In vivo tracking and protective properties of Yersinia-specific intestinal T cells

Abstract

After invasion via M cells enteropathogenic Yersinia enterocolitica subsequently establish an infection at three different sites: (i) Peyer's patches (PP), (ii) mesenteric lymph nodes (MLN), and after systemic dissemination in (iii) spleen, liver and lung. In order to characterize protective properties of intestinal T cells at the different sites of Y. enterocolitica infection, PP and MLN T cells were isolated from Y. enterocolitica-infected C57B1/6 mice and Yersinia-specific T cell lines were generated. These T cells exhibited the phenotype of CD4 Th1 cells. The adoptive transfer of Yersinia-specific Th1 cells from PP and MLN conferred protection against a lethal orogastric inoculum with Y. enterocolitica as revealed by survival post-infection. However, determination of bacterial counts in infected organs revealed that the transfer of PP T cells conferred protection in spleen but not in MLN and PP, whereas the transfer of T cells from MLN reduced bacterial counts in both spleen and MLN but not in PP. To elucidate the different protection pattern we wanted to track the transferred cells in vivo. For this purpose the cells were labelled with the stable green fluorescent cell linker PKH2-GL prior to the adoptive transfer. In vivo tracking of these cells revealed that the distribution pattern of transferred T cells in spleen, MLN and PP correlated closely with the protection pattern observed after Yersinia infection. Thus, most cells were recovered from the spleen, while only few cells were recovered from MLN and PP. In keeping with these results a rapid and significant increase in interferon-gamma (IFN-gamma) production in the spleen of mice after adoptive transfer of T cell lines was observed. Taken together, the present results demonstrate that intestinal CD4 Th1 cells from PP and MLN may be involved in the defence against Y. enterocolitica at different sites of the infection, and that PKH2-GL labelling is a suitable tool to characterize T cell functions in vivo.

Fig. 1

Survival of mice after adoptive transfer of LK 5.93 and GLP 2.92 T cells and subsequent (day 0) infection with Yersinia enterocolitica. Control mice received ovalbumin-specific T cells.

Fig. 2

Adoptive transfer of PKH2-GL-labelled or non-labelled Yersinia-specific T cell lines (a) GLP 2.92 and (b) LK 5.93 1 day before orogastric infection with 7 × 10⁸Yersinia enterocolitica. Five days after infection, mice were killed and bacterial counts in Peyer's patches (PP), mesenteric lymph nodes (MLN) and spleen determined as described in Materials and Methods. The values given are the means of 10 mice per group. *Values that differ from the control group at P < 0.05.

Fig. 3

Proliferative responses of PKH2-GL-labelled and non-labelled T cells (GLP 2.92) 24, 48 and 72 h after stimulation with concanavalin A (Con A). Proliferation of cells was determined by 3H-thymidine uptake as described in Materials and Methods. *Values that differ from the non-labelled control cells at P < 0.05. ▪, Control cells; □, PKH2-GL-labelled cells.

Fig. 4

IFN-γ production of PKH2-GL-labelled and non-labelled T cells (GLP 2.92) 24, 48 and 72 h after stimulation with concanavalin A (Con A). IFN-γ production was determined in cell culture supernatants by capture ELISA as described in Materials and Methods. *Values that differ from the non-labelled control cells at P < 0.05. ▪, Control cells; □, PKH2-GL-labelled cells.

Fig. 5

Tracking of PKH2-GL-labelled T cells (GLP 2.92) by FACS analysis in spleen 4 days after adoptive transfer of 2 × 10⁷ GLP 2.92 T cells. Fifty thousand cells per sample were analysed. Non-labelled T cells (GLP 2.92, left), PKH2-GL-labelled T cells (GLP 2.92, middle) and recovered PKH2-GL T cells (GLP 2.92, spleen, right). Ordinate, number of cells; abscissa, relative fluorescence signal.

Fig. 6

Double staining of PKH2-GL-labelled GLP 2.92 T cells recovered from the lung 4 days after adoptive transfer. The ordinate indicates the relative fluorescence intensity for CD4, the abscissa indicates the relative fluorescence intensity of PKH2-GL-labelled T cells.

Fig. 7

Fluorescence microscopy of PKH2-GL-labelled T cells 1 day (a) and 4 days (b) after staining with PKH2-GL and culture in vitro. Cryosections of spleen (c) and mesenteric lymph nodes (MLN) (e) 4 days after transfer of 2 × 10⁷ PKH2-GL-labelled LK 5.93 T cells and cryosections of spleen (d) and MLN (f) 4 days after a transfer of 2 × 107 PKH2-GL-labelled GLP 2.92 T cells.