Homeostatic proliferation rates of primary (1°) and secondary (2°) antigen-specific memory CD4-T cells are similar.

<p>Homeostatic proliferation was assessed based on BrdU incorporation by antigen-specific 1° and 2° antigen-specific CD4 T cells evaluated simultaneously in the same host at the indicated memory time-points following LCMV and <i>actA⁻</i>LmOva infection. Each histogram plot is gated on GP61 or LLO190-specific CD4 T cells identified in the spleens and shows the fraction of BrdU⁺ cells (black line). Isotype control staining is depicted by the shaded grey histograms. Data are representative of results obtained from 3 mice analyzed per group.</p

Virus-neutralization using sera obtained from LCMV-infected mice that received naïve, primary-(1°) or secondary-(2°) memory-SMARTA cells.

<p>Serum samples were obtained from each mouse at the indicated time points and analyzed by performing PRNT assays. The bar graphs depict the percent change in virus-titers following incubation of LCMV-Armstrong with a 1∶20 dilution of serum obtained from each mouse (n = 3–5 mice per group) compared with virus-titers measured following incubation of LCMV-Armstrong with 1∶20 dilution of serum obtained from naïve mice normalized to 100%. Data generated using pooled serum from immune mice (>180 days post-LCMV-Arm infection) that did not receive SMARTA cells and assayed at each time point was averaged for the entire data set and all data are expressed as mean±SEM and statistical significance was analyzed using the unpaired Student’s t test (p-value <0.05).</p

Kinetics of primary and secondary antigen-specific CD4 T cell responses following homologous prime-boost.

<p>Purified CD4 T cells derived from spleens of LCMV- and <i>actA⁻</i>LmOva-immune B6 donor (Thy1.1⁺) mice using negative selection were transferred into naïve recipients B6 (Thy1.2⁺) that were infected the following day as indicated (A–B). Total numbers of antigen-specific primary (endogenous: Thy1.1<i>⁻</i>IFNγ⁺) and secondary (transferred: Thy1.1⁺IFNγ⁺) responders were determined in the spleens of the recipients at the indicated time-points and are depicted in the line-graphs (C–D).</p

CD62L and IL-2 expression-patterns are selectively altered on memory CD4-T cell subsets following a heterologous-prime-challenge.

<p>(A). Bulk CD4 T cells were purified from spleens of LCMV-infected donor B6 (CD45.1⁺) mice using negative selection at the indicated time-point after infection and transferred into naïve B6 recipients (CD45.2⁺) that were challenged the following day with <i>actA⁻</i>LmGP33/61. (B) Surface expression of CD62L, CCR7 and elaboration of IL-2 was evaluated on primary (endogenous) responders (grey filled histograms) and secondary (transferred) responders (black line). In the converse heterologous prime-challenge experiment, bulk CD4 T cells were purified from spleens of <i>actA⁻</i>LmGP33/61-infected donor B6 (CD45.2⁺) mice using negative selection at the indicated time-point after infection and transferred into naïve B6 recipients (CD45.1⁺) that were challenged the following day with LCMV-Arm (C). (D) Surface expression of CD62L, CCR7 and elaboration of IL-2 was evaluated on primary (endogenous) responders (grey filled histograms) and secondary (transferred) responders (black line).</p

Assessment of CD62L, CCR7 expression and IL-2 production by antigen-specific CD4-T cells in homologous-prime-challenged mice.

<p>Antigen-specific T cells were identified by IFNγ staining in recipient mice as previously described. Representative histograms are gated on GP61 and LLO190-specific CD4 T cells T cells in the spleens and depict the expression patterns of the indicated molecule following antibody staining at the indicated time-points. Each plot consists of a histogram depicting the expression pattern of the relevant molecule on the primary (1°; endogenous) responders (grey filled histograms) and the secondary (2°; transferred) responders (black line).</p

Tracking CD8+ cells in naïve and immunized mouse livers.

<p>CD8+ T cells were identified by anti-CD8a-PE labeling in the livers of naïve mice (circles) or mice immunized with Py-RAS (triangles), Pyuis4(<b>−</b>) (diamonds), or Pyfabb/f(<b>−</b>) (squares) and monitored via IVM. Velocities (<b>A</b>) and arrest coefficients (<b>B</b>) were calculated from the tracks of individual CD8+ T cell from at least 2 infected mice per group. One-way ANOVA on Ranks shows that the velocities of the CD8+ T cells from all immunized mice are significantly higher and the arrest coefficients significantly lower than those of the CD8+ T cells from naive mice (p<0.05). The same statistical analysis does not reveal any significant difference between the CD8+ T cell velocities between the groups of immunized mice (p>0.05). *  =  p<0.005, ns  =  not significant. CD8+ T cells of immunized mice were monitored at least 2 weeks after the second booster.</p

<i>In vivo</i> CD8+ T Cell Dynamics in the Liver of <i>Plasmodium yoelii</i> Immunized and Infected Mice

