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

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 in vitro and in vivo. 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.


Introduction
An escalating dose of high-affinity myelin basic protein (MBP)-Ac1-9[4Y] peptide, administered subcutaneously (s.c.) to T cell receptor (TCR) transgenic Tg4 mice, induces peripheral tolerance characterized by the induction of IL-10-secreting CD4 + T cells [1]. These IL-10 + T cells differentiate along the T helper 1 (Th1) pathway, but convert to a regulatory phenotype as part of a negative-feedback loop maintaining peripheral tolerance [2]. Several factors including peptide affinity, dose and solubility and the route of administration used have been shown to play an important role in the successful induction of IL-10 + T cells [3].
In CD4 + T cells protein kinase C theta (PKCθ) is phosphorylated and activated following ligation of the TCR by peptide-MHC. Active PKCθ is required for initiation of NFκB-a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 dependent transcription therefore naïve CD4 + T cells from PKCθ deficient mice fail to proliferate in response to antigen [4]. PKCθ has also been shown to be specifically required for the differentiation of autoimmunity-associated T helper 17 (Th17) cells [5] and to inhibit the function of FoxP3 + regulatory T cells (Treg) [6]. These observations have led some to suggest that PKCθ represents an attractive drug target for inducing immune suppression in the context of transplant and autoimmunity [7][8][9]. However, recent trials of the PKCθ inhibitor sotrastaurin (AEB071), in renal transplant patients, have demonstrated inferior efficacy compared to existing regimes [10,11].
As peptide affinity, and thus strength-of-signal from the TCR, are crucial in determining efficiency of induction of IL-10-secreting T cells from Th1 effector cells [12,13], we hypothesized that altering TCR-mediated signaling by disrupting PKCθ would impact the generation of IL-10 + T cells, and thus the maintenance of peripheral tolerance, following therapeutic peptide administration. We utilized PKCθ-deficient Tg4 mice (Tg4 KO ) to study the role of this signaling pathway on the generation of IL-10-secreting T cells and the induction of a tolerant immune environment following MBP-Ac1-9[4Y] administration.

Ethical statement
All animal experiments were carried out under the UK Home Office Project Licence number 30/2705 held by David Wraith and the study was approved by the University of Bristol ethical review committee.

Mice and peptide treatments
PKCθ-deficient Tg4 mice were generated by cross breeding Tg4 mice [14] with B6.129P2-Prkcq tm1Litt /J mice [4] (originally generated by D. Littman, a gift of A. Poole, Bristol) for at least 8 generations. Expression of the transgenic Vβ8 TCR and H-2 u was confirmed by flow cytometry and deletion of PKCθ was assessed by PCR of genomic DNA isolated from tail or ear tissue as described [4]. Tg4 WT , Tg4 KO and B10.PL mice were bred and maintained under SPF conditions at the University of Bristol with constant access to water and standard lab chow. Male and female mice aged 6-14 weeks were used and were equally distributed between groups based on age and sex. Mice were injected s.c. with PBS or [4Y] peptide in a volume of 200μl of sterile PBS every 3-4 days.
FlowCytomix TM Multiplex (eBioscience) according to the manufacturers instructions. Data was acquired on an LSRII (BD) flow cytometer and analyzed using Flow Cytomix Pro 2.4 software (eBioscience).

Cell isolation
Spleens were disaggregated and red blood cells removed by osmotic lysis. Where indicated, CD4 + T cells were isolated using negative magnetic separation with CD4 + T cell Isolation Kit II (Miltenyi Biotech) or MagniSort™ Mouse CD4 + T cell Enrichment Kit (eBioscience).

RT-PCR
3-5x10 6 isolated CD4 + T cells were stimulated for 18 hours with plate-bound anti-CD3 and anti-CD28 prior to mRNA isolation using an RNeasy Mini Kit, including DNase treatment (QIAGEN). RNA quality and quantity was assessed using a Nanodrop TM 2000 (Thermo Fisher Scientific). Reverse transcription and amplification was carried out using Super-Script III First-strand Synthesis SuperMix for qRT-PCR (Invitrogen). Real-time PCR was performed

Statistical analysis
Graphs were constructed and data tested for statistical significance in GraphPad Prism version 6.0.

