T cell-intrinsic TLR2 stimulation promotes IL-10 expression and suppressive activity by CD45RbHi T cells.

While Toll-like receptors (TLRs) represent one of the best characterized innate immune pathways, evidence suggests that TLRs are not restricted to innate leukocytes and some epithelial cells, but are also expressed in T cells. Specifically, published evidence focusing on FoxP3+ regulatory T cells demonstrate that they express functional TLR2, which is already known among the TLR family for its association with immune suppression; however, little is known about the relationship between T cell-intrinsic TLR2 binding and cytokine production, T cell differentiation, or T cell receptor (TCR) stimulation. Here, we demonstrate that TCR and TLR2 co-stimulation provides a T cell-intrinsic signal which generates a dramatic, synergistic cytokine response dominated by IL-10. Importantly, the response was not seen in either CD4+CD25+ or CD4+FoxP3+ Tregs, yet resulted in the expansion of a suppressive CD4+CD25+CD62L-CD44+CD45Rbhi effector/memory T cell subset not typically associated with immune inhibition. This study reveals the striking ability of a prototypical innate immune receptor to trigger a potent and suppressive IL-10 response in effector/memory T cells, supporting the notion that TLR2 is a co-regulatory receptor on T cells

TLR2 stimulation promotes a T cell-suppressive environment.

<p>(A) WT and IL-10^-/- CD4⁺ T cells isolated using MACS were stimulated at day 0 as before. After 24 hours, cells were washed to remove soluble agonists, and then allowed to incubate for an additional 3 days. Conditioned media supernatants, which were analyzed for IL-10 by ELISA (B), were then transferred to fresh recipient CD4⁺ T cells stimulated by αCD3 on day 4. On day 7, the IFNγ response of the recipient cells was quantified. Normalized to the response without transferred supernatant (100%; dotted line) and subtracting IFNγ concentrations in the donor supernatants, we found that supernatants from TCR/TLR2 co-stimulated WT T cells inhibited the recipient IFNγ response by 50%, whereas supernatants from TLR2-only stimulated T cells augmented recipient IFNγ production by over 2 fold (C). In contrast, supernatants generated from all stimulated IL-10^-/- donor T cells promoted IFNγ by the recipients by nearly 2 fold (D). All data are n≥3, unless otherwise noted; *<i>p</i><0.05; #<i>p</i>>0.05.</p

Luminex analysis of stimulated CD4⁺ T cells.

<p>MACS-sorted CD4⁺ T cells were stimulated as indicated for 72 hours. Conditioned media was harvested and analysis by 32-plex cytokine/chemokine Luminex at Eve Technologies. Analytes were grouped by the pattern of response by the co-stimulated cells. (n = 3 per stimulation).</p

TCR and TLR co-stimulation promotes synergistic IL-10 production.

<p>Splenic CD4⁺ T cells isolated from an IL-10 (IRES-GFP) reporter were stimulated with TCR (αCD3) and TLR2 receptor agonists (Pam3Csk4, P3C) independently or in combination at 2μg/ml each. IL-10 expression was measured on day 3 by flow cytometry as % GFP positive within CD4⁺ gated cells. Representative histograms (A) and replicates (B) show synergistic IL-10 response to simultaneous TCR/TLR2 co-stimulation above either stimulus independently. (C) WT C57Bl/6 splenic CD4⁺ T cells were cultured as above and IL-10 quantified by ELISA on day 3, showing synergistic IL-10 secretion. (D) WT CD4⁺ T cells cultured with αCD3 and/or P3C as before, with and without αCD28. IL-10 synergy is not significantly impacted by the addition of CD28 stimulation. (E) Magnetic bead-isolated (MACS) CD4⁺ T cells used in panels A-D by flow cytometry showed 91.2% purity. (F) Sterile flow cytometry-sorted (FACS) CD4⁺ T cells showed 99.2% purity. (G) IL-10 secretion of ultra-pure FACS sorted CD4⁺ T cells (see panel F) stimulated as before, showing indistinguishable synergy from MACS-isolated T cells (see panel C). (H) mRNA transcript levels of the IL-10 gene from stimulated WT CD4⁺ T cells was measured by microarray. Normalized expression levels compared to the canonical T cell marker Lck are shown, showing that αCD3 induces transcription of the IL-10 to an equal extent as with added P3C, suggesting P3C controls IL-10 concentration through regulation of IL-10 translation. All data are n≥3, unless otherwise noted; *<i>p</i><0.05; #<i>p</i>>0.05.</p

TLR2-Responding Cells are CD25^- and FoxP3^- effector/memory T cells.

<p>In order to identify the TCR/TLR2 responding populations, cells isolated from WT, FoxP3-reporter, or IL-10/FoxP3-dual reporter mice were purified by FACS into varied populations, stimulated as before for 3 days and analyzed by IL-10 ELISA (A-D) or by flow cytometry (E-G). Neither CD25⁺ (A), regulatory FoxP3⁺ (B), nor naïve CD62L⁺CD44^- (C) CD4⁺ T cells produced IL-10 in response to stimulation, whereas CD25^-, FoxP3^- and CD62L^-CD44⁺ antigen experienced CD4⁺ T cells showed IL-10 synergy in response to TCR/TLR2 co-stimulation. (D) Post-stimulation analysis at day 3 of CD4⁺ and CD4⁺FoxP3^- T cells showed a modest 10% induction of FoxP3 expression upon P3C stimulation, but only 5% of cells expressed FoxP3 under synergistic IL-10-producing conditions (αCD3+P3C). (E) After stimulation, dual reporter CD4⁺ T cells were analyzed by flow cytometry. CD4⁺ cells showed an increase in the number of IL-10⁺/GFP⁺ T cells (E, left), and among these cells, the addition of P3C selectively increased the number of IL-10⁺FoxP3^- cells (E, right, arrow). Quantitation of the percentage (F) and MFI (G) of CD4⁺FoxP3^-IL-10⁺ among all CD4⁺ T cells (n = 5) is shown. All data are n≥3, unless otherwise noted; *<i>p</i><0.05; #<i>p</i>>0.05.</p

