Construction of a bacterial autoinducer detection system in mammalian cells

Quorum sensing (QS) is a cell density-dependent signaling system used by bacteria to coordinate gene expression within a population. QS systems in Gram negative bacteria consist of transcription factors of the LuxR family and their acyl homoserine lactone (AHL) ligands. We describe here a method for examining QS signaling systems in mammalian cells that uses engineered LuxR-type proteins from the opportunistic pathogen, Pseudomonas aeruginosa, which can function as AHL-dependent transcription factors. The engineered proteins respond to their cognate ligands and display sequence specific DNA binding properties. This system has several potential biotechnological and biological applications. It may be used to characterize any LuxR-type protein, screen animal and plant cell extracts or exudates for compounds that mimic or interfere with AHL signaling or to screen different cell types for AHL inactivating activities

CD4+ T cell subset differentiation and avidity setpoint are dictated by the interplay of cytokine and antigen mediated signals.

CD4(+) T cell differentiation has been shown to be regulated by the cytokine milieu present during activation as well as peptide MHC levels. However, the extent to which these two important regulatory signals work in concert to shape CD4(+) T cell function has not been investigated. Using a murine OT-II transgenic TCR model of in vitro differentiation, we demonstrate that the ability of CD4(+) T cells to commit to a distinct lineage, i.e. Th1 vs. Th2 vs. Th17, is restricted by the amount of peptide antigen present in the stimulating environment. In addition, whether cells succumb to inhibitory effects associated with high dose antigen is dependent on the array of cytokine signals encountered. Specifically, stimulation with high dose antigen in Th1 or Th17 conditions promoted efficient generation of functional cells, while Th2 polarizing conditions did not. Finally, we found that the peptide sensitivity of an effector cell was determined by the combined actions of cytokine and peptide level, with Th1 cells exhibiting the highest avidity, followed by Th17 and Th2 cells. Together, these data show that the interplay of antigen and cytokine signals shape both the differentiation fate and avidity setpoint of CD4(+) T cells

Proliferation and survival are dependent on the combined effects of peptide and cytokine environment.

<p>OT-II splenocytes from naïve mice were stimulated with a high, intermediate or low concentration of peptide in the presence of Th1, Th2, or Th17 differentiating conditions. A. Cell recovery for each condition was assessed on d7 post-primary and d7 post-secondary stimulation. Data are presented as recovery on d7 post stimulation divided by input cell number at initiation of each stimulation culture. All cultures began with the same number of input cells. Averaged data are from 2–4 independent experiments, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100175#pone-0100175-g001" target="_blank">Figure 1</a>. B. CFSE-labeled naïve OT-II cells were cultured for 72h in the presence of differentiating cytokines and peptide. OT-II cells cultured in the absence of peptide are shown as a control. Values shown in the histograms are the percent of cells in the divided gate. C. At 72h following initiation of primary culture, cells were incubated with 7-AAD as a measure of cell death. OT-II cells were identified by CD4 and CD45.2 positivity. All data are representative of three independent experiments. *p≤0.05, ***p≤0.001.</p

CD25 and CD69 are differentially regulated among the lines generated with high, intermediate, or low dose peptide.

<p>OT-II cells were stimulated with the high, intermediate or low concentration of peptide in the presence of Th1, Th2 or Th17 differentiating conditions and CD45.1⁺ splenocyte stimulators. At 0, 24, 48 and 72 hours, the expression of CD25 and CD69 was determined on CD4⁺CD45.2⁺ (OT-II cells). Cells were assessed for marker positivity (A and B) and the level of marker expression (C and D). Data shown are the average of 3 (A and B) or 2 (B and D) independent experiments. One experiment utilized different fluorophores for the detection of CD25 and CD69 thereby precluding averaging in C and D; however, the patterns of expression were similar. * p≤0.05, **p≤0.01, ***p≤0.001.</p

The avidity setpoint is determined by the combined effect of cytokine and pMHC level.

<p>The EC₅₀ for each line was determined by stimulation with titrated concentrations of peptide. Peptide dose response curves are shown in A and averaged EC₅₀ values are shown in B. Data are from 3 independent experiments in all cases, except for the 10⁻⁵M Th1 line (n = 4). * p≤0.05, **p≤0.01, ***p≤0.001.</p

The differentiation of CD4⁺ T cells is affected by both cytokine and antigen dose.

<p>OT-II lines were generated by repeated weekly stimulation in the presence of cytokine that polarized the cells toward differentiation into Th1, Th2 or Th17 cells together with high (10⁻⁵M), intermediate (10⁻⁷M) or low (10⁻⁹M) concentrations of peptide. Established OT-II cell lines were assayed for cytokine production following stimulation with OVA_323–339 peptide (10⁻⁴M). The differentiation state of the cell lines was determined by measuring the production of IFN-γ (Th1), IL-4 (Th2) or IL-17 (Th17). A. Representative dotplots of cytokine production for each cell line are shown. Data shown are gated on CD4⁺CD45.2⁺ cells. B. The percentage of cells that produce cytokine following peptide stimulation is shown for each stimulation condition. Th17 cells did not survive on low dose peptide. Data are the average of independently generated lines: −5M Th1 (n = 3), −7M Th1 (n = 3), −9M Th1 (n = 4), −5M Th2 (n = 3), −7M Th2 (n = 3), −9M Th2 (n = 3), −5M Th17 (n = 4), −7M Th17 (n = 3), and −9M Th17 (n = 2). * p≤0.05, *** p≤0.0005.</p

The lack of peptide responsiveness in Th2 cells generated on high dose peptide can be overcome by stimulation with PMA+ionomycin, but not anti-CD3 antibody.

<p>Established Th2 cell lines were stimulated for 5 hours in the presence of 100 µM peptide, PMA (50 ng/ml)+ionomycin (500 ng/ml), or immobilized anti-CD3 antibody (plate coated overnight with 50 µg/ml). OT-II cells were identified by CD4 and CD45.2 staining. Cytokine production was assessed by ICCS. Data shown are the average of 3 independently generated lines. *** p≤0.001.</p

Differences in functional avidity cannot be explained by the level of CD4, TCR, or CD44.

<p>The established Th1, Th2 and Th17 cell lines were assessed for the expression of CD4, TCRβ and CD44 on d7 post routine stimulation (to circumvent any activation induced changes in level). OT-II cells were identified by CD4 and CD45.2 staining. The mean fluorescence intensity (MFI) for each marker averaged across independently generated lines is shown, n = 3 for all cases, except −5M Th1 (n = 4) and −9M Th1 (n = 2).</p