The long-term presence of CD62L⁻ LCMV-specific CD8 T cells in the MedLN.

<p>Naïve Thy1.1⁺ P14 CD8 T cells were adoptively transferred into naïve Thy1.2⁺ recipients that were subsequently infected i.p. with LCMV 24 h later. At the indicated times p.i. tissues were harvested and P14 cells were analyzed for expression of CD62L. (A) Representative histograms depicting CD62L staining (open histograms) as compared to isotype control staining (filled histogram). (B) Kinetic analysis of CD62L expression on P14 CD8 T cells. (C), Representative dot plots depicting surface expression of CD127 and KLRG-1 on P14 CD8 T cells in various tissues following LCMV infection. (D) Kinetic analysis of CD127 and KLRG-1 expression of P14 CD8 T cells in the indicated tissues following LCMV infection. *, MedLN is significantly decreased (<i>p</i><0.05) as compared to ILN or CLN. ^x, MesLN is significantly decreased (<i>p</i><0.05) as compared to ILN or CLN. Combined data representing four independent experiments with 3–4 mice per group is shown. In some experiments, LNs from >day 15 p.i. were pooled and analyzed as a pooled sample.</p

Intraperitoneal infection with LCMV results in rapid antigen display in the MedLN.

<p>CFSE-labeled Thy1.1⁺ P14 TCR transgenic CD8 T cells were adoptively transferred into naïve Thy1.2⁺ C57BL/6 mice that were subsequently infected i.p. with LCMV 24 h later. At the indicated times, various organs were harvested and P14 CD8 T cells were assessed for CFSE dilution and expression of activation markers. (A) Representative dot plots showing CFSE dilution profile and CD25 expression. (B) Quantification of CFSE dilution as well as CD25, CD43^glyco and CD62L expression. Representative data from one of three individual experiments with three mice at each time point is shown. CD62L data is from two individual experiments with three mice at each time point. *, MedLN is significantly different (<i>p</i><0.05) as compared to all other tissues as determined by <i>ANOVA</i> with a Tukey post-test. Error bars represent the standard error of the mean for three mice per group.</p

No residual viral RNA in the draining LN following an i.p. LCMV infection.

<p>RNA was isolated from naïve, day 4 and day 34 LCMV infected mice, and quantified by RT-PCR. N.D. not detected. Combined data from two individual experiments with four mice per group. Error bars represent the standard error of the mean.</p

Long-term reactivity of the MedLN following an i.p. infection with LCMV.

<p>C57BL/6 mice were infected i.p. with LCMV and at various times following infection the indicated tissues were harvested and analyzed for the presence of GC B cells by B220 and PNA staining. (A) Representative dot plots showing the frequency of GC B cells at various times p.i. (B) Kinetic analysis of GC B cells following an i.p. infection with LCMV. *, MedLN is significantly greater (<i>p</i><0.05) as compared to all other tissues as determined by <i>ANOVA</i>. (C) Comparison in the frequency of GC B cells in LNs before or 34 days following LCMV infection. (D) Total cell number in LNs either prior to or 34 days following LCMV infection. *, significantly greater (<i>p</i><0.05) than naïve mice as determined by Student t-test. These data represent more than 4–5 independent experiments, where in some experiments MedLN and CLN were pooled from three mice. Error bars represent the standard error of the mean for greater than four samples per time point.</p

No prolonged presence of viral antigens in the MedLN following an i.p. LCMV infection.

<p>CFSE-labeled memory P14 CD8 T cells were co-cultured with single-cell suspensions of either splenocytes or LN cells from naïve (1∶100 memory P14∶spleen/LN cell) and day 34 LCMV-infected mice (1∶100 memory P14∶spleen/LN cell). Memory CFSE-labeled P14 cells were also cultured with either naïve splenocytes or LN cells pulsed with 1 µM GP_33–41 peptide as a positive control. Cells were co-cultured for 3 days and subsequently analyzed for CFSE dilution, and either CD25 or CD62L expression. (A) Representative dot plots showing CD62L and CFSE profiles gated on memory P14 CD8 T cells. (B) Frequency of CFSE^hi P14 CD8 T cells after 3 days of co-culture with either splenocytes or LN cells from either naïve or LCMV-infected mice as described above. (C) Frequency of CD62L⁺ P14 CD8 T cells after 3 days of co-culture with either splenocytes or LN cells from naïve or LCMV-infected mice as described above. *, significantly decreased (<i>p</i><0.05) as compared to P14 memory cells co-cultured with either control naïve splenocytes or LN cells as determined by Student t-test. Data are representative of three individual experiments with 3–4 mice per group. Error bars represent the standard error of the mean.</p

High frequencies of CD62L⁻ LCMV-specific CD8 T cells in the draining LN is not unique to the MedLN.