Tg4 KO mice have lower serum IL-10 levels following s.c. [4Y] treatment
We administered a previously-described regimen of escalating s.c. doses of [4Y] peptide to Tg4 WT and Tg4 KO mice ( Fig 1A) and measured the concentration of selected cytokines in peripheral blood serum two hours after each treatment, the point of peak cytokine production following peptide administration [1,15]. Most strikingly, concentrations of IL-10 were significantly (up to 100-fold) lower in Tg4 KO mice than Tg4 WT ( Fig 1B). The serum concentration of both IFNγ and IL-2 was equivalent in Tg4 WT and Tg4 KO mice, both showing a peak at the early 80μg doses which was reduced by the final dose, as shown previously [1] (Fig 1C and  1D). In contrast, serum IL-17A and TNFα concentrations were significantly lower in Tg4 KO mice (Fig 1E and 1F). We observed no significant difference in the number of CD4 + T cells or the proportion of non-viable cells recovered from spleens of [4Y] treated Tg4 WT or Tg4 KO mice (S1 Fig), arguing against differences in T cell expansion or cell death between Tg4 WT and Tg4 KO mice as the cause of reduced serum IL-10 concentrations in Tg4 KO mice.

Lower proportion of IL-10 + CD4 + T cells and reduced expression of tolerance-associated markers in [4Y] treated Tg4 KO mice
We next sought to further characterize the T cell compartment in Tg4 WT and Tg4 KO mice treated with [4Y] or PBS. Intracellular cytokine staining showed no difference in the proportion of splenic CD4 + T cells expressing IL-10, IFNγ, IL-2, IL-4, GM-CSF or IL-17A in Tg4 WT and Tg4 KO following PBS treatment. There was also no significant difference in the proportion of IFNγ + , IL-2 + , IL-4 + , GM-CSF + or IL-17A + CD4 + T cells in the spleen of either [4Y] treated Tg4 WT or Tg4 KO mice (Fig 2C-2G). In contrast, the proportion of IL-10 + CD4 + T cells among Tg4 KO splenocytes was significantly lower than in Tg4 WT (4.1% +/-0.7 verses 12.6% +/-1.0), as was the mRNA expression of il10 in isolated Tg4 KO CD4 + T cells (Fig 2B and 2K). We and others have previously identified transcription factors and cell-surface proteins associated with tolerance and specifically with IL-10 + CD4 + T cells [1,16]. Following [4Y] treatment, there was a significantly lower proportion of CD4 + T cells expressing the toleranceassociated transcription factors cMaf and FoxP3 in Tg4 KO splenocytes relative to Tg4 WT ( Fig  2H-2J). Expression of cmaf and nfil3 (e4bp4) mRNA was also approximately 10-fold lower in isolated CD4 + T cells from [4Y] treated Tg4 KO relative to Tg4 WT (Fig 2L and 2M).

