Role of Wnt3a expressed by dendritic cells in the activation of canonical Wnt signaling and generation of memory T cells during primary immune responses

doi:10.1016/j.cellimm.2016.08.005

Cellular Immunology

Volume 310, December 2016, Pages 99-107

https://doi.org/10.1016/j.cellimm.2016.08.005 Get rights and content

Highlights

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mDCs had a higher expression level of Wnt3a compared to imDCs.
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Wnt3a expressed in DCs influenced Wnt canonical pathway activation in TNs.
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Wnt3a expressed in DCs influenced the TNs differentiate towards the TMs subtype.
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Wnt3a expressed in DCs influenced its cytokines production.

Abstract

The presence of memory T cells (T_Ms) hinders transplant survival. Dendritic cells (DCs) induce the generation of T_Ms during primary immune responses. However, the specific mechanisms are unclear. In this study, we constructed a Wnt3a-expressing adenovirus and used small interfering RNA (siRNA) targeting Wnt3a to investigate the influence of Wnt3a expression in DCs on the generation of T_Ms during primary immune responses. Our results demonstrated that the Wnt3a expression levels in DCs influenced the generation of T_Ms after 5 days in co-culture with naïve T cells through activation of the Wnt canonical pathway. Interleukin-7 secretion levels in supernatants of DC/T_Ns co-cultures showed a similar pattern of Wnt3a expression levels in DCs. These findings provide a better understanding of T_Ms generation mechanisms that might be useful to improve transplant outcomes.

Introduction

A severe shortage of donor organs and the use of immunosuppressive agents that cannot ensure long-term functional survival of transplants in recipients are still major challenges in solid organ transplantation. Hence, induction of transplant immunological tolerance is the ultimate goal, which has been pursued by clinicians for decades [1].

Approximately 40–50% of T cells have a memory phenotype in adult peripheral blood [2]. Memory T cells (T_Ms) can be generated during transplantation and are important for protective immunity because of their capacity to initiate rapid and potent recall responses against invading bacterial and viral pathogens [3], [4], which should provide an evolutionary advantage for transplant survival. However, T_Ms can cross-react with allogeneic antigens, inducing antigen-specific protective immunity [5], [6] and exerting a potentially contradictory effect on transplant tolerance and outcomes. Unlike naïve T cells (T_Ns), activation of alloreactive T_Ms is less dependent on co-stimulation signals and has lower thresholds [7]. Importantly, T_Ms are less susceptible to most conventional immunosuppressants [8], which hinders long-term transplant survival. The mechanisms for the generation of T_Ms from T_Ns or effector T cells (T_Es) are not yet fully understood. Our previous study described four models for T_Ms generation [9]. The most likely direct source of alloreactive T_Ms is T_Ns stimulated by alloantigens during primary immune responses [10].

Wnt ligands are a family of 19 secreted glycoproteins that play key roles in cell fate determination, proliferation, differentiation, and migration [11]. Dendritic cells (DCs) are the most effective antigen-presenting cells (APCs) of the immune system [12]. DC properties of either initiating immune responses or inducing immune tolerance mainly depend on two general functional states, immature (imDCs) and mature (mDCs) [13]. A recent study by Oderup et al. demonstrated that exogenous Wnt3a treatment of DCs triggers activation of β-catenin and the canonical Wnt signaling pathway [14]. Valencia et al. found that human T_Ns express Wnt receptors and respond to Wnt5a during stimulation [15]. Furthermore, Suryawanshi et al. concluded that low density-related lipoprotein receptor 5/6 (LRP5/6)-mediated activation of β-catenin in DCs regulates differentiation of T_Ns into Th1 and Th17 cells, which may be achieved through regulation of DC-specific production of cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-10 [16]. Blockade of β-catenin accumulation inhibits T cell proliferation and interferon (IFN)-γ secretion during DC/T cell co-culture [17]. Thus, Wnt signaling plays an important role in the proliferation and differentiation of T_Ns. Because T_Ms can be generated from T_Ns during primary immune responses by stimulation of APCs loaded with alloantigens [9], [10], we assumed that Wnt signaling in DCs might play a role in this process.

