Protein kinase C theta is required for efficient induction of IL-10-secreting T cells

<div><p>Secretion of interleukin-10 (IL-10) by CD4⁺ T cells is an essential immunoregulatory mechanism. The work presented here assesses the role of the signaling molecule protein kinase C theta (PKCθ) in the induction of IL-10 expression in CD4⁺ T cells. Using wildtype and PKCθ-deficient Tg4 T cell receptor transgenic mice, we implemented a well-described protocol of repeated doses of myelin basic protein (MBP)Ac1-9[4Y] antigen to induce Tr1-like IL-10⁺ T cells. We find that PKCθ is required for the efficient induction of IL-10 following antigen administration. Both serum concentrations of IL-10 and the proportion of IL-10⁺ T cells were reduced in PKCθ-deficient mice relative to wildtype mice following [4Y] treatment. We further characterized the T cells of [4Y] treated PKCθ-deficient Tg4 mice and found reduced expression of the transcription factors cMaf, Nfil3 and FoxP3 and the surface receptors PD-1 and Tim3, all of which have been associated with the differentiation or function of IL-10⁺ T cells. Finally, we demonstrated that, unlike [4Y] treated wildtype Tg4 T cells, cells from PKCθ-deficient mice were unable to suppress the priming of naïve T cells <i>in vitro</i> and <i>in vivo</i>. In summary, we present data demonstrating a role for PKCθ in the induction of suppressive, IL-10-secreting T cells induced in TCR-transgenic mice following chronic antigen administration. This should be considered when contemplating PKCθ as a suitable drug target for inducing immune suppression and graft tolerance.</p></div

Glycogen synthase kinase-3 controls IL-10 expression in CD4⁺effector T-cell subsets through epigenetic modification of the IL-10 promoter

The serine/threonine kinase glycogen synthase kinase-3 (GSK3) plays an important role in balancing pro- and anti-inflammatory cytokines. We have examined the role of GSK3 in production of IL-10 by subsets of CD4(+) T helper cells. Treatment of naive murine CD4(+) T cells with GSK3 inhibitors did not affect their production of IL-10. However, treatment of Th1 and Th2 cells with GSK3 inhibitors dramatically increased production of IL-10. GSK3 inhibition also led to upregulation of IL-10 among Th1, Th2, and Th17 subsets isolated from human blood. The encephalitogenic potential of GSK3 inhibitor treated murine Th1 cells was significantly reduced in adoptive transfer experiments by an IL-10-dependent mechanism. Analysis of the murine IL-10 promoter in response to inhibition of GSK3 in Th1 cells showed modification to a transcriptionally active state indicated by changes in histone H3 acetylation and methylation. Additionally, GSK3 inhibition increased expression of the transcription factors c-Maf, Nfil3, and GATA3, correlating with the increase in IL-10. These findings are important in the context of autoimmune disease since they show that it is possible to reprogram disease-causing cells through GSK3 inhibition

PKCθ links proximal T cell and Notch signaling through localized regulation of the actin cytoskeleton

Notch is a critical regulator of T cell differentiation and is activated through proteolytic cleavage in response to ligand engagement. Using murine myelin-reactive CD4 T cells, we demonstrate that proximal T cell signaling modulates Notch activation by a spatiotemporally constrained mechanism. The protein kinase PKCθ is a critical mediator of signaling by the T cell antigen receptor and the principal costimulatory receptor CD28. PKCθ selectively inactivates the negative regulator of F-actin generation, Coronin 1A, at the center of the T cell interface with the antigen presenting cell (APC). This allows for effective generation of the large actin-based lamellum required for recruitment of the Notch-processing membrane metalloproteinase ADAM10. Such enhancement of Notch activation is critical for efficient T cell proliferation and Th17 differentiation. We reveal a novel mechanism that, through modulation of the cytoskeleton, controls Notch activation at the T cell:APC interface thereby linking T cell receptor and Notch signaling pathways. DOI: http://dx.doi.org/10.7554/eLife.20003.00

