In vivo compartmentalization of functionally distinct, rapidly responsive antigen-specific T-cell populations in DNA-immunized or Salmonella enterica serovar Typhimurium-infected mice

Abstract

The location and functional properties of antigen-specific memory T-cell populations in lymphoid and nonlymphoid compartments following DNA immunization or infection with Salmonella were investigated. Epitope-specific CD8+ -T-cell expansion and retention during the memory phase were analyzed for DNA-immunized mice by use of a 5-h peptide restimulation assay. These data revealed that epitope-specific gamma interferon (IFN-gamma)-positive CD8+ T cells occur at higher frequencies in the spleen, liver, and blood than in draining or peripheral lymph nodes during the expansion phase. Moreover, this distribution is maintained into long-term memory. The location and function of both CD4+ and CD8+ Salmonella-specific memory T cells in mice who were given a single dose of Salmonella enterica serovar Typhimurium was also quantitated by an ex vivo restimulation with bacterial lysate to detect the total Salmonella-specific memory pool. Mice immunized up to 6 months previously with S. enterica serovar Typhimurium had bacterium-specific CD4+ T cells that were capable of producing IFN-gamma or tumor necrosis factor alpha (TNF-alpha) at each site analyzed. Similar findings were observed for CD8+ T cells that were capable of producing IFN-gamma, while a much lower frequency and more restricted distribution were associated with TNF-alpha-producing CD8+ T cells. This study is the first to assess the frequencies, locations, and functions of both CD4+ and CD8+ memory T-cell populations in the same Salmonella-infected individuals and demonstrates the organ-specific functional compartmentalization of memory T cells after Salmonella infection.

FIG. 1.

Detection of low frequencies of K^bS-specific CD8⁺ T cells. Single-cell suspensions of splenocytes were incubated for 5 h in medium alone or with either the K^bOVA peptide or K^bS peptide, as indicated in the dot plots. BFA was included for the final 4 h of incubation. The cells were gated for the expression of TCRβ, and IFN-γ expression within the TCRβ⁺ CD8⁺ population was examined. The data shown are for a mouse that was immunized 14 days previously with plasmid pCI/S. Similar results were obtained in seven independent experiments in which a total of over 60 pCI/S-immunized and 12 control (6 pCI/C-immunized and 6 naïve mice) mice were examined. Over 100,000 TCRβ⁺ CD8⁺ cells are shown in each case. The percentage of cells within the indicated gate is shown for each restimulation. All axes represent log fluorescence intensities. Staining with an appropriate isotype control monoclonal antibody (MAb) did not result in cells within the indicated gate (data not shown).

FIG. 2.

Kinetics of K^bS-specific CD8⁺-T-cell response to pCI/S immunization in different organs. Mice were immunized with plasmid pCI/S, and the mean frequencies of IFN-γ-responding K^bS-specific CD8⁺ T cells were determined, as described in Materials and Methods, for multiple time points. The graph shows this response in the spleen (•), liver (○), and draining (inguinal) LN (▴). Data were obtained from 62 pCI/S-immunized mice in seven independent experiments. n = 3 to 6 for each time point for each organ.

FIG. 3.

Frequencies of epitope-specific IFN-γ-responsive cells in different organs after immunization with pCI/S. Plots show 1/the frequency of cytokine-positive CD8⁺ cells within the gated TCRβ⁺ CD8⁺ population for each organ from naïve mice (a) or pCI/S-immunized mice at day 14 (b) or day 42 (c) postimmunization. Each symbol represents the value for an individual mouse. Horizontal lines indicate the mean values for each organ. Frequencies were quantitated by intracellular cytokine staining and flow cytometry after a 5-h restimulation with HBsAg (208-215) peptide as described in Materials and Methods. Organs were taken from 6 to 21 individuals in two to five separate experiments. For all organs, at least six individuals were examined, except for the blood of a naïve mouse (a), for which a single individual was analyzed. ILN, inguinal lymph node; SILN, superficial inguinal lymph node; MLN, mesenteric lymph node; CLN, cervical lymph node. P values obtained by the two-tailed Student's t test for unequal variances for each organ for infected mice compared to naïve mice are shown.

FIG. 4.

