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

Modification of the FoxP3 Transcription Factor Principally Affects Inducible T Regulatory Cells in a Model of Experimental Autoimmune Encephalomyelitis

T regulatory (Treg) cells expressing the transcription factor FoxP3 play a key role in protection against autoimmune disease. GFP-FoxP3 reporter mice have been used widely to study the induction, function and stability of both thymically- and peripherally-induced Treg cells. The N-terminal modification of FoxP3, however, affects its interaction with transcriptional co-factors; this can alter Treg cell development and function in certain self-antigen specific animal models. Interestingly, Treg cell function can be negatively or positively affected, depending on the nature of the model. In this study, we focused on the effect of the GFP-FoxP3 reporter on Treg cell development and function in the Tg4 mouse model. In this model, T cells express a transgenic T cell receptor (TCR) specific for the Myelin Basic Protein (MBP) peptide Ac1-9, making the animals susceptible to experimental autoimmune encephalomyelitis (EAE), a disease akin to multiple sclerosis in humans. Unlike diabetes-susceptible mice, Tg4 FoxP3(gfp) mice did not develop spontaneous autoimmune disease and did not demonstrate augmented susceptibility to induced disease. Concurrently, thymic generation of natural Treg cells was not negatively affected. The induction of FoxP3 expression in naive peripheral T cells was, however, significantly impaired as a result of the transgene. This study shows that the requirements for the interaction of FoxP3 with co-factors, which governs its regulatory ability, differ not only between natural and inducible Treg cells but also between animal models of diseases such as diabetes and EAE

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

Regulation of adaptive immunity; the role of interleukin-10

Since the discovery of interleukin-10 (IL-10) in the 1980s, a large body of work has led to its recognition as a pleiotropic immunomodulatory cytokine that affects both the innate and adaptive immune systems. IL-10 is produced by a wide range of cell types, but for the purposes of this review we shall focus on IL-10 secreted by CD4+ T cells. Here we describe the importance of IL-10 as a mediator of suppression used by both FoxP3+ and FoxP3- T regulatory cells. Moreover, we discuss the molecular events leading to the induction of IL-10 secretion in T helper cell subsets, where it acts as a pivotal negative feedback mechanism. Finally we discuss how a greater understanding of this principle has allowed for the design of more efficient, antigen specific immunotherapy strategies to exploit this natural phenomenon clinically

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

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

CD4⁺ T cells from [4Y] treated Tg4^KO mice do not suppress the proliferation of naïve Tg4^WT T cell <i>in vitro</i>.

<p>(<b>A</b>) Experimental design. Splenocytes from Tg4^WT and Tg4^KO mice, treated with [4Y] or PBS, were expanded <i>in vitro</i> with MBPAc1-9[4K] and rhIL-2 for seven days before co-culture with CellTrace-labeled CD4⁺ T cells isolated from naïve Tg4^WT mice. (<b>B</b>) Examples of the secretion of IL-10 and IFNγ by expanded CD4⁺ cells from the indicated mice, following restimulation with PMA and ionomycin. (<b>C, D</b>) Representative flow cytometry data and the computed Division Indices of naïve Tg4^WT cells when stimulated with 0.1, 1 or 10μg/ml [4K] and co-cultured with CD4⁺ splenocytes from Tg4^WT and Tg4^KO mice which had been treated with [4Y] or PBS. Gated on live, CD4⁺, CellTrace⁺ cells. All plots show the mean value +/- SEM. Each point represents data from one [4Y] or PBS-treated mouse assayed individually <i>in vitro</i> and is representative of two experiments. *p<0.05, **p<0.01, ns p>0.05 assessed by ANOVA with Tukey’s correction for multiple comparisons.</p