Post-thymic maturation status of naïve P14 transgenic T cells determines their TNF producing capability.

<p>Female CD45.1⁺ P14-CD8⁺ thymocytes were transferred into female CD45.2⁺ B6 congenic mice. Host spleens were recovered after the indicated time periods and were stimulated in vitro for 4 hours with GP33+αCD28 and donor CD45.1⁺ T cells were stained for intracellular TNF as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#s4" target="_blank"><i>Materials and Methods</i></a>. Dead cells were excluded using Live Dead Aqua Dead cell stain for this experiment. For analysis, cells were gated on the live donor SP P14-CD8⁺ T cells and the maturation profile of donor cells that are TNF⁺ (indicated by arrows in the boxed quadrants in plots iii,iv,v,vi) were compared at all the time points shown (corresponding histograms). Additionally, some CD45.1⁺ P14-CD8⁺ thymocytes were stimulated before transfer in the context of CD45.2⁺ B6 splenocytes in vitro for 4 hours with GP33+αCD28 and their maturation profile was compared to CD45.1⁺ P14-CD8⁺ thymocytes and splenocytes stimulated alone in vitro (plots i, ii and vii).</p

Maturation state of SP thymocytes reflects their TNF producing capability^a.

a<p>The average MFI of maturation markers CD24 (n = 6), CD45RB (n = 6) and Qa2 (n = 3 for group1 and 2 and n = 6 for for group 3 and 4) in TNF producing donor SP P14-CD8⁺ are shown. The averages were analysed using One-way ANOVA with a Tukey post-test as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#s4" target="_blank"><i>Materials and Methods</i></a>. Error indicates SD. N/A, Not Applicable.</p>b<p>p<0.05 vs Table II, group 1.</p>c<p>p<0.05 vs Table II, group 1.</p>d<p>p<0.05 vs Table II, group 1.</p>e<p>p<0.05 vs Table II, group 1.</p>f<p>p<0.05 vs Table II, group 1.</p>g<p>p<0.05 vs Table II, group 1.</p>h<p>p<0.05 vs Table II, group 1 TNF+.</p>i<p>p<0.05 vs Table II, group 1 TNF+.</p>j<p>p<0.05 vs Table II, group 1 TNF+.</p

SP thymocytes acquire the ability to produce TNF as a function of time in the periphery^a.

a<p>The recovery of the donor (CD44^lo) SP P14-CD8⁺ thymocytes from recipient spleens and the MFI of TNF expression at the indicated time points post-transfer are shown. The average recovery and the MFI of TNF expression by donor thymocytes (n = 6 per time point) were analysed using One-way ANOVA with a Tukey post-test as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#s4" target="_blank"><i>Materials and Methods</i></a>. Error indicates SD. N/A, Not Applicable.</p>b<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 1.</p>c<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 1.</p>d<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 1.</p>e<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 1.</p>f<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 2.</p>g<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 2.</p>h<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 3.</p

Reduced upregulation of TNF message in SP thymocytes relative to naïve splenic T cells upon TCR stimulation.

<p>SP P14-CD8⁺ thymocytes and (CD44^lo) naïve splenic CD8⁺ T cells were purified by cell sorting and stimulated in the presence of GP33 and GP33+αCD28 for 4 hours followed by RNA isolation and cDNA synthesis from 50 ng of RNA and then amplified using TNF specific primers by quantitative real time PCR from the indicated populations as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#s4" target="_blank"><i>Materials and Methods</i></a>. A, The basal steady state level of TNF transcripts in 50 ng of total RNA (normalized to a β-actin control) isolated from unstimulated SP P14-CD8⁺ thymocytes and (CD44^lo) naïve splenic T cells are shown. B, The increase in TNF message in these subsets upon stimulation with GP33 and GP33+αCD28 in terms of fold induction with respect to unstimulated SP thymocytes been (normalized to a β-actin control) are shown. This profile is representative of 3 individual experiments.</p

SP thymocytes are poor TNF producers relative to naïve splenic T cells during TCR activation in vitro.