Dosing strategy for effective peptide immunotherapy of experimental autoimmune disease

Previous work has shown that repetitive intranasal (i.n.) administration of the myelin basic protein peptide MBP Acl-9[4Y] induces tolerance in the TCR transgenic Tg4 EAE model. Chronic antigen stimulation of C04+ T cells was found to induce an anergic IL-l0-secreting regulatory phenotype in Th-I cells, protecting animals from EAE. Alternative routes for peptide administration were explored in the Tg4 model, to increase our understanding of the factors influencing peptide immunotherapy. Epicutaneously (e. c.) administered MBP Acl- 9[4Y] was slowly trafficked to the lymph nodes and spleen where C04+ T cells were activated. However, repetitive e.c. MBP Acl-9[4Y] administration did not reliably induce characteristics of C04+ T cell tolerance, attributed to the difficulty of controlling the e.c. administered MBP Acl-9 [4 Y] dose. Use of the subcutaneous (s.c.) route circumvented this problem, and titrated MBP Acl-9[4Y] doses were administered s.c. to Tg4 mice. Tolerance induced was proportional to the s.c. MBP Acl-9[4Y] dose administered, with higher doses better inducing an anergic, suppressive C04+ T cell phenotype, upregulation of IL-l0 secretion and protection from EAE. However, further increasing the s.c. peptide dose induced severe adverse effects in Tg4 (and Tg4 Rag-l -!-) mice, concomitant with high systemic inflammatory cytokine levels. Novel application of the technique of dose escalation to self-peptide immunotherapy allowed delivery of high peptide doses s.c. to Tg4 Rag-l +/+ and -/- mice without adverse effects. Escalating to higher s.c. peptide doses better induced tolerance, providing long-term protection of Tg4 Rag-l -/- mice from the spontaneous development of EAE. This demonstrates that low peptide doses administered s.c. during the escalation stage of treatment modulated the response of a monoclonal C04+ T cell population to subsequent high s.c. peptide doses. Collectively, these results show that route of antigen administration contributes to the outcome of peptide immunotherapy, which is also closely related to the peptide dose administered. Furthermore, we propose that dose escalation is essential for the safe and effective translation of peptide immunotherapy of auto immune disease into the clinic.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

PKCθ is required for induction of a suppressive environment <i>in vivo</i>.

<p>(<b>A</b>) Experimental design. Cell Proliferation Dye-labeled CD4⁺ T cells from naïve Tg4WT mice were adoptively transferred to Tg4^WT and Tg4^KO mice, pretreated with [4Y] or PBS. After 48 hours, mice were challenged with 80μg of [4Y] and the division of the transferred Tg4^WT cells was measured by flow cytometry after a further 48 hours. (<b>B</b>) Example flow cytometry data and (<b>C</b>) plotted data from all mice showing the proportion of transferred Tg4^WT cells which remained undivided following [4Y] challenge under each pre-treatment condition. Shown is the mean +/- SEM. Each data point represents one [4Y] or PBS-treated recipient mouse which were assayed in a single experiment. *p<0.05 assessed by ANOVA with Tukey’s correction for multiple comparisons.</p

Serum cytokine concentrations in Tg4^WT and Tg4^KO mice over the course of [4Y] treatment.

<p><b>(A)</b> Experimental design. Escalating doses of MBPAc1-9[4Y] peptide were administered subcutaneously to mice every 3–4 days. (<b>B-F</b>) Concentrations of IL-10, IL-2, IFNγ, TNFα and IL-17A in serum from peripheral blood taken two hours after each [4Y] treatment in Tg4^WT (open circles) and Tg4^KO (closed circles) mice. Plots show the average of four animals +/- standard error of the mean (SEM) representing one experiment of three performed. *p<0.01 **p<0.001 by Student’s two-tailed T test.</p