CD4 + T cells from [4Y] treated Tg4 KO mice do not suppress naïve T cell activation
A characteristic of IL-10 + T cells induced by treatment with [4Y] is the ability to suppress the priming of naïve CD4 + T cells [1,15]   ml rhIL-2 (as illustrated in Fig 4A). The addition of exogenous IL-2 reverses the anergy of the tolerant T cells, promoting IL-10 secretion, and previous studies have shown that restimulation is necessary to observe optimal suppressive effects in vitro [15]. The proliferation of [4Y] treated Tg4 WT and Tg4 KO cells following restimulation in the presence of IL-2 was equivalent (S2 Fig). The proportion of IL-10 + CD4 + T cells after expansion is shown in Fig 4B and in each condition is comparable to the proportion see directly ex vivo (Fig 2B).
The ability of each T cell population to suppress naive T cells responses was assessed by coculturing CellTrace Violet labeled naïve Tg4 WT CD4 + T cells, expanded CD4 + T cells from treated mice and irradiated B10.PL (wildtype) splenocytes as a source of antigen presenting cells at a ratio of 1:1:2 with 0.1, 1 or 10μg/ml [4K] peptide. After 72 hours, the proliferation of the Tg4 WT responders was assessed by flow cytometry (as illustrated in Fig 4A). Tg4 WT [4Y] treated cells significantly suppressed the proliferation of the responder cells relative to Tg4 WT PBS treated cells at all concentrations of antigen tested (Fig 4C and 4D). In contrast, [4Y] treated Tg4 KO cells failed to suppress responder cell proliferation, regardless of the concentration of antigen. We considered that Tg4 KO cells could fail to suppress if they had died upon restimulation. Analysis of the proportion of live cells recovered at the end of the co-culture demonstrated that the [4Y] treated Tg4 WT and Tg4 KO cells were similarly viable following restimulation (S2B Fig). Previous work has demonstrated discrepancies between mechanisms of Treg-mediated suppression when assayed in vitro and in vivo [15]. We therefore tested the suppressive capacity of [4Y] treated Tg4 KO cells in vivo. Cell Proliferation Dye-labeled naïve Tg4 WT cells were    the transferred responder cells and the proportion of undivided cells in the [4Y] treated Tg4 KO mice was not significantly higher than in PBS-treated Tg4 KO mice and was significantly lower than in [4Y] treated Tg4 WT mice (Fig 5B and 5C).

