Widespread and efficient deletion of IFNαβR in vivo following tamoxifen activation of Cre-ERT2.

<p>(A) LCMV immune UBC-Cre-ERT2⁺IFNαβR^fl/fl and UBC-Cre-ERT2^-IFNαβR^fl/fl mice were injected with tamoxifen, and the indicated analyses were carried out. (B) The efficiency of deletion at the DNA level was assessed by PCR analysis of genomic DNA extracted from the indicated tissues; M = markers; Splns = splenocytes; LN = lymph nodes; x = empty lane. 1144bp = floxed allele, 309bp = deleted allele. (C & D) The efficiency of Cre activation in T cells of LCMV-immune mice was determined. Cre⁺ or Cre^- IFNαβR^f/fzsGreen^+/wt mice were injected with tamoxifen and, two weeks later, splenocytes were harvested and incubated with each of the four indicated peptides, and virus-specific T cells were identified using standard intracellular cytokine staining for IFNγ. zsGreen expression also was evaluated. The red numbers indicate zsGreen⁺ cells as a percentage of all CD8⁺ T cells (No Peptide groups) or of virus-specific (i.e, IFNγ⁺) cells (peptide-stimulated groups). (C) Gated on CD8⁺ T cells and (D) gated on CD4⁺ T cells. (E) The efficiency with which Cre activation resulted in ablation of IFNαβR biological function was determined, by measuring Stat 1 phosphorylation after <i>in vitro</i> incubation with IFNβ. ZsGreen+CD8⁺ T cells from Cre⁺ mice were compared to CD8⁺ T cells from Cre^- animals. Grey histograms = isotype control antibody. Mouse numbers: C & D, n = 6–7; E, n = 4.</p

Kinetics of naive and memory CD4⁺ T cells in the same mouse.

<p>Wildtype mice containing 1.3×10³ naïve SMARTA (Thy1.1) and 1.3×10³ memory SMARTA (Ly5a) cells were given LCMV, and the relative abundance of the two SMARTA cell populations was determined by flow cytometry at various times post infection (two mice per time point). A. After gating on CD4⁺ T cells, the host CD4⁺ T cells (H), and the naïve and memory SMARTA cells (N & M respectively) were distinguished by Thy1.1 and Ly5a staining. The numbers indicate the frequencies of naïve and memory SMARTA cells as a percentage of all CD4⁺ T cells. B. The average±SE of the percentage of each population among all CD4⁺ T cells is shown over time. C. The total number of memory or naïve SMARTA CD4⁺ T cells per spleen is shown (average±SE).</p

The delay in naïve T cell division is organism-wide.

<p>Mice containing approximately 1.4×10⁵ CFSE-labeled SMARTA CD4⁺ T cells were infected with LCMV. At the indicated times after infection, lymphocytes were isolated (2 mice per time point) and the donor cells were identified by flow cytometry. A. The ovals in the dot plots identify the SMARTA CD4⁺ T cells, and the numbers indicate their percentage among leukocytes isolated from each tissue. The histograms show the CFSE-fluorescence of the SMARTA CD4⁺ T cells; the numbers indicate the percentages of SMARTA CD4⁺ T cells that have divided. B. The line graphs show the percentages of SMARTA CD4⁺ T cells among all isolated leukocytes at various times after infection. For each tissue, the dashed line indicates the number of SMARTA cells in uninfected mice.</p

Changing the microenvironment reduces the in vivo delay in T cell division.