Polyclonal CD4⁺ memory T-cell responses to Salmonella. Single-cell suspensions of organs from Salmonella-immunized animals were restimulated either with medium alone or with Salmonella lysate and were assessed for IFN-γ and TNF-α expression as described in Materials and Methods. Plots represent the CD4⁺-T-cell responses of individual mice who were immunized 28 weeks previously with Salmonella. Data are representative of at least six individuals for each organ. Similar results were obtained in two to four independent experiments. Plots show gated viable TCRβ⁺ CD4⁺ cells. All axes represent log fluorescence intensities. Staining with appropriate isotype control MAbs did not result in cells within the indicated gate (data not shown).

FIG. 5.

Polyclonal CD8⁺ memory T-cell responses to Salmonella. Single-cell suspensions of organs from Salmonella-immunized animals were restimulated either with medium alone or with Salmonella lysate and were assessed for IFN-γ and TNF-α expression. Plots represent the CD8⁺-T-cell responses of individual mice who were immunized 28 weeks previously with Salmonella. Data are representative of at least six individuals for each organ. Similar results were obtained in two to four independent experiments. Plots show gated viable TCRβ⁺ CD8⁺ cells. All axes represent log fluorescence intensities. Staining with appropriate isotype control MAbs did not result in cells within the indicated gate (data not shown).

FIG. 6.

Frequencies of bacterium-specific IFN-γ- and TNF-α-producing CD4⁺ cells in different organs after immunization with S. enterica serovar Typhimurium. Plots show 1/the frequency of IFN-γ (a)- and TNF-α (b)-positive CD4⁺ cells within the gated TCRβ⁺ CD4⁺ population for each organ of naïve (○) or Salmonella-immunized mice (•) after restimulation with a Salmonella lysate. Each symbol represents the value for an individual mouse. Specific frequencies were quantitated as described in Materials and Methods. Horizontal lines indicate the mean values for each organ. The asterisk in panel b indicates that no cytokine-positive cells were detected in these samples (blood) for any naïve individual and therefore represents the mean number of cells analyzed. Organs were taken from 6 to 18 individuals in four separate experiments, with the exception of inguinal LN responses in immunized mice, which were examined in two independent experiments. Naïve inguinal LN responses were not determined. MLN, mesenteric lymph node; ILN, inguinal lymph node; SILN, superficial inguinal lymph node; CLN, cervical lymph node. P values obtained by a two-tailed Student's t test for unequal variances for each organ for infected mice compared to naïve mice are shown.

FIG. 7.

Frequencies of bacterium-specific IFN-γ- and TNF-α-producing CD8⁺ cells in different organs after immunization with S. enterica serovar Typhimurium. Plots show 1/the frequency of IFN-γ (a)- and TNF-α (b)-positive CD8⁺ cells within the gated TCRβ⁺ CD8⁺ population for each organ of naïve (○) or Salmonella-immunized mice (•) after restimulation with a Salmonella lysate. Each symbol represents the value for an individual mouse. Specific frequencies were quantitated as described in Materials and Methods. Horizontal lines indicate the mean values for each organ. The asterisks in panel b indicate that no cytokine-positive cells were detected in these organs for any naïve individual and therefore represent the mean numbers of cells analyzed. Organs were taken from 6 to 18 individuals for four separate experiments, except for inguinal LN responses in immunized mice, which were examined in two independent experiments. Naïve inguinal LN responses were not determined. MLN, mesenteric lymph node; ILN, inguinal lymph node; SILN, superficial inguinal lymph node; CLN, cervical lymph node. P values obtained by a two-tailed Student's t test for unequal variances for each organ for infected mice compared to naïve mice are shown.

FIG. 8.

Frequencies of IFN-γ- and TNF-α-producing T cells in the spleens of Salmonella-immunized mice restimulated with LPS. The plot shows 1/the frequency of IFN-γ- and TNF-α-positive TCRβ⁺ CD4⁺ or TCRβ⁺ CD8⁺ cells in the spleens of naïve mice (open symbols) or mice who were orally immunized >15 weeks earlier with Salmonella (filled symbols) after an ex vivo restimulation with Salmonella lysate (○ or •) or with purified Salmonella LPS at the same concentration as that in the lysate (▵ or ▴). Organs were from four to seven individuals, and each symbol represents the value for an individual mouse. Specific frequencies were quantitated as described in Materials and Methods. Horizontal lines indicate the mean values for each organ.