Table 1.
Bacterial strains and plasmids.
Figure 1.
OVA-specific T-cell responses to OVA produced intracellularly in E. coli.
E. coli HB101 was transformed with the pIAβ8A-OVA plasmid encoding full-length ovalbumin (OVA), or an empty pIAβ8A-control plasmid. A, Western blot showing presence of OVA in sonicates of the transformed bacteria; OVA = soluble OVA standard. B, Proliferative responses of OVA-specific transgenic DO11.10 T cells co-cultured for 4 d with irradiated splenocytes pulsed with E. coli-OVA, E. coli-control or soluble OVA (n = 6 mice for 5×107/ml bacteria and 100 µg/ml OVA; n = 2 for the other concentrations). C, Production of cytokines in 4 d co-cultures of OVA-specific T cells and CD11c+-enriched splenocytes pulsed with bacterial or soluble OVA (n = 6). D, Proliferative responses by 4 d co-cultures of OVA-specific T cells and APCs pulsed with bacterial or soluble OVA. The APCs used were CD11c+ MACS-bead enriched cells (CD11c+ enriched), or cells further purified by FACS-sorting into CD11c+B220− cells (cDCs) CD11clowB220+Ly6C+CD19− cells (pDC) or cells lacking CD11c+B220− (CD11c+ enriched cells without cDCs) (n = 6 for CD11c+-enriched cells and cDCs; n = 4 for other APCs). * P<0.05; ** P<0.01; *** P<0.001.
Figure 2.
OVA-specific T-cell responses to OVA fragment 319–386 produced intracellularly in E. coli, lactobacilli and lactococci.
Lactobacillus sakei, Lactobacillus plantarum, Lactococcus lactis, and E. coli were transformed with vectors encoding an immunodominant OVA fragment (OVAf). A, Western blot showing production of OVAf by L. sakei and E. coli; expression is lower in E. coli due to use of a vector optimal for G+ bacteria. His-tagged OVAf was used as a molecular mass marker (one representative experiment of three). Proliferative responses of DO11.10 T cells stimulated with irradiated splenocytes pulsed with OVAf-transformed E. coli (B), or lactobacilli/lactococci (C). Despite lower expression of OVAf in E. coli, OVA-specific proliferation is much more pronounced in cultures with transformed E. coli than in cultures with transformed lactobacilli/lactococci; note the different scales in the respective graphs (n = 5 for E. coli and L. sakei; n = 3 for L. plantarum and L. lactis). * P<0.05; ** P<0.01.
Table 2.
Relative expression levels of OVA fragment and GFP by transgenic Gram-negative and Gram-positive bacteria.
Figure 3.
T cell proliferation and cytokine production in response to E. coli and L. sakei expressing OVAf.
Proliferative response and cytokine production by 4 d co-cultures of OVA-specific DO11.10 T cells and CD11c+-enriched splenocytes pulsed with OVAf-expressing E. coli (A) or L. sakei (B) or the respective control bacteria transformed by an empty plasmid (n = 5). ** P<0.01.
Figure 4.
Bacteria trigger expression of activation markers on APCs, and the lactobacilli do not reduce the T cell stimulatory capacity of DCs.
A, Splenocytes were stimulated overnight with 5×107/ml of UV-inactivated L. sakei or E. coli and the expression of CD40 and CD86 on CD11c+ DCs was determined by flow cytometry. The results of one representative experiment of three performed are shown. B, CD11c+-enriched splenocytes were pulsed with 5×107/ml UV-inactivated E. coli-OVA (or E. coli-control) in the absence or presence of graded doses of UV-inactivated L. sakei or L. plantarum. The antigen-presenting cells were thereafter co-cultured with OVA-specific DO11.10 T cells and proliferation was measured day 5 (n = 5).
Table 3.
Cytokine production by bacteria-stimulated splenocytes (n = 5, mean ± SEM).