<p>C57BL/6 mice were infected in the footpad with LCMV. (A) Viral titers in the Ips PopLN (solid upwards triangle), Con PopLN (downwards triangle) and spleen (solid square) were measured via plaque assay at 24, 48 and 72 h p.i.. *, Ips PopLN is significantly greater (<i>p</i><0.05) than all other as determined by <i>ANOVA</i>. (B and D) Naïve Thy1.1⁺ P14 CD8 T cells were adoptively transferred into naïve Thy1.2⁺ recipients and infected via the footpad with LCMV the following day 34–40 p.i., the indicated tissues were harvested and assessed for the frequency (B) GC B cells (as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003054#ppat-1003054-g005" target="_blank">Figure 5</a>) and (D) CD62L⁺ P14 CD8 T cells. (C) The total number of cells in each LN or spleen was assessed in naïve mice or in mice previously infected (day 34–40) with LCMV via the footpad. (B and C) *, Day 34 Ips PopLN is significantly greater (<i>p</i><0.05) than naïve Ips PopLN determined by <i>ANOVA</i>. (D) *, Ips PopLN is significantly lower (<i>p</i><0.05) than Con PopLN as determined by <i>ANOVA</i>. Combined data from 2–3 individual experiments with four mice per group. Error bars represent the standard error of the mean.</p

Priming of the LCMV-specific CD8 T cell response occurs in the MedLN.

<p>Thy1.1⁺ P14 CD8 T cells were adoptively transferred into naïve Thy1.2⁺ C57BL/6 recipients that were subsequently infected i.p. with LCMV 24 h later. Starting one day after infection, mice were treated i.p. with either 50 µg of FTY720 or vehicle daily. Spleens, ILN, MesLN and MedLN were harvested on day 5 p.i. and analyzed for (A) the frequency of P14 CD8 T cells and (B) the total number of P14 CD8 T cells. *, significantly different (<i>p</i><0.05) as determined by Student t-test. n.s., not significant. Representative data from one of four individual experiments with 3–5 mice per group is shown.</p

CD62L⁻ effector memory CD8 T cells preferentially traffic to the draining MedLN.

<p>CD62L⁺ and CD62L⁻ memory P14 cells were isolated from LCMV-immune mice. CD62L⁺ cells were CFSE-labeled and mixed with unlabeled CD62L⁻ P14 cells and transferred into either naïve or day 34 LCMV infected mice. 72 h post-transfer, LNs were harvested and transferred cells (CD8⁺Thy1.1⁺) were examined for their ratios in naïve mice or day 34 LCMV-infected mice. (A) Representative histograms from naïve (top) and day 34 LCMV-infected (bottom) mice is shown from one of three experiments with 5 mice total per group. (B) Graphs depicting combined data from all experiments presented in a logarithmic scale. Na?¨ve mice are shown the open bars and day 34-LCMV mice are shown in the closed bars. **, MedLN is significantly lower (<i>p</i><0.01) than the ILN, CLN and MesLN as determined by <i>ANOVA</i>.</p

Viral titers in secondary lymphoid tissues early following LCMV infection.

<p>C57BL/6 mice were infected with LCMV i.p.. At the indicated times p.i. the spleens, ILNs, MesLNs, CLNs and MedLNs were harvested and assayed for viral titers by plaque assay (A). Data represent pooled data from 3–4 experiments with 3–4 mice per group, except for the ILN and CLN at 0.5 days p.i. where the data represent six mice from two individual experiments. * significantly greater (<i>p</i><0.05) than spleen ⁺, significantly increased (<i>p</i><0.05) as compared to ILN. ^# significantly increased (<i>p</i><0.05) as compared to MesLN. ^$, significantly increased as compared to CLN. All statistical analysis was done by <i>ANOVA</i> with a Tukey post-test.</p

LT-α-KO mice exhibit a dramatically reduced LCMV-specific CD8 T cell response.

<p>WT, LT-α-KO and splenectomized mice were infected with LCMV i.p.. The LCMV (A) GP₃₃-, (B) NP₃₉₆- and (C) GP₂₇₆-specific CD8 T cell responses from the indicated tissues were measured at day 8 post-LCMV infection via ICS for IFN-γ. (D) GP₃₃-, NP₃₉₆- and GP₂₇₆-specific CD8 T cell responses in the peripheral blood. *, significantly greater (<i>p</i><0.05) than WT control mice as determined by Student t-test. ^x, significantly decreased (<i>p</i><0.05) as compared to WT control mice as determined by Student t-test. Representative data from one of three individual experiments with 3–5 mice per group per experiment. Error bars represent the standard error of the mean. N.D. = none detected.</p