Rapid loss of early antigen-presenting activity of lymph node dendritic cells against Ag85A protein following Mycobacterium bovis BCG infection

Abstract

Background: Control of Mycobacterium tuberculosis (Mtb) infection requires CD4⁺ T-cell responses and major histocompatibility complex class II (MHC II) presentation of Mtb antigens (Ags). Dendritic cells (DCs) are the most potent of the Ag-presenting cells and are central to the initiation of T-cell immune responses. Much research has indicated that DCs play an important role in anti-mycobacterial immune responses at early infection time points, but the kinetics of Ag presentation by these cells during these events are incompletely understood.

Results: In the present study, we evaluated in vivo dynamics of early Ag presentation by murine lymph-node (LN) DCs in response to Mycobacterium bovis bacillus Calmette-Guérin (BCG) Ag85A protein. Results showed that the early Ag-presenting activity of murine DCs induced by M. bovis BCG Ag85A protein in vivo was transient, appearing at 4 h and being barely detectable at 72 h. The transcription levels of CIITA, MHC II and the expression of MHC II molecule on the cell surface increased following BCG infection. Moreover, BCG was found to survive within the inguinal LN DC pool, representing a continuing source of mycobacterial Ag85A protein, with which LN DCs formed Ag85A peptide-MHCII complexes in vivo.

Conclusions: Our results demonstrate that a decrease in Ag85A peptide production as a result of the inhibition of Ag processing to is largely responsible for the short duration of Ag presentation by LN DCs during BCG infection in vivo.

Keywords: Ag-presenting activity; Dendritic cell; In vivo; M. bovis BCG; Major histocompatibility complex class II.

Fig. 1

Detection of IFN-γ production following BCG infection. Four groups of C57BL/6 mice (n = 6) s.c. vaccinated with 1 × 10⁸ CFU BCG were sacrificed at different time points, and their spleens and inguinal LNs were removed. Increased IFN-γ levels were detected in the culture supernatants of splenocytes (a) and inguinal LN cells (b). Results are representative of three independent experiments and presented as means ± SEM. Statistical significance was determined using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). CM, culture medium

Fig. 2

Detection of murine LN DCs Ag-presenting activity ex vivo. Suspensions of inguinal LN cells from C57BL/6 mice were stained with anti-CD11c MicroBeads and separated by autoMACS, resulting in a population of 94.7% CD11c⁺ cells (a). To investigate the dynamics of LN DC Ag-presenting activity, we harvested and sorted LN DCs from the inguinal LN at various time points groups after s.c. injection of mice (n = 6) with BCG or heat-killed BCG. Then, these cells were serially diluted and used to directly stimulate DE10 T-cell hybridomas. In vivo formation of Ag85A peptide-MHC complexes on LN DCs from mice injected with BCG (b) or heat-killed BCG (c) were estimated by measuring IL-2 production in DE10 T-cell hybridoma culture supernatants ex vivo. The experiment was repeated at least three times

Fig. 3

Expression of MHC II, CIITA, and co-stimulatory molecules on DCs following BCG infection. Inguinal LNs were obtained at different time points following s.c. infection of mice with 1 × 10⁸ CFU BCG (n = 6) and were sorted and stained with a panel of mAbs to detect cell-surface expression of CD40 (a), CD54 (b), CD80 (c), and CD86 (d) by FACS. Inguinal LNs were obtained from five groups of mice (n = 6) at different time points following s.c. injection of 1 × 10⁸ CFU BCG. Transcription levels of MHC II, total CIITA (CIITA T), and CIITA type I (CIITA I) were analyzed using real-time PCR (e), and DCs were sorted and stained with mAbs to detect MHC II by FACS (f). The results are representative of three independent experiments and presented as means ± SEM. Statistical significance was determined using Student’s t-test (*P < 0.05)

Fig. 4

BCG infection kinetics of murine LN DCs. Six groups of mice (n = 6) were s.c. injected with 1 × 10⁸ CFU rBCG-GFP, and LN cells were harvested at different time points. DCs were sorted and analyzed for the presence of rBCG-GFP. Infection of murine LN DCs with BCG (a). Six groups of mice (n = 6) were s.c. injected with 1 × 10⁸ CFU BCG, and BCG in DCs was quantified in CFUs by culturing on Middlebrook 7H10 agar (b). The results are representative of three independent experiments and presented as means ± SEM. Statistical significance was determined using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001)