N=4). (D) Sorted H-2K^b+CD45.2⁺CD8⁺ donor T cells from liver MNC were stimulated with immobilized anti-CD3, and ³H-TdR incorporation was measured. The mean ± SE of 4 replicate experiments is shown.