B7-H1 expression on old CD8+ T cells negatively regulates the activation of immune responses in aged animals

Abstract

T cell responses are compromised in the elderly. The B7-CD28 family receptors are critical in the regulation of immune responses. We evaluated whether the B7-family and CD28-family receptors were differentially expressed in dendritic cells, macrophages, and CD4(+) and CD8(+) T cells from young and old mice, which could contribute to the immune dysfunction in the old. Although most of the receptors were equally expressed in all cells, >85% of the old naive CD8(+) T cells expressed B7-H1 compared with 25% in the young. Considering that B7-H1 negatively regulates immune responses, we hypothesized that expression of B7-H1 would downregulate the function of old CD8(+) T cells. Old CD8(+) T cells showed reduced ability to proliferate, but blockade of B7-H1 restored the proliferative capacity of old CD8(+) T cells to a level similar to young CD8(+) T cells. In vivo blockade of B7-H1 restored antitumor responses against the B7-H1(-) BM-185-enhanced GFP tumor, such that old animals responded with the same efficiency as young mice. Our data also indicate that old CD8(+) T cells express lower levels of TCR compared with young CD8(+) T cells. However, following antigenic stimulation in the presence of B7-H1 blockade, the levels of TCR expression were restored in old CD8(+) T cells, which correlated with stronger T cell activation. These studies demonstrated that expression of B7-H1 in old CD8(+) T cells impairs the proper activation of these cells and that blockade of B7-H1 could be critical to optimally stimulate a CD8 T cell response in the old.

FIGURE 1

Staining of young and old macrophages, DCs, and CD4 and CD8 T cells for the expression of B7 family and CD28 family receptors. A, Macrophages were stained for anti–CD11b-allophycocyanin and anti-F4/80-PE. B, DCs were stained with anti–CD11c-PE. C, CD4⁺ T cells were stained with anti–CD4-PE. D, CD8⁺ T cells were stained with anti–CD8-PE. The prevalence of B7 family and CD28 family receptors in these subpopulations was analyzed by staining the samples with FITC-conjugated Abs to anti-B7.1, anti-B7.2, anti–B7-H1, anti–B7-H2, anti–B7-H3, anti–B7-H4, anti–B7-DC, anti-ICOS, anti–PD-1, anti-CD28, and anti-CTLA4. The results shown are the average percentages of positive cells in the spleens from a group of young and old animals. Three animals were included per group. *p < 0.01; young versus old mice. The results shown are representative of three independent experiments.

FIGURE 2

Kinetics of B7-H1 and PD-1 expression in naive and memory young and old CD8 T cells. Naive (CD44^loCD62L^hi) and memory (CD44^hi CD62L^lo) CD8⁺ T cells from young and old mice were sorted and stimulated with anti-CD3 plus anti-CD28. The expression of B7-H1 (A) and PD-1 (B) was analyzed at time zero and at 24, 48, and 72 h poststimulation. The results shown are the average percentages of positive cells from naive and memory CD8⁺ T cells from spleens of three young and old animals. The results shown are representative of two independent experiments.

FIGURE 3

Expression of B7-H1 in proliferating and nonproliferating young and old CD8⁺ T cells. A, Treatment regimen used on young and old animals to evaluate the expression of B7-H1 on CD8⁺ T cells in vivo. Details of the experimental designed are described in Materials and Methods. B, The expression of B7-H1 was evaluated in BrdU⁺ (proliferating) and BrdU⁻ (nonproliferating) CD8⁺ T cells from young and old animals implanted or not with BM-185 EGFP tumors. *p < 0.01; old versus young mice. Four animals were included per group. The results shown are representative of two independent experiments.

FIGURE 4

Blockade of B7-H1 restores the proliferative activity in old CD8⁺ T cells. Enriched young and old CD8⁺ T cells were labeled with CFSE. A, Stimulation with anti-CD3+anti-CD28 beads. B, Stimulation with anti-CD3+anti-CD28+Ig-PD-1 beads. C, Stimulation with anti-CD3+anti-CD28 beads in the presence of anti-B7-H1 (10 μg/ml). D, Stimulation with anti-CD3+anti-CD28+Ig-PD-1 beads in the presence of anti–B7-H1 (10 μg/ml). Cell division was evaluated after 72 h by flow cytometry. E, Young and old enriched CD8⁺ T cells were stimulated with plate bound anti-CD3+anti-CD28 in the presence or absence of anti–B7-H1 (10 μg/ml). After 72 h, the proliferation of CD8⁺ T cells was evaluated by thymidine incorporation. Each value represents the mean of triplicate wells ± SE. p < 0.001; young versus old CD8⁺ T cells. Cell division (represented by the colored curves) and proliferation data are from a single experiment that is representative of at least three independent experiments.

FIGURE 5

Blockade of B7-H1 restores the immune responses in vivo in old mice. A, Assessment of B7-H1 expression on BM-185-EGFP tumor cells. B, BM-185-EGFP tumor cells (10⁵) were implanted on day 0 in old BALB/c mice. Starting on day 5, 12 and 19 animals were injected i.p. with 100 μg/injection of anti–B7-H1 mAb or isotype control Ab or were not treated. As a positive control, a group of young BALB/c mice were injected with BM-185 EGFP tumor cells. Seven animals were included per group. Survival percentages were determined. Data are representative of two independent experiments. C, BM-185 EGFP tumors were implanted in young and old BALB/c mice treated with or without 100 μg/injection of anti-B7-H1 mAb or isotype control Ab. Animals were sacrificed 2 wk after immunization, and splenocytes were stimulated with BM-185-EGFPCD80 for 5 d in complete media. The cytotoxic activity of restimulated cultures from young mice, old mice treated with isotype control Ab, and old mice treated with anti-B7-H1 mAb was measured against the BM-185 WT and BM-185 EGFP in a standard 4-h [⁵¹Cr]-release assay at the indicated E:T ratios. The results shown are representative of three independent experiments.

FIGURE 6

Effect of blocking B7-H1 on TCR expression and subunits associations in old CD8⁺ T cells. A, Freshly isolated enriched young and old CD8⁺ T cells were stimulated or not with anti-CD3+anti-CD28 mAb in the presence of isotype control Ab or anti–B7-H1 (10 μg/ml). The levels of TCRβ expression were evaluated after 72 h. B, IP-FCM method. C, Freshly isolated young and old CD8⁺ T cells (ex vivo) were analyzed for TCR–subunit interactions. Intact TCR subunits were immunoprecipitated with the H146 anti-ζ mAb; probed with anti-ε, anti-ζ, and anti-β mAb; and the interaction between the subunits was measured by flow cytometry, as described in Materials and Methods. D, Enriched young and old CD8⁺ T cells were cultured in the presence of anti-CD3+anti-CD28 mAb in the presence of isotype control Ab or anti–B7-H1 (10 μg/ml). Native TCR complexes were immunoprecipitated with the H57 anti-β mAb and probed with Abs to ζ and ε subunits mAb; the interaction between the subunits was measured by flow cytometry. Data are from a single experiment that is representative of at least three independent experiments.