In vivo costimulatory role of B7-DC in tuning T helper cell 1 and cytotoxic T lymphocyte responses

Abstract

B7-DC, one of the recently described B7 family members, has the capacity to inhibit T cell responses via engagement of the immunoreceptor tyrosine-based inhibitory motif-containing inhibitory PD-1 receptor as well as enhance responses via an as yet unidentified costimulatory receptor. B7-DC is highly homologous to a coinhibitory B7 family member, B7-H1, which also binds PD-1. It is currently unclear which B7-DC function-costimulation or inhibition-predominates in vivo. To study in vivo functions of B7-DC, we evaluated immune responses in B7-DC knockout (KO) mice. Although not eliminated, interferon-gamma (IFN-gamma) production by CD4 T cells and IFN-gamma-dependent humoral responses were reduced in B7-DC KO mice relative to wild type mice. Antigen-specific CD8 T cell responses and cytotoxic T lymphocyte (CTL) activity were also diminished in B7-DC KO mice. Hepatic tumors grew more quickly in B7-DC KO mice, associated with a decrease in intrahepatic tumor-specific CD8 T cells. These results highlight the contrasting in vivo roles of B7-DC and B7-H1 and indicate that B7-DC functions as a tuning molecule, selectively augmenting T helper 1 and CTL responses.

Figure 1.

Generation and characterization of B7-DC gene KO mice. (a) The targeting map of B7-DC genomic locus. The signal peptide with the ATG initiation codon of B7-DC was replaced with EGFP and NEO cassette. After mating with CMV-Cre mouse, the NEO cassette was deleted. The 5′ probe indicated was used for Southern blot analysis of mouse genomic DNA. (b) Flow cytometry analysis of B7-DC, B7-H1, B7-1, B7-2, and MHC class II (H-2K^d) expression on the surface of BMDCs from WT and B7-DC KO mice on the BALB/c background. Mouse BM cells were cultured with GM-CSF for 6 d and with GM-CSF and IL-4 for additional 2 d. Data are representative of six experiments. (c) Absolute numbers of BMDCs from WT or B7-DC KO mice (n = 13/group).

Figure 2.

In vitro T cell proliferation. (a) Purified T cells from WT and B7-DC KO mice were cultured with the indicated concentration of ConA for 3 d. 16 h before the end of culture, ³H-thymidine was added, and its uptake was measured. (b) Purified DO11.10 CD4 T cells were labeled with CFSE and cultured for 3 d with BMDCs from WT and B7-DC KO mice in the presence of various concentrations of OVA_323-339 peptide. CFSE dilution as a measure of cell division of CD4⁺KJ1-26⁺ T cells was analyzed by FACS. Data are representative of three independent experiments.

Figure 3.

In vitro Th1 and Th2 cell generation by dendritic cells. 1 × 10⁴ purified DO11.10 CD4 T cells were cultured with 3 ×10⁴ BMDCs from WT or B7-DC KO mice with OVA_323-339 peptide for 5 d in the presence of recombinant IL-12 and anti-IL-4 mAb for Th1 generation or recombinant IL-4 and anti-IFN-γ mAb for Th2 generation. 6 h before end of culture, additional OVA_323-339 peptide with or without anti-CD3 mAb was added for restimulation. After surface CD4 was stained, cells were fixed and permeabilized, and intracellular IFN-γ and IL-4 were stained. The data shown are gated on CD4⁺ cells. Data are representative of four independent experiments.

Figure 4.

In vivo CD4 T cell stimulation after DC immunization. 5 ×10⁶ purified CFSE-labeled DO11.10 CD4 T cells were transferred into WT or B7-DC KO mice on d −1. Mice were then immunized with 1 ×10⁶ OVA_323-339 peptide–pulsed BMDCs on d 0. Peptide-pulsed BMDCs from WT mice were injected into WT mice, and peptide-pulsed BMDCs from B7-DC KO mice were injected into B7-DC KO mice, respectively. (a and b) Proportion and (c and d) cell division (CFSE dilution) of CD4⁺KJ1-26⁺ T cells in spleen, PLN, liver, and HLN were analyzed by FACS on d 3 (a and c) and d 5 (b and d). The CFSE-labeled CD4⁺KJ1-26⁺ T cells in liver, immunized with BMDCs without OVA_323-339 peptide, are also shown in Fig. S2 as negative controls. (e) The absolute number of DO11.10 CD4 T cells in spleen and liver was calculated on d 3 and 5 by the formula: total cell number × percent CD4⁺ cells × percent CD4⁺ cells staining with KJ1-26. *, Significant difference (P < 0.001) observed in liver of six pairs of B7-DC KO mice and WT mice on d 3 and 5. Data are representative of three independent experiments.

Figure 4.

