<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 Py-RAS and Pyfabb/f(−) activated CD8+ T cells in the livers of simultaneously infected mice.

<p>Mice were injected intravenously with 1–2 million PyXNL-GFP sporozoites and then immediately inoculated with 1 million labeled CD8+ T cells purified from the spleens of Py-RAS (circles), Pyfabb/f(−) (triangles), or Py-CS_280–288 (squares) immunized mice 2 weeks after the second booster. Mean velocities (<b>A</b>) and arrest coefficients (<b>B</b>) of CD8+ T cells from mice immunized with Py-RAS versus Pyfabb/f(−) and Pyfabb/f(−) versus Py-CS_280–288 differed significantly from each other (one-way ANOVA on Ranks). No significant difference was found for PyRAS versus Py-CS_280–288. Adoptive transfer of CD8+ T cells into infected BALB/c mice was done immediately after PyXNL-GFP sporozoite inoculation. IVM was performed at 18 h post-infection. At least three mice were analyzed per experimental condition. *  =  p<0.05; NS  =  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

Behavior of adoptively transferred CD8+ T cells in the liver of infected mice.

<p>At least 2 weeks after the second booster, CD8+ T cells were purified from the spleens of Py-RAS immunized mice and loaded with CellTracker Red. One million CD8+ T cells were transferred into recipient mice 18 h (<b>A</b>) or 42 h (<b>B</b>) post infection with PyXNL-GFP, and immediately imaged by IVM. CD8+ T cells (red) remained immobile and failed to migrate to or make contact with hepatocytes infected with PyXNL-GFP LS (green). Hepatocyte autofluorescence is shown in green in (<b>A</b>) and red in (<b>B</b>). Nuclei were stained with Hoechst (blue) in (<b>A</b>). Scale bars 20 μm.</p

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