<div><p><i>Plasmodium falciparum</i> malaria remains one of the most serious health problems globally and a protective malaria vaccine is desperately needed. Vaccination with attenuated parasites elicits multiple cellular effector mechanisms that lead to <i>Plasmodium</i> liver stage elimination. While granule-mediated cytotoxicity requires contact between CD8+ effector T cells and infected hepatocytes, cytokine secretion should allow parasite killing over longer distances. To better understand the mechanism of parasite elimination <i>in vivo</i>, we monitored the dynamics of CD8+ T cells in the livers of naïve, immunized and sporozoite-infected mice by intravital microscopy. We found that immunization of BALB/c mice with attenuated <i>P. yoelii</i> 17XNL sporozoites significantly increases the velocity of CD8+ T cells patrolling the hepatic microvasculature from 2.69±0.34 μm/min in naïve mice to 5.74±0.66 μm/min, 9.26±0.92 μm/min, and 7.11±0.73 μm/min in mice immunized with irradiated, early genetically attenuated (Pyuis4-deficient), and late genetically attenuated (Pyfabb/f-deficient) parasites, respectively. Sporozoite infection of immunized mice revealed a 97% and 63% reduction in liver stage density and volume, respectively, compared to naïve controls. To examine cellular mechanisms of immunity in <i>situ</i>, naïve mice were passively immunized with hepatic or splenic CD8+ T cells. Unexpectedly, adoptive transfer rendered the motile CD8+ T cells from immunized mice immotile in the liver of <i>P. yoelii</i> infected mice. Similarly, when mice were simultaneously inoculated with viable sporozoites and CD8+ T cells, velocities 18 h later were also significantly reduced to 0.68±0.10 μm/min, 1.53±0.22 μm/min, and 1.06±0.26 μm/min for CD8+ T cells from mice immunized with irradiated wild type sporozoites, Pyfabb/f-deficient parasites, and <i>P. yoelii</i> CS_280–288 peptide, respectively. Because immobilized CD8+ T cells are unable to make contact with infected hepatocytes, soluble mediators could potentially play a key role in parasite elimination under these experimental conditions.</p></div

Tracking adoptively transferred Pyuis4(−) activated CD8+ T cells in the livers of infected mice.

<p>Mice were infected with 1–2 million PyXNL-GFP sporozoites. Activated CMTPX labeled CD8+ T cells were then purified 2 weeks after the second booster from the spleens of Py-RAS immunized mice and 1 million was adoptively transferred into the infected mice at 18 h (triangles) or 42 h (squares) after infection with PyXNL-GFP. Anti-mouse CD8a labeled T cells in Pyuis4(−) immunized mice (circles) were used as controls. ANOVA on Ranks show that the mean velocities (<b>A</b>) and arrest coefficients (<b>B</b>) of all adoptively transferred CD8+ T cells differ significantly from those of the CD8+ T cells in Pyuis4(−) immunized mice. At least three infected mice were used per experimental condition. *  =  p<0.05, ns  =  not significant.</p

Inhibition of LS development after infection of immunized mice.

<p>Naïve or Pyfabb/f(<b>−</b>) immunized BALB/c mice were infected with 3×10⁵ PyXNL-GFP sporozoites. Immunized mice were infected 2 weeks after the second booster. At 18 h or 42 h post-infection, livers were removed and LS parasites were quantified in fresh unfixed liver tissue. Using vibratome sections of known thickness and a region of interest of a defined size, the number of LS was counted and normalized to a volume of 1.3 cm³ (approximate liver volume of adult mice). Panels (<b>A</b>) and (<b>B</b>) show the reduction in LS number and volume, respectively, in Pyfabb/f(<b>−</b>) immunized mice compared to naïve mice. *  =  p<0.05.</p

Behavior of CD8+ T cells in the liver of naïve and immunized mice.

<p>Representative IVM images of CD8+ T cells observed in naïve and <i>P. yoelii</i> immunized mouse livers. Note that in contrast to naïve mice, the T cells from immunized mice exhibit the characteristic amoeboid shape of activated effector cells and patrol the sinusoids with a leading edge and a trailing pseudopod. (<b>A</b>) 3D representation of a naïve Tie2-GFP mouse liver with fluorescent endothelia (green), anti-CD8a-PE labeled CD8+ T cells (red), and Hoechst stained nuclei (blue). (<b>B</b> and <b>C</b>) 3D projections of anti-CD8a-PE labeled CD8+ T cells (green) in the livers of mice immunized with (<b>B</b>) Pyfabb/f(<b>−</b>)or (<b>C</b>) Py-RAS. Hepatocyte mitochondria were labeled with MitoTracker (red) and nuclei were stained with Hoechst (blue). (<b>D</b>) 2D snapshot of the liver of a mouse immunized with Pyuis4(<b>−</b>) showing CD8+ T cells labeled with anti-CD8a-PE (red). Hepatocyte mitochondria labeled with MitoTracker (green). CD8+ T cells of immunized mice were monitored 2 weeks after the second booster. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070842#pone.0070842.s003" target="_blank">Videos S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070842#pone.0070842.s004" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070842#pone.0070842.s005" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070842#pone.0070842.s006" target="_blank">S4</a></b> for the corresponding movies. Scale bars 20 μm.</p