Elsevier

Immunology Letters

Volume 143, Issue 1, 30 March 2012, Pages 116-121
Immunology Letters

Isolation, expansion and characterisation of alloreactive human Th17 and Th1 cells

https://doi.org/10.1016/j.imlet.2011.12.007Get rights and content

Abstract

Interleukin 17 producing T helper cells (Th17) and IFNγ producing Th1 cells are distinct subsets of effector memory CD4+ T cells that are crucial to host immunity and have been linked to the pathology of certain inflammatory autoimmune diseases. We have developed a method for the isolation and long term culture of human Th17 and Th1 cells. Using allogeneic stimulation we have cultured homogeneous populations of Th17 and Th1 cells to large cell numbers. These alloreactive cell lines were established from CD4+CD45RO+ memory T cells expressing, or lacking, CCR6 and CCR4. The Th17 cells were derived only from cells expressing both CCR6 and CCR4 whereas the Th1 cells, secreting IFNγ, were derived from cells lacking CCR6 and CCR4. The CCR6+ and CCR4+ memory T cells also gave rise to a third population of polyfunctional cells expressing both IL-17 and IFNγ. All cell populations expressed the TCR αβ and the Th17 cells characteristically expressed CCR6, CCR4 and CD161. The use of this protocol will ultimately allow for the comparative analysis of the Th17 and Th1 cells.

Highlights

► Long term, homogeneous cultures of Th17 and Th1 cells were established from CD4+ CD45RO+ memory T cells. ► Th17 are distinguished by expression of CD161, CCR6 and CCR4 whereas Th1 cells lack CCR6 and CCR4 but express CXCR3. ► The Th17 and Th1 lines all exclusively express αβ T cell receptor.

Introduction

CD4+ T helper cells are functionally distinct effector T cells crucial in the orchestration of adaptive immune responses. Subsets of these cells, in particular T helper 1 (Th1) and T helper 2 (Th2), have been extensively characterised since their original discovery [1]. Classically the Th1 cells are characterised by the transcription factor, T-bet and the production of IFNγ, playing a key role in the protection against intracellular pathogens and in the development of autoimmunity [2]. Th2 cells are characterised by transcriptional regulation by GATA-3; as well the secretion of IL-4, IL-5 and IL-13 being central to protection against extracellular pathogens and development of allergic disease [2]. More recently an additional subset of CD4+ T helper cells was identified; the Th17 cells, which are characterised by the transcription factor RORγt and secretion of IL-17A [3], [4], [5], [6]. Th17 cells are critical in normal immune responses to certain gut bacterial and fungal species [7], [8] with the gastrointestinal tract being a key site of Th17 regulation [9]. In addition, murine models of autoimmune inflammation have indicated that Th17 cells are key pathogenic determinants in experimental autoimmune encephalomyelitis [