<p>Thymocytes and splenocytes from P14-CD8⁺, OT-1-CD8⁺, SMARTA-CD4⁺ and OT-2-CD4⁺ TCR transgenic mice were stimulated in vitro as indicated for 4 hours and then stained for intracellular TNF cytokine, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#s4" target="_blank"><i>Materials and Methods</i></a>. For analysis, the cells were gated on either SP CD8⁺ CD4⁻ or SP CD4⁺ CD8⁻ cells. A, The percentages of TCR transgenic thymocytes and splenic T cells (both CD44^lo and CD44^hi) staining positive for TNF are shown. B, Resting P14-CD8⁺ thymocytes and naïve splenocytes were stained with mAbs to Vα2 and Vβ8.1. The profile for the SP thymocytes is shown in gray solid histograms and for splenic T cells in black line histograms. C, P14-CD8⁺ thymocytes and naïve splenocytes were either unstimulated (gray solid histograms) or stimulated (black line histograms) for 4 hours with GP33+αCD28 in vitro and stained with mAbs to the indicated surface molecules. D, CD45.1⁺ SP P14-CD8⁺ thymocytes were enriched and stimulated with GP33+αCD28 for 4 hours either alone or in the presence of live or irradiated (3000cGy) H-2D^b WT or H-2D^b KO splenocytes respectively. Cells were then stained for intracellular TNF. The percentages of CD45.1⁺ CD8⁺ thymocytes (CD44^lo and CD44^hi) staining positive for TNF are shown.</p

TNF producing SP thymocytes exhibit a lower maturation profile relative to their splenic counterparts.

<p>A and B, CD45.1⁺ P14-CD8⁺ thymocytes were stimulated with GP33+αCD28 for 4 hours in vitro and then stained for maturation markers and intracellular TNF, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#s4" target="_blank"><i>Materials and Methods</i></a>. The TNF producers and non producers of the thymic and the splenic subsets were each classified into 4 subgroups based on their CD24 and Qa2 expression as shown namely Subgroup 1 (CD24^hi Qa2^lo) followed by Subgroup 2 (CD24^hi-int Qa2^lo), Subgroup 3 (CD24^lo Qa2^lo) and finally Subgroup 4 (CD24^lo Qa2^hi). C, shows the histogram comparison of the small population of TNF producing SP thymocytes (dotted line histograms), the majority of SP thymocytes that are TNF non-producers (gray histograms) and TNF producing splenic T cells (solid dark line histograms) and TNF non-producing splenic T cells (black histograms). The MFIs of each of the maturation markers in TNF producing and non-producing thymic and splenic T cells are indicated in the left hand side of the histograms respectively. D, shows the maturation profile of the total SP CD8⁺ thymocytes based on their CD24 and Qa2 expression. The proportion of cells capable of making TNF in the 4 subgroups is shown with respect to CD45RB expression.</p

Post-thymic maturation status of naïve polyclonal T cells determines their TNF producing capability.

<p>A, The percentages of non-transgenic CD8⁺ and CD4⁺ cells (both CD44^lo and CD44^hi) from thymi and spleens of B6 mice staining positive for TNF cytokine are shown. B, Thymocytes and splenocytes from NG-BAC transgenic mice were stimulated with αCD3+αCD28 for 4 hours and then stained for maturation markers and intracellular TNF. The GFP profile of SP thymocytes, RTEs and MN T cells in the CD8⁺ and CD4⁺ compartments is shown. B and C, The percentages of CD44^lo TNF producing cells in the 3 different T cell subsets and their respective average MFI for TNF expression are shown. The average MFI of TNF expression were analysed by one-way ANOVA with a Tukey post-test. The data are representative of 4 individual mice. The error bars indicate SD.</p

SP thymocytes and splenic T cells exhibit a differential ability to produce TNF during in vivo TCR activation.

<p>A and B, CD45.1⁺ P14-CD8⁺ and SMARTA-CD4⁺ splenocytes were mixed at 1∶1 ratio, treated with bfA and transferred into CD45.2⁺ congenic hosts that were uninfected or infected with either WT LCMV Armstrong or the LCMV variant GP1V. CD45.1⁺ P14-CD8⁺ and SMARTA-CD4⁺ thymocytes were treated with bfA and transferred separately into CD45.2⁺ congenic hosts that were infected with either WT LCMV Armstrong or the viral variant GP1V. Spleens from all recipient mice were harvested 4 hours later and directly stained for intracellular TNF. The percentages of donor CD45.1⁺ P14-CD8⁺ and SMARTA-CD4⁺ splenic T cells and SP thymocytes that are TNF positive and have down-regulated CD62L are shown. C, The average percentages of CD62L^loTNF⁺ donor T cells detected ex vivo are shown. One-way ANOVA with Tukey post-test was used to compare the mice that received P14 thymocytes or splenocytes and were either uninfected or infected with WT LCMV strain or the viral mutant strain (p<0.05). These data are a pool of 6 experiments. Error bars indicate SD.</p