In vivo CD4 T cell stimulation after DC immunization. 5 ×10⁶ purified CFSE-labeled DO11.10 CD4 T cells were transferred into WT or B7-DC KO mice on d −1. Mice were then immunized with 1 ×10⁶ OVA_323-339 peptide–pulsed BMDCs on d 0. Peptide-pulsed BMDCs from WT mice were injected into WT mice, and peptide-pulsed BMDCs from B7-DC KO mice were injected into B7-DC KO mice, respectively. (a and b) Proportion and (c and d) cell division (CFSE dilution) of CD4⁺KJ1-26⁺ T cells in spleen, PLN, liver, and HLN were analyzed by FACS on d 3 (a and c) and d 5 (b and d). The CFSE-labeled CD4⁺KJ1-26⁺ T cells in liver, immunized with BMDCs without OVA_323-339 peptide, are also shown in Fig. S2 as negative controls. (e) The absolute number of DO11.10 CD4 T cells in spleen and liver was calculated on d 3 and 5 by the formula: total cell number × percent CD4⁺ cells × percent CD4⁺ cells staining with KJ1-26. *, Significant difference (P < 0.001) observed in liver of six pairs of B7-DC KO mice and WT mice on d 3 and 5. Data are representative of three independent experiments.

Figure 5.

In vivo Th1 response in B7-DC KO mice. 5 ×10⁶ purified DO11.10 CD4 T cells were adoptively transferred into WT and B7-DC KO mice on d −1 as in Fig. 4. Mice were then immunized with 10⁶ OVA_323-339 peptide-pulsed BMDCs on d 0. Peptide-pulsed BMDCs from WT mice were injected into WT mice, and peptide-pulsed BMDCs from B7-DC KO mice were injected into B7-DC KO mice, respectively. On d 5, CD4⁺KJ1-26⁺ T cells were sorted out, and their mRNA was extracted. The expression level of each cytokine was detected by real-time RT-PCR, and relative transcripts were calculated by the formula given in Material and methods. Data are representative of three independent experiments.

Figure 6.

Class switching of antibodies in B7-DC KO mice. 100 μg of NP¹⁷-KLH with alum was injected twice (2-wk interval between injections) into WT mice (n = 7) or B7-DC KO mice (n = 7). Sera were collected, and the titers of isotype of NP¹⁷-specific antibody were evaluated by ELISA. Each dot indicates the titer of a single mouse. *, Significantly different (P < 0.001) from WT mice.

Figure 7.

CD8 T cell responses and CTL activity in B7-DC KO mice. 2 × 10⁶ T cells from H-2K^d+ HA-specific TCR transgenic mice (clone 4 mice) were transferred into BALB/c WT or B7-DC KO mice on d −1 followed by immunization with 1 ×10⁶ peptide (HA_518-526)-loaded BMDCs. As in the CD4 T cell experiments in Figs. 4 and 5, WT mice were immunized with WT BMDCs, and B7-DC KO mice were immunized with B7-DC KO BMDCs. (a) Expansion of HA-specific CD8 T cells was measured by FACS analysis of hepatic, lymph node, and splenic lymphocytes. Because there is no clonotypic antibody for this TCR, donor clone 4 T cells were marked with Thy1.1, and recipients were Thy1.2. (b) To measure CTL activity directly, an in vivo CTL assay was done in which two populations of differentially CFSE- labeled Balb/c splenocytes, one of which was loaded with HA_518-526, were transferred into the immunized mice. As a control, some immunized mice received splenocytes in which neither the CFSE^hi or CFSE^lo cells were peptide loaded. In vivo CTL activity is determined based on the selective reduction in the ratio of peptide-loaded cells (CFSE^lo) to unloaded cells (CFSE^hi) reisolated from the spleen 10 h after injection, calculated as described in Materials and methods. Data are representative of three independent experiments.

Figure 8.

Antitumor responses in B7-DC KO mice. Hepatic metastases of CT26 were established by intrahemisplenic injection of 1 × 10⁵ CT26 cells into either WT or B7-DC KO mice on d 0 (n = 10). (a) Mouse survival was observed (P < 0.01). (b) Livers were excised from WT and B7-DC KO mice on d 15, and liver weight and number of tumor nodules was measured. (c) Hepatic lymphocytes were isolated on d 3 and stained with AH-1 peptide–loaded Ld tetramer or control (β-galactosidase–loaded Ld tetramer). (d) Total number of AH-1–specific CD8 T cells were analyzed by FACS staining. Data in b, c, and d are representative of three independent experiments.

Figure 8.

Antitumor responses in B7-DC KO mice. Hepatic metastases of CT26 were established by intrahemisplenic injection of 1 × 10⁵ CT26 cells into either WT or B7-DC KO mice on d 0 (n = 10). (a) Mouse survival was observed (P < 0.01). (b) Livers were excised from WT and B7-DC KO mice on d 15, and liver weight and number of tumor nodules was measured. (c) Hepatic lymphocytes were isolated on d 3 and stained with AH-1 peptide–loaded Ld tetramer or control (β-galactosidase–loaded Ld tetramer). (d) Total number of AH-1–specific CD8 T cells were analyzed by FACS staining. Data in b, c, and d are representative of three independent experiments.