Rapamycin programs memory CTLs in the presence of IL-12 in early activation.

<p>Sorted OT1 cells were stimulated with antigen+B7+IL-12 in the presence of rapamycin at different concentrations. Programmed CTLs were harvested at 72 hrs, and transfer into naïve B 6 mice at 10⁶/mouse through tail injection. (A) Memory OT1 cells in spleen at day 30 after transfer. (B–D) Comparison of molecular expression in memory CTLs programmed by rapamycin (at 250 ng/ml) with control (no rapamycin). (E) Spleen cells containing 10⁵ memory OT1 from (B) were transferred into naïve B6 mice, which were challenged the next day with 5×10⁵ CFU LM-OVA through i.v. Bacterium counts were examined 3 days after LM-OVA challenge in spleen. Every group was compared to control group (OT1 naïve–10⁵ naive OT1 transfer group). These are representatives of two independent experiments with similar results. Every group was compared to control group (rapamycin 0) in A and D. Asterisks indicate statistical significance. *, P<0.05; **, P<0.01; ***, P<0.001. ns: not significant.</p

Rapamycin regulates memory CTLs and CD62L expression in different time windows.

<p>Sorted OT1 cells were stimulated with antigen+B7+IL-12 in the presence of rapamycin at 250 ng/ml for different windows. Programmed CTLs were harvested at 72 hrs, and transfer into naïve B6 mice at 10⁶/mouse. Memory OT1 cells were examined in spleen at day 30 after transfer for analysis of memory size (A), expression of CD62L (B–C) and production of IFNγ (D). Every group was compared to control group (0–72) in A and C.</p

Rapamycin delays CTL proliferation during early activation.

<p>Sorted OT1 cells were labeled with CFSE, then stimulated with 2 SI (antigen+B7) or 3 SI (2 SI plus IL-12) in the presence of rapamycin at different concentrations. (A–B) Cell proliferation (CFSE dilution) was evaluated at day 2 and 3. (C) Fold expansion of CTLs 3 days after in vitro stimulation was calculated according to original input. (D) CD62L expression in CFSE labeled cells at day 3 after stimulation. Numbers beside or above each gate indicates the percentage of gated cells. MFI: Mean Fluorescence Intensity of the total population. These are representatives of at least three independent experiments with similar results. (E) Transcriptional regulation of CD62L by rapamycin. OT1 cells were stimulated for 3 days under 3 SI in the presence or absence of rapamycin, and then were harvested for real-time PCR examination. CD62L and housekeeping gene GAPDH were analyzed in triplicate in real-time RT-PCR assays. Relative mRNA amounts were normalized with respect to expression levels in Naïve OT1 control (fold change = 1). The results are expressed as mean+SEM of three independent experiments.</p

Rapamycin experienced CTLs have increased proliferation and maintain higher CD62L expression during memory differentiation.

<p>Experimental setting was the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025177#pone-0025177-g002" target="_blank">Figure 2</a>. Activated CTLs were harvested at day 3 after stimulation in vitro, and were transferred into naïve B6 mice at 10⁶/mouse. Blood samples were drawn at day 5, 15 and 30 after transfer. (A) Numbers of OT1 cells in spleen 5 days after transfer. Every group was compared to control group (rapamycin 0). (B) Representative CD62L expression in OT1 cells in blood at day 5 after transfer. (C) Comparison of CD62L expression in OT1 cells during memory differentiation. “In vitro” represents samples harvested at day 3 after stimulation in vitro. (D) In a separated experiment, rapamycin+3-SI programmed and control CTLs under 3-SI stimulation were harvested at 72 hrs, and labeled with CFSE before transferred into B6 mice. CFSE dilution was examined in OT1 cells from spleens at days 1 and 2 after transfer. Experiments are representatives of at least three independent experiments with similar results (A–C) or one experiment (D). Each value represents the mean plus the SD of 5 mice per group in C.</p

Injection of rapamycin in recipients can enhance memory CTL programmed by IL-12 after transfer.