<p>Naive SMARTA cells were CFSE-labeled and transferred either to mice that had been infected with LCMV two days previously, or to uninfected mice some of which were immediately infected with LCMV. A. 2, 3 or 4 days after cell transfer (as indicated), the spleens of the recipient mice were isolated and the donor SMARTA CD4⁺ T cells were identified by flow cytometry (ovals). Individual mice are shown, and the numbers indicate the proportion of SMARTA cells as a percentage of all spleen cells. Mouse numbers in each of the 4 groups: 1, 1, 3, 3. B. The bar graph shows cumulative data, as percentages of SMARTA CD4⁺ T cells. C. The histograms show the CFSE fluorescence of the indicated SMARTA CD4⁺ T cells. Note that the 3-day delay in proliferation is shortened to 2 days if the mice were pre-infected.</p

Viral epitopes are presented within hours of infection, and stimulate memory T cell effector functions.

<p>Mice that contained approximately 3×10³ SMARTA/Ly5a CD4⁺ T cells were infected with LCMV and, 354 days later, were re-challenged intraperitoneally with 2×10⁶ PFU LCMV-Armstong. Six hours post-infection, the mice were given 0.25 mg Brefeldin A i.v., and 6 hours later the spleens were harvested and immediately surface stained for CD4, Ly5a, or CD8, then permeabilized and stained for intracellular IFNγ. The cells were not re-stimulated <i>ex vivo</i> with peptide antigen. A. ∼5% of all CD8⁺ T cells, and ∼1% of all CD4⁺ T cells, are actively producing IFNγ in response to infection. B. Using the SMARTA cells transferred ∼1 year previously as an indicator of the responsiveness of virus-specific CD4⁺ memory T cells, ∼14% of LCMV-specific CD4⁺ memory T cells actively produce IFNγ within 12 hours of virus infection. Data shown are from an individual mouse, and are representative of independent datasets. C. A separate set of naive mice were given CFSE-labeled pooled SMARTA cells (4×10⁵ naive SMARTA/Thy1.1 cells and 2×10⁴ memory SMARTA/Ly5a T cells). 4 days later, some of the recipient mice were given LCMV. Six hours later, BFA was administered to all mice, and after a further 6 hours splenocytes were harvested. The cells were immediately stained (without peptide re-stimulation) for CD4, Thy1.1, Ly5a and IFNγ, and were analyzed by flow cytometry. Approximately 2% memory SMARTA cells had begun to synthesize IFNγ in response to LCMV infection (top row) but none of those responding memory cells showed any dilution of CFSE signal. The naïve SMARTA cells (bottom row) failed to produce IFNγ at this early time point post-infection, and no sign of cell division was seen. Data are from one of two independent experiments.</p

Naive antiviral CD4⁺ and CD8⁺ T cell division has a lag phase of 2–3 days.

<p>Equal numbers of CFSE-labeled P14 cells (TcR-transgenic CD8⁺ T cells specific for LCMV GP_33–41, expressing Thy1.1) and CFSE-labeled SMARTA cells (TcR transgenic CD4⁺ T cells specific for LCMV GP_61–80, expressing Ly5a) were pooled, and inoculated into wildtype C57BL/6 mice, which then were infected with LCMV. A. At the indicated times after infection, each donor population was identified by flow cytometry (ovals). B. The numbers of P14 and SMARTA T cells in the spleen are shown (mean±SE) at the indicated times after infection (two separate experiments, two mice per experiment). C. After gating to identify the P14 or the SMARTA T cells, the histograms show these cells' CFSE fluorescence. Note that both T cell subsets begin proliferating at the same time (day 3).</p

IFNαβR deletion prior to secondary viral infection does not limit LCMV-specific T cell attrition or the expression of MHC Class I & Qa-1 in vivo.

<p>(A) LCMV immune UBC-CreERT2⁺ and UBC-Cre-ERT2^-IFNαβR^fl/fl mice were tamoxifen injected and rechallenged with LCMV or given a sham injection. LCMV-specific (B) CD8⁺ and (C) CD4⁺ T cells were quantified via standard intracellular cytokine staining at the indicated times p.i. (D & E) Qa-1 and (F & G) MHC Class I expression was measured upon D^bGP_33-41⁺ (left column) and D^bNP_396-404⁺ (right column). Shown in panels D & F are representative histograms (gated on D^bGP_33-41⁺ CD8⁺ T cells). Data in B, C, E, & G are summations of 2 independent experiments (n = 4–7). Significance was determined via two-way ANOVA with Sidak correction (**** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05).</p

Delayed accumulation of naive and memory CD4⁺ T cells occurs also in non-lymphoid tissues.

