Preexisting inflammation due to Mycobacterium bovis BCG infection differentially modulates T-cell priming against a replicating or nonreplicating immunogen

Abstract

Induction of T-cell memory by vaccination ensures long-term protection against pathogens. We determined whether on-going inflammatory responses during vaccination influenced T-cell priming. A preexposure of mice to Mycobacterium bovis BCG impaired their subsequent ability to prime T cells against Listeria monocytogenes. This was characterized by a decrease in L. monocytogenes-specific gamma interferon (IFN-gamma)-secreting CD4(+) and CD8(+) T cells. The intensity of T-cell priming towards L. monocytogenes depended on the extent of L. monocytogenes expansion, and a cessation of this expansion caused by M. bovis BCG-induced inflammation resulted in impairment in T-cell priming. A challenge of M. bovis BCG-infected mice with a higher L. monocytogenes dose increased L. monocytogenes survival and restored T-cell priming towards L. monocytogenes. Impairment in T-cell priming towards L. monocytogenes due to M. bovis BCG-induced inflammation resulted in a compromised protective efficacy in the long term after mice were rechallenged with L. monocytogenes. Preexisting inflammation selectively impaired T-cell priming for replicating immunogens as CD8(+) T-cell response to ovalbumin administered as an inert antigen (ovalbumin-archaeosomes) was enhanced by M. bovis BCG preimmunization, whereas priming towards ovalbumin administered as a live immunogen (L. monocytogenes-ovalbumin) was impaired. Thus, depending on the nature of the immunogen, the presence of prior inflammatory responses may either impede or boost vaccine efficacy.

FIG. 1.

Preimmunization with M. bovis BCG impairs T-cell priming for L. monocytogenes. BALB/c mice were injected with PBS or M. bovis BCG on day 1, and 30 days later, mice were challenged with L. monocytogenes. Spleens were harvested at day 37 (7 days after L. monocytogenes [LM] challenge) and day 60 (30 days after L. monocytogenes challenge), and the number of IFN-γ secreting cells was enumerated after stimulating cells with (a) L. monocytogenes antigens or (b) LLO_91-99. The number of IFN-γ secreting cells per 10⁶ spleen cells is indicated. The dotted line indicates the threshold of detection.

FIG. 2.

M. bovis BCG inhibits the development of cytolytic CD8⁺ T-cell response to L. monocytogenes in a dose-dependent manner. BALB/c mice were injected with PBS or with 10⁴ M. bovis BCG or 10⁵ M. bovis BCG. Thirty days later mice were injected with L. monocytogenes (LM). On day 37, pooled spleen cells from various groups of mice were restimulated with irradiated pHem3.3 cells (expressing LLO_91-99) for 5 days in the presence of IL-2 (0.1 ng/ml) as described in the Materials and Methods section. Cells were harvested and washed, and effectors were tested for their cytolytic activity on ⁵¹Cr-labeled P815 (open symbols) or P815+LLO_91-99 (solid symbols) target cells. Means ± standard deviations for triplicate cultures are shown.

FIG. 3.

M. bovis BCG infection results in an accumulation of inflammatory cells and causes increased clearance of L. monocytogenes. BALB/c mice were injected with PBS or M. bovis BCG. On day 30, spleen cells were isolated, and the numbers of cells expressing various markers were analyzed as described in the Materials and Methods section (a). Mice injected with PBS or M. bovis BCG were also challenged with L. monocytogenes (LM) on day 30, and the numbers of viable L. monocytogenes in the spleens of mice were enumerated on day 33 (b). Each symbol represents the data for an individual mouse. The dotted line indicates the threshold of detection.

FIG. 4.

Potent inflammatory response in M. bovis BCG-infected mice. PBS or M. bovis BCG was injected into BALB/c mice on day 1. On day 30, spleen cells from the PBS (solid squares) or M. bovis BCG (solid triangles)-injected mice were incubated (5 × 10⁵/well) with medium or with various numbers of heat-killed L. monocytogenes (HK LM) in vitro, and the production of various molecules was determined in 72-h supernatants. Means ± standard deviations for triplicate cultures are shown.

FIG. 5.

Rapid clearance of L. monocytogenes by M. bovis BCG results in impairment in T-cell priming. BALB/c mice were injected with PBS or M. bovis BCG on day 1. On day 30, PBS-injected mice were challenged with 5 × 10³ L. monocytogenes (LM), and the M. bovis BCG-injected mice were challenged with either 5 × 10³ or 5 × 10⁵ L. monocytogenes. Three days later (day 33), the number of viable L. monocytogenes organisms in the spleens of individual mice was enumerated (a). Dotted line indicates the threshold of detection. On day 37, spleen cells from the various experimental groups were incubated with medium (control) or with LLO_91-99, and the production of IFN-γ was measured in 72-h supernatants (b). Means ± standard deviations for triplicate cultures are shown. Statistically significant values by Student's t test (P < 0.05) for PBS control mice versus M. bovis BCG-infected mice challenged with 5 × 10³ L. monocytogenes (★) and for M. bovis BCG-infected mice challenged with 5 × 10³ versus 5 × 10⁵ L. monocytogenes (⋆).

FIG. 6.

Development of T-cell memory to L. monocytogenes correlates to the expansion of L. monocytogenes. BALB/c mice were infected with various numbers of L. monocytogenes (LM) as indicated in the figure. At days 3 and 7, the number of viable L. monocytogenes organisms in the spleens of individual mice in various experimental groups was enumerated (a). At day 30, the number of IFN-γ-secreting cells was enumerated after stimulating the spleen cells with medium (control) or with LLO_91-99. The number of IFN-γ-secreting cells per 10⁶ spleen cells in triplicates is indicated. The dotted line indicates the threshold of detection.

FIG. 7.

Impairment in the development of a T-cell response due to inflammation compromises long-term protection against a rechallenge with L. monocytogenes. BALB/c mice were injected with PBS or M. bovis BCG on day 1, and 30 days later mice were infected with L. monocytogenes (LM). On day 180, mice were rechallenged with L. monocytogenes, and the numbers of viable L. monocytogenes organisms in the spleens were enumerated at day 183. Each symbol represents data for an individual mouse. The dotted line indicates the threshold of detection.

FIG. 8.

M. bovis BCG-induced inflammation has opposite effects on T-cell priming depending on the nature of the immunogen. C57BL/6 mice were injected with PBS or with M. bovis BCG. On day 30, mice were injected intraperitoneally with 5 × 10⁵ L. monocytogenes-ovalbumin (LMOva) (a and b) or with 10 μg of ovalbumin-archaeosomes (Ova) (c). On day 33, the number of viable L. monocytogenes-ovalbumin in the spleens of individual mice was enumerated (a). Dotted line indicates the threshold of detection. On day 37, spleen cells were harvested and incubated with medium (control) or OVA_257-264. The production of IFN-γ after injection with L. monocytogenes-ovalbumin (b) or with ovalbumin-archaeosomes (c) was measured in 72-h supernatants. Means ± standard deviations for triplicate cultures are shown.