Progranulin Inhibits Human T Lymphocyte Proliferation by Inducing the Formation of Regulatory T Lymphocytes

We have examined the effect of progranulin (PGRN) on human T cell proliferation and its underlying mechanism. We show that PGRN inhibits the PHA-induced multiplication of T lymphocytes. It increases the number of iTregs when T lymphocytes are activated by PHA but does not do so in the absence of PHA. PGRN-mediated inhibition of T lymphocyte proliferation, as well as the induction of iTregs, was completely reversed by a TGF-β inhibitor or a Treg inhibitor. PGRN induced TGF-β secretion in the presence of PHA whereas it did not in the absence of PHA. Our findings indicate that PGRN suppresses T lymphocyte proliferation by enhancing the formation of iTregs from activated T lymphocytes in response to TGF-β.


Introduction
Regulatory T lymphocytes (Tregs) play a pivotal role in preventing autoimmune reactions and regulating immune responses [1,2]. Tregs are classified into two types. Naturally occurring Tregs (nTregs) are thymic in origin and suppress self-recognition T cells in secondary lymphoid tissues [3]. On the other hand, induced Tregs (iTregs) are formed in peripheral lymphoid tissues under the influence of TGF- [4]. It has been reported that nTregs are generated to prevent autoimmune diseases, whereas iTregs are thought to control chronic inflammation [5]. Since iTregs are critical for modulating immune responses, understanding of how they are derived from naïve T lymphocytes and how they exert their effects is important because of their therapeutic potential [6][7][8].
Progranulin (PGRN) is a secreted glycoprotein that was first isolated from a teratoma prostate cancer cell line [9][10][11]. PGRN is a cysteine-rich protein that consists of 593 amino acids and can be converted into granulin (GRN) by extracellular proteases, including proteinase 3 and neutrophil elastase [12][13][14]. It is made up of 7.5 repeats of the GRN domain, and GRN was actually discovered before PGRN was cloned [14].
PGRN is widely expressed in cells such as hematopoietic cells, neurons, and immune cells such as macrophages, microglia, dendritic cells, and T lymphocytes [15]. PGRN is a growth factor and also promotes regeneration and tumorigenesis [16][17][18]. It plays important roles in wound healing [19], host defense, and inflammatory responses [20] and has neurotrophic properties [11,21]. Full-length PGRN has an antiinflammatory effect, while GRN is thought to have proinflammatory properties [14,22]. Nevertheless, PGRN also has proinflammatory effects in specific diseases like obesity and insulin-resistant diabetes. In such cases, its proinflammatory effect is exerted by the production of IL-6 [23]. Therefore, the exact effect of PGRN varies depending on the pathological context.
The role of PGRN in the immune system is not clearly understood, although there is evidence that it is critical for modulating both innate and adaptive immunity [19,[24][25][26][27]. Thus, PGRN reduces TNF--induced IL-8 secretion [22] and binds to the tumor necrosis factor receptor (TNFR) blocking the TNF signal. Hence PGRN protects Tregs from negative regulation by TNF-in humans [26]. Moreover, PGRN induces the formations of Tregs in mice [27]. However, exact inhibitory role of PGRN in human immune cells remains uncertain, and in the present work we examined its immune       Cell proliferation (% of control)

Statistical Analysis.
All experiments were performed at least in triplicate, and the results are shown as means ± standard deviations. The statistical significance of differences was tested with GraphPad Prism 5 software (GraphPad software, La Jolla, CA, USA), using one-way ANOVA and Tukey's post hoc HSD test. values less than 0.05 were considered statistically significant. Similar results were obtained in three to five independent experiments.

PGRN Inhibits Human T Cell Proliferation.
To see whether PGRN suppresses T cell proliferation, hPBMCs were treated with PHA in the presence or absence of PGRN. As shown in Figure 1(a), PGRN inhibited T cell proliferation in a concentration-dependent manner. To confirm that this inhibition occurs in both total T cells and CD4 + T cells, we purified CD3 + or CD4 + T lymphocytes and stimulated them with PHA in the absence or presence of PGRN. As shown in Figures 1(b) and 1(c), PGRN reduced the proliferation of CD3 + and CD4 + T lymphocytes.