<p>(A) Sorted OT1 cells were stimulated with antigen+B7+rapamycin (at different concentration) in the absence of IL-12. Stimulated CTLs were harvested at 72 hrs, and transfer into naïve B6 mice at 10⁶/mouse. Memory OT1 cells in spleen were examined at day 30 after transfer of CTLs. These are representatives of three independent experiments with similar results. (B) Sorted OT1 cells were stimulated with antigen+B7+IL-12, and were transferred into recipient B6 mice after 3 days. Half of the recipients received daily injection of rapamycin at 75 ug/kg from −1 to day 10 after transfer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025177#pone.0025177-Araki1" target="_blank">[26]</a>. Memory CTLs in spleen were examined at day 30 after transfer. These are representatives of two independent experiments with similar results. (C) Sorted OT1 cells were stimulated with antigen+B7+IL-12 for 3 days, and then were labeled with CFSE before being transferred into recipient B6 mice at 10⁶/mouse. Half recipients received daily injection of rapamycin at 75 ug/kg from −1 to day 2 after transfer. CFSE dilution was examined at days 1 and 2 after transfer. Control: CFSE-labeled naïve OT1 cells. These are representatives of five animals in each treatment.</p

Rapamycin suppresses effector function of CTLs in wide range of concentration.

<p>Sorted OT1 cells were stimulated with antigen+B7+IL-12 in the presence of rapamycin at different concentrations. Programmed CTLs were harvested at 48 hrs. (A) The expression of IFNγ. (B) The expression of granzyme B (GZB). (C) The expression of P-SK6. (D) The expression of 4E-BP. These are representatives of three independent experiments with similar results.</p

Nicotine Inhibits Memory CTL Programming

<div><p>Nicotine is the main tobacco component responsible for tobacco addiction and is used extensively in smoking and smoking cessation therapies. However, little is known about its effects on the immune system. We confirmed that multiple nicotinic receptors are expressed on mouse and human cytotoxic T lymphocytes (CTLs) and demonstrated that nicotinic receptors on mouse CTLs are regulated during activation. Acute nicotine presence during activation increases primary CTL expansion <i>in vitro</i>, but impairs <i>in vivo</i> expansion after transfer and subsequent memory CTL differentiation, which reduces protection against subsequent pathogen challenges. Furthermore, nicotine abolishes the regulatory effect of rapamycin on memory CTL programming, which can be attributed to the fact that rapamycin enhances expression of nicotinic receptors. Interestingly, naïve CTLs from chronic nicotine-treated mice have normal memory programming, which is impaired by nicotine during activation <i>in vitro</i>. In conclusion, simultaneous exposure to nicotine and antigen during CTL activation negatively affects memory development.</p></div

Nicotine effects on T-bet expression and mTOR signaling.

<p>Sorted OT-I cells were stimulated with 3SI (antigen+B7+IL-12) in the presence of nicotine at different concentrations. Programmed CTLs were harvested at 3 days post-stimulation and analyzed for expression of T-bet, Eomes, S6, mTOR nad 4EBP. These are representatives of three independent experiments with similar results.</p

Expression of nAChRs in CTLs from human and mice.

<p>Purified RNA from human and mouse CD8 T cells were examined for nAChR subunits using PCR. A. PCR results (40 cycles, all PCR products were confirmed by sequencing). Memory OT-I cells were obtained as described in the text and as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068183#pone.0068183-Xiao2" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068183#pone.0068183-Basu1" target="_blank">[83]</a>. B–C. Quantitative PCR was performed on RNA from human CD8 T cells (B) or murine naive CD8 T cells (C). Expression of all subunits was relative to β2 (the highest expression) in murine naïve CTLs (C), which was designated as 1. D. Purified naïve OT-I cells were stimulated with 2SI or 3SI (2SI plus IL-12) for 3 days, and expression of nAChRs was compared relatively to β2 in each treatment (2SI and 3SI). E. The expression of 3 dominant nAChR subunits in 2SI and 3SI treatments was compared to naïve OT-I using the housekeeping gene GAPDH as internal control. Subunits α1, α7 and β3 were not detectable in D–E. F. CD8 OT-I cells were purified from naïve OT-I mice or OT-I mice treated with nicotine water at 200 µg/ml for 60 days to quantify the expression of nAChR subunits. Data are representatives of at least three experiments with similar results.</p

Nicotine does not affect CTL activation.

<p>Purified OT-I cells were cultured for 3 days with 3SI in the presence of nicotine at different concentrations. A. Comparison of fold expansion was calculated as the cell yield at day 3 divided by initial input cells at day 0. B. Cells were harvested to examine activation markers. C. Comparison of IFNγ, granzyme B (GZB) and other surface molecules. Data are representatives of at least five experiments with similar results. Asterisks indicate statistical significance. *, P<0.05; **, P<0.01; ***, P<0.001.</p