<p>Mice containing 1.3×10⁴ naïve (Thy1.1) and 1.3×10⁴ memory (Ly5a) SMARTA CD4⁺ T cells were infected with LCMV and, at the indicated times after infection, lymphocytes from several lymphoid and non-lymphoid tissues were isolated and analyzed by flow cytometry. A. Dot plots show gated CD4⁺ T cells isolated from the tissues. The ovals identify the SMARTA cells (N, M  =  naïve & memory respectively), and the numbers indicate their percentage among all CD4 T cells (H  =  host CD4⁺ T cells). B. For each tissue, naïve and memory SMARTA CD4⁺ T cells are shown as percentages of all CD4⁺ T cells (two mice per time point). Note that both naïve and memory cells become prominent after day 4; however, the memory cells dominate the response in the non-lymphoid tissues.</p

Naive and memory CD4⁺ T cells show near-identical delays in onset of division.

<p>Mice containing 2×10³ naive SMARTA CD4⁺ T cells (Ly5a) were infected with LCMV and allowed to become immune. A. Six months after infection, memory SMARTA CD4⁺ T cells were isolated from the spleen and analyzed by flow cytometry. The first dot plot identifies the memory SMARTA CD4⁺ T cells (oval). After gating on these cells, the histogram shows their expression of CD44, and the remaining two dot plots evaluate IFNγ and IL-2 production after brief <i>in vitro</i> stimulation with GP_61–80 peptide. B. The memory SMARTA cells (Ly5a) were mixed with naive SMARTA cells (Thy1.1), labeled with CFSE, and then transferred to naive mice. The recipient mice were given approximately 5×10⁴ memory SMARTA CD4 T cells and 5×10⁵ naive SMARTA CD4 T cells. 3 days later, the recipients were infected with LCMV. The dot plots show spleen cells isolated from recipient mice at the indicated times after infection, and the ovals identify the memory SMARTA CD4 T cells (top two rows) and the naive SMARTA CD4 T cells (bottom two rows). The histograms show the CFSE fluorescence of the SMARTA cells, and the numbers in the histograms indicate the percentage of SMARTA CD4⁺ T cells that have not divided. Data are representative of two independent experiments.</p

Reporter-negative (IFNαβR-intact) CD8+ or CD4+ memory T cells do not preferentially expand during the recall response.

<p>As described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005861#ppat.1005861.g003" target="_blank">Fig 3A</a>, LCMV-immune Cre reporter (zsGreen) mice were generated, treated with tamoxifen, and subjected to secondary LCMV infection. Mice were sacrificed, and virus-specific CD8⁺ and CD4⁺ T cells were enumerated using intracellular cytokine staining, and evaluated for zsGreen expression, days 0, 2, 5 & 23 following the secondary infection. Representative epitope-specific responses in individual mice are shown. Cells were harvested at day 23 following secondary infection, and are gated on (A) CD8⁺ T cells or (B) CD4⁺ T cells. Numbers represent the proportion of the gated cells in each quadrant. (C-F) Cumulative data showing the proportion of zsGreen-positive cells at the indicated time points over the course of the recall response, in each of the four epitope-specific T cell populations. (G) At the indicated time points, splenocytes from Cre⁺ mice were exposed in vitro to IFNβ, and levels of pSTAT expression were evaluated. Representative plots are shown, gated on CD8 and zsGreen. The rightmost panel is a positive control, showing the responsiveness to IFNβ of day 23 post-secondary CD8⁺ T cells from Cre^- animals. Grey histograms = isotype control antibody.</p