T cell-intrinsic TLR2 stimulation promotes IL-10 expression and suppressive activity by CD45Rb^Hi T cells

<div><p>While Toll-like receptors (TLRs) represent one of the best characterized innate immune pathways, evidence suggests that TLRs are not restricted to innate leukocytes and some epithelial cells, but are also expressed in T cells. Specifically, published evidence focusing on FoxP3⁺ regulatory T cells demonstrate that they express functional TLR2, which is already known among the TLR family for its association with immune suppression; however, little is known about the relationship between T cell-intrinsic TLR2 binding and cytokine production, T cell differentiation, or T cell receptor (TCR) stimulation. Here, we demonstrate that TCR and TLR2 co-stimulation provides a T cell-intrinsic signal which generates a dramatic, synergistic cytokine response dominated by IL-10. Importantly, the response was not seen in either CD4⁺CD25⁺ or CD4⁺FoxP3⁺ Tregs, yet resulted in the expansion of a suppressive CD4⁺CD25⁺CD62L^-CD44⁺CD45Rb^hi effector/memory T cell subset not typically associated with immune inhibition. This study reveals the striking ability of a prototypical innate immune receptor to trigger a potent and suppressive IL-10 response in effector/memory T cells, supporting the notion that TLR2 is a co-regulatory receptor on T cells.</p></div

TLR2 stimulation induces expansion of CD45Rb^Hi effector/memory T cells.

<p>To further characterize the phenotype and viability of the T cells after stimulation, we measured expression of CD25 and CD45Rb (A) and CD62L and CD44 (B) by flow cytometry of cells isolated using MACS after 3 days of stimulation. P3C-stimulated cells are most similar to freshly isolated T cells without stimulation (No Stim), while αCD3 induced a robust shift to CD25⁺CD45R^hi (85.4%) which was increased even further to 94.5% in response to P3C. Likewise, the majority of cells following stimulation with αCD3 were CD62L^-CD44⁺, with P3C co-stimulation having little impact on this pattern. (C) A majority of T cells without stimulation were dead or dying, while all stimulation conditions lead to increased viability as measured by propidium iodide exclusion.</p

TLR2 stimulation shows differential effects on T cell cytokine production.

<p>Multianalyte analysis was performed using Luminex on 35 cytokines and chemokines on WT CD4⁺ T cells isolated using MACS stimulated as before. TLR2-dependent synergistic increases in IL-10 (A), IL-6 (B), CXCL-10 (C), and IL-13 (D) were seen in TCR/TLR2 co-stimulated cells. In contrast, IL-1β (E) and TGFβ (F) showed no response to any stimulus. MIP-1α (G) was strongly induced by αCD3, but TLR2 stimulation failed to change the level of production, whereas MIP-1β showed modest synergy, albeit less than 2 fold (H). MIP-2 showed strong responsiveness to P3C alone (I), and IL-2 was highest in αCD3-stimulated cells from WT mice (J). All data are n≥3, unless otherwise noted; *<i>p</i><0.05; #<i>p</i>>0.05.</p

TLR2 stimulation promotes a T cell-suppressive environment.

<p>(A) WT and IL-10^-/- CD4⁺ T cells isolated using MACS were stimulated at day 0 as before. After 24 hours, cells were washed to remove soluble agonists, and then allowed to incubate for an additional 3 days. Conditioned media supernatants, which were analyzed for IL-10 by ELISA (B), were then transferred to fresh recipient CD4⁺ T cells stimulated by αCD3 on day 4. On day 7, the IFNγ response of the recipient cells was quantified. Normalized to the response without transferred supernatant (100%; dotted line) and subtracting IFNγ concentrations in the donor supernatants, we found that supernatants from TCR/TLR2 co-stimulated WT T cells inhibited the recipient IFNγ response by 50%, whereas supernatants from TLR2-only stimulated T cells augmented recipient IFNγ production by over 2 fold (C). In contrast, supernatants generated from all stimulated IL-10^-/- donor T cells promoted IFNγ by the recipients by nearly 2 fold (D). All data are n≥3, unless otherwise noted; *<i>p</i><0.05; #<i>p</i>>0.05.</p

TLR2 and TLR9 show similar IL-10-induction activity.

<p>Co-stimulation of WT CD4⁺ T cells, isolated using MACS, with different TLR agonists at varying concentrations was performed for 3 days and IL-10 quantified by ELISA. The TLR2/6-dependent agonist lipoteichoic acid (LTA) failed to increase IL-10 (A), whereas the TLR2/1-dependent LpqH (B) and P3C (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180688#pone.0180688.g001" target="_blank">Fig 1</a>) showed IL-10 synergy. Similar to LTA, TLR3-dependent Poly I:C (C), TLR4-dependent LPS (D), and TLR5-dependent flagellin (E) all failed to increase IL-10. As seen with P3C and LpqH, the TLR9-dependent CpG also induced robust IL-10 synergy (F). mRNA transcript levels of the TLR genes from freshly isolated unstimulated WT CD4⁺ T cells was measured by microarray. Normalized expression levels compared to the canonical T cell marker Lck (G) are shown. TLR1, expressed at essentially the same level as Lck, is the most highly expressed TLR gene. All data are n≥3, unless otherwise noted; only LpqH and CpG showed significant increases in IL-10 over baseline (<i>p</i><0.05); *<i>p</i><0.05; #<i>p</i>>0.05.</p