Discussion
The aim of this study was to determine if the signaling protein PKCθ plays a role in the induction of suppressive IL-10 + T cells during a tolerance induction protocol of self-antigen administration. IL-10 plays an essential role in the regulation of immune responses and it is therefore important to understand the mechanisms by which it is induced [17]. Furthermore, if PKCθ is to be considered a suitable drug target in immune-mediated diseases [7,9], it is essential to understand the implications that inhibiting this kinase may have on all immunoregulatory processes.
We demonstrate that PKCθ knock out TCR-transgenic mice fail to upregulate IL-10 following the sequential administration of escalating doses of a high-affinity variant of the relevant cognate antigen, a well-described protocol for the induction of IL-10 + T cells [1,2,18]. Tg4 KO mice show significantly reduced serum concentrations of IL-10 and IL-17A following peptide treatment, but concentrations of the Th1-associated cytokines IFNγ and IL-2 are equivalent in Tg4 WT and Tg4 KO mice. We also observe a reduced proportion of IL-10 + CD4 + splenocytes in Tg4 KO mice compared to Tg4 WT , but proportions of IFNγ + and IL-2 + CD4 + T cells are unaffected by PKCθ deficiency. This is consistent with observations that PKCθ is required for the differentiation of Th17 but not Th1 cells [5,19]. The transcription factor cMaf is essential for IL-10 transcription in macrophages and upregulation of cMaf is associated with IL-10 expression in CD4 + T cells [1,20]. Similarly, FoxP3 and Nfil3 have both been shown to regulate IL-10 transcription [21]. cMaf is strongly induced in [4Y] treated Tg4 WT mice, but is not upregulated in Tg4 KO mice CD4 + T cells. FoxP3 is induced in a small proportion of Tg4 WT cells, but not in Tg4 KO cells. Similarly, mRNA expression of Nfil3 is approximately 4-fold lower in [4Y] treated Tg4 KO cells compared to Tg4 WT . As il10 mRNA levels are also significantly reduced in Tg4 KO cells, it suggests that the transcription factors required to drive expression of IL-10 are not induced in Tg4 KO cells and that this contributes to the reduced levels of IL-10 observed.
The mechanism by which Nfil3 is induced in CD4 + T cells remains unknown. In natural killer cells, induction of Nfil3 is dependent on 3 0 -phosphoinositide-dependent kinase 1 (PDK1) [22], a known substrate of PKCθ [22,23].This suggests a possible pathway downstream of PKCθ that could lead to Nfil3 expression in CD4 + T cells. It is similarly unclear what links PKCθ activation to the induction of cMaf expression. Under conditions favoring the development of Th17 cells, induction of cMaf in CD4 + cells is dependent upon Stat3 [24] and PKCθ has been shown to induce expression of Stat3 via NFκB and AP-1 [5], providing a possible link between PKCθ and cMaf.
We also observed altered expression of tolerance-associated co-inhibitory molecules in Tg4 KO CD4 + cells following [4Y] treatment. Although PD-1 was induced on Tg4 KO CD4 + T cells, it was on a lower proportion of cells than in Tg4 WT mice. Tim3 was not induced on Tg4 KO cells but expression of Lag3 and TIGIT was, on average, unaffected by PKCθ deficiency. The mechanisms of action and the relative roles of these co-inhibitory receptors is poorly defined [25], but this data suggest that each may be induced via different stimuli, some dependent of PKCθ and some not. The failure of Tg4 KO cells to upregulate Tim3 may be explained by the deficient Nfil3 expression in these cells as Nfil3 has been shown to be required for Tim3 expression in IL-27-induced regulatory cells [26].
We have previously demonstrated that [4Y] treatment protects Tg4 WT and B10.PL non-TCR transgenic mice from experimental autoimmune encephalomyelitis (EAE) [1]. Due to the impact of PKCθ deficiency on CD4 + T cell priming and Th17 differentiation, PKCθ knockout mice are generally resistant to EAE induction [27]. These confounding factors make it difficult to test the ability of Tg4 KO [4Y]-induced IL-10 + T cells to suppress T cell activation in this disease model. Instead we chose to assess the capacity of these cells to prevent Tg4 WT cell activation in in vitro co-cultures and in an adoptive transfer model of suppression. We demonstrate that [4Y] treated Tg4 KO T cells are unable to suppress the proliferation of naïve Tg4 WT cells in both in vitro in co-cultures and in vivo following adoptive transfer of responder cells to pre-treated Tg4 KO mice. PKCθ is required for efficient FoxP3 + Treg induction [28] and functions to modulate the activity of thymically-derived Treg [6]. Given these observations we cannot exclude the possibility that altered induction or function of FoxP3 + Treg is responsible for the reduced suppressive activity we observe in T cells from 4Y-treated Tg4 KO mice. However, we have previously excluded a role for FoxP3+ Treg in suppression following 4Y treatment [29], supporting our hypothesis that defective IL-10 induction from Tr1-like cells is the cause of reduced suppression in Tg4 KO mice.
We observe that CD4 + T cells from [4Y] treated Tg4 KO mice lack only some of the tolerance-associated characteristics of [4Y] treated Tg4 WT cells (of those measured, specifically IL-10, cMaf, Nfil3 and Tim3) and this may provide information about which molecules mediate the cells suppressive ability. Induction of Lag3 and TIGIT is unaffected by PKCθ deficiency, but the cells are unable to suppress proliferation of the responders, suggesting that these proteins do not play a significant role in the suppression of naïve T cell responses in this system.
When interpreting the experiments presented here it is important to consider the role that PKCθ plays in all aspects of immune biology and how this may impact the induction of IL-10 + T cells. For example, PKCθ is required for efficient positive selection of T cells in the thymus [30]. Even in the context of a transgenic TCRαβ a second endogenous α-chain can be selected and variation in this repertoire as a result of PKCθ deficiency may alter the behavior of CD4 + T cells in the periphery. Until recently, PKCθ expression was believed to be restricted to the lymphoid lineage. A recent study has described expression of PKCθ mRNA in macrophages and defined a role for PKCθ in the response of macrophages to infection [31]. This opens the previously unappreciated possibility that PKCθ-deficiency may impact other aspects of the myeloid lineage, innate-derived cytokine production and potentially antigen presentation.
This work presented here demonstrates that PKCθ is required for the efficient induction of IL-10 in effector T cells following administration of an escalating dose of self-antigen to TCRtransgenic mice. This should be considered when contemplating PKCθ as a suitable drug target for promoting immune tolerance and the impact of PKCθ inhibitors, including sotrastaurin, on the induction of IL-10 + CD4 T cells should be investigated.