PGRN Increases the Number of iTregs.
To test whether PGRN promotes the production of Tregs as reported in mice [27], we examined its effect on numbers of iTregs produced from activated hPBMC in response to PHA. PGRN increased the number of CD4 + Foxp3 + iTregs in a concentrationdependent manner, but it did not induce the frequency of iTreg in the whole CD4 + T population (Figure 2(a)). It is noteworthy that PGRN had no effect on iTreg formation from hPBMCs in the absence of PHA (Figure 2(b)).

PGRN Suppresses the Proliferation of T Lymphocytes through iTregs.
To examine whether the iTreg formation stimulated by PGRN was responsible for the inhibition of T cell proliferation, we incubated CD4 + T lymphocytes with PHA plus PGRN in the presence or absence of various concentrations of a Treg inhibitor or a TGF-inhibitor. Peptide P60, a Treg inhibitor, and the TGF-inhibitor almost completely reversed the PGRN-induced inhibition of CD4 + T cell proliferation as well as Treg formation (Figures 3(a)  and 3(b)). This indicates that the iTregs induced by PGRN suppress PHA-mediated CD4 + T cell proliferation. Our data also suggest that PGRN stimulates the formation of iTregs from activated CD4 + T cells. To confirm that PGRN enhances TGF-secretion, we incubated CD4 + cells with or without PGRN in the presence or absence of PHA and measured TGFlevels in the culture media. As shown in Figure 4, PGRNinduced TGF-secretion in the presence of PHA whereas it did not in the absence of PHA. These data indicated that CD4 + cell stimulation was prerequisite for PGRN-mediated TGF-secretion. In Figure 5, incubating CD4 + T cells with TGF-inhibited CD4 + cell proliferation and induced iTreg formation as comparable as with PGRN.

Discussion
In this work we first demonstrated that PGRN suppressed the proliferation of human T lymphocytes by enhancing their conversion into iTregs and went on to show that inhibition of iTreg formation with Peptide P60 completely reversed the immunosuppressive effect of PGRN. Moreover blocking TGF-signaling, which is pivotal for iTreg differentiation, also completely abolished the effect of PGRN on T lymphocytes. Also, PGRN did not promote iTreg formation from T lymphocytes that were not incubated with PHA. This indicates that PGRN does not act on naïve T cells or on fully differentiated Tregs. Based on these results, we conclude that PGRN acts on activated T lymphocytes to enhance their    conversion into iTregs rather than on already-differentiated iTregs. This idea is supported by a previous study showing that PGRN promoted the TGF--mediated formation of mouse iTregs [27].
PGRN-binding proteins have been studied in different tissues under various conditions. Sortilin has been reported as a binding protein for PGRN. The interaction between sortilin and PGRN is thought to be crucial to regulate PGRN trafficking in neurons [28,29]. In addition, Zhou et al. found that prosaposin interacted with PGRN and facilitated sortilin-independent PGRN trafficking via the cationindependent mannose 6-phosphate receptor [30].
Moreover, it has been reported that PGRN binds to TNF receptors [26]. Although Chen et al. failed to demonstrate these interactions in their assay, recent studies have provided independent evidences that confirm the interactions of PGRN with TNFR in various cell types, including human lymphocytes [31][32][33][34].
Therefore, the identity of the target of PGRN and how it acts remain to be uncovered. Although we do not know the exact mechanism(s) by which PGRN promotes iTreg formation, we suggest the following model: (1) the PGRN receptor (PGRNR) is expressed on T lymphocytes only when they are activated or on iTregs induced by PHA; (2) PGRN/PGRNRmediated intracellular signaling increases TGF-expression and secretion; (3) this, in turn, induces Treg formation. Identification of the PGRNR and its intracellular signal transduction pathway will be critical for understanding its immunosuppressive action.
In conclusion, we have shown that PGRN has the ability to inhibit the proliferation of human CD4 + T cells by inducing them to differentiate into iTregs as a result of TGF-production. Our findings suggest that PGRN might be used to treat autoimmune diseases or chronic inflammatory disease or to facilitate allogeneic stem cell transplantation.