Thus far, there have been no studies on Wnt3a expression in imDCs and mDCs, and its effect on activation of the Wnt signaling pathway in T cells and the generation of T_Ms during co-culture of DCs and T_Ns. In this study, we generated a Wnt3a-expressing adenovirus and small interfering RNA (siRNA) targeting Wnt3a to investigate whether Wnt3a expression in DCs induces activation of the canonical Wnt signaling pathway in T cells and influences the differentiation of T_Ns into T_Ms.

Section snippets

Animals

C57BL/6 (H-2Kb) and BALB/c (H-2Kd) mice weighing 25–30 g and aged 8–10 weeks were obtained from the Experimental Animal Center of Sichuan University (Sichuan, China). All animal experiments were performed according to protocols approved by the Institution Animal Care and Use Committee.

Cell isolation and culture

imDCs were generated from bone marrow (BM) cells of BALB/c mice. CD4⁺ T_Ns (CD44^-CD62L⁺) derived from C57BL/6 mice were isolated by negative magnetic selection using a MagCellect Mouse Naive CD4⁺ T Cell Isolation Kit

Ad.CMV.Wnt3a induces stable expression of Wnt3a in imDCs

Wnt3a is a prototypical activator of Wnt/β-catenin signaling. An adenovirus expressing Wnt3a was generated to up-regulate the expression of Wnt3a in imDCs. Our results showed that imDCs transduced with Ad.CMV.Wnt3a had nearly 4-fold higher Wnt3a mRNA levels compared with other imDC groups (untransduced or transduced with Ad.CMV.LacZ), which was even higher than that in the mDC group at 3 days after transduction (p < 0.05) (Fig. 1A). In addition, after 5 days of transduction, immunofluorescence

Discussion

To date, transplantation remains the only effective therapeutic regimen for patients with end-stage organ failure. T_Ns and T_Ms are major T cell subsets that play important roles in the development of transplantation rejection, autoimmunity, and transplant survival. There has been increasing interest in T_Ms as formidable barriers of transplant tolerance and long-term graft survival [23]. A previous study has described three possible mechanisms for the generation of alloreactive T_Ms in the

Authors’ contributions

L.L. and Z.Y.L. conceived the study, established the design, and carried out the experimental work. G.H.L., Y.T.Z., and J.Y. participated in the data analysis and provided critical comments on the study design and manuscript. H.C. drafted the manuscript. All authors read and approved the final manuscript.

Conflicts of interest

None of the authors has financial interests or commercial associations that might create conflicts of interest.

Acknowledgments

This work was supported by the Guizhou Province Science and Technology Agency Fund (grant number LH[2014]7034); and Natural Science Foundation of China (grant number 31360222).

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¹: Co-first authors.

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Research paperRole of Wnt3a expressed by dendritic cells in the activation of canonical Wnt signaling and generation of memory T cells during primary immune responses

Highlights

Abstract

Introduction

Section snippets

Animals

Cell isolation and culture

Ad.CMV.Wnt3a induces stable expression of Wnt3a in imDCs

Discussion

Authors’ contributions

Conflicts of interest

Acknowledgments

Am. J. Transplant.

Immunol. Lett.

Immunol. Lett.

Immunity

Immunol. Lett.

Cell. Immunol.

Cell

Cell

Immunity

Curr. Opin. Immunol.

Transplantation 50 years later–progress, challenges, and promises

N. Engl. J. Med.

Identification of a human recent thymic emigrant phenotype

Proc. Natl. Acad. Sci. U.S.A.

Donor-reactive T-cell stimulation history and precursor frequency: barriers to tolerance induction

Transplantation

Are prime alloresponses truly primary?

Int. Immunol.

Critical, but conditional, role of OX40 in memory T cell-mediated rejection

J. Immunol.

Memory T Cells in Transplantation

Curr. Transplant Rep.

Transcriptional control of effector and memory CD8+ T cell differentiation

Nat. Rev. Immunol.

Towards an integrated view of Wnt signaling in development

Development

Research paper
Role of Wnt3a expressed by dendritic cells in the activation of canonical Wnt signaling and generation of memory T cells during primary immune responses