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

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

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

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

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 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

Minimal T cell activation following infection with wild-type or recombinant CVB3.

<p>Mice were infected with wtCVB3, rCVB3.6, or LCMV as a positive control, and the extent of T cell activation was analyzed on day 8. (A) CD44 and CD62L expression profiles of CD8⁺ or CD4⁺ splenocytes were compared for naïve and virus-infected mice. Representative histograms are gated on CD8⁺ or CD4⁺ cells, and the numbers indicate the proportion of cells that are CD44^hi or CD62L^lo. The frequency of CD44^hi (B) or CD62L^lo (C) CD4⁺ or CD8⁺ T cells in the spleen was determined for each group of mice. (D) The capacity of CD4⁺ and CD8⁺ T cells to produce IFNγ was evaluated following simulation with PMA and ionomycin and ICCS. The percentage of CD4⁺ or CD8⁺ T cells that produce IFNγ is shown. All data are representative of 2 independent experiments, and show the mean+SE for 3 mice per group. * p<0.05, ** p<0.01, *** p<0.001, compared to the naïve (uninfected) control.</p

The absence of CVB3-induced CD8⁺ T cell responses cannot be attributed to redistribution into non-lymphoid target organs.

<p>Equal numbers (10⁴) of P14 and SMARTA cells from uninfected mice were combined and transferred into uninfected recipients. Four days post transfer, mice were infected with rCVB3.6 or LCMV and, 8 days later, mononuclear cells were isolated from the spleen, heart, and pancreas. (A) P14 and SMARTA responses were analyzed by flow cytometry. The gates shown in the dot plots identify P14, SMARTA, or CD4⁺ SMARTA⁻ cells, and the numbers indicate their percentage among all mononuclear cells. (B) Frequency of P14 and SMARTA cells as a percentage of relevant cells (CD8⁺ or CD4⁺) in the spleen. (C) Total number of P14 and SMARTA cells in the spleen. Data are shown as the mean+SD of 3 or 4 mice per group. (D) The expression of CD44, CD62L, and CD127 on SMARTA cells from the spleen, heart, and pancreas was compared in rCVB3.6-infected and LCMV-infected mice. The dot plots shown are gated on CD4⁺ cells, and the numbers indicate the proportion of cells in each quadrant, as a percentage of all CD4⁺ cells. CD45.1⁺ SMARTA cells are present in the upper right and left quadrants. All data are representative of 2 independent experiments.</p

Kinetics of CD4⁺ and CD8⁺ transgenic T cell responses in rCVB3-infected and LCMV-infected mice.

<p>10⁴ P14 cells and 10⁴ SMARTA transgenic T cells from uninfected mice were combined and transferred into uninfected congenic recipients. One day post transfer, mice were infected with rCVB3.6 or LCMV. On the indicated days p.i., the frequencies of P14 and SMARTA cells were determined by flow cytometry, and are presented as a percentage of total PBMCs. Each symbol represents the time-course in an individual mouse. Note that the y-axes differ among the graphs; this was done to facilitate the comparison of the kinetics of T cell responses that differ substantially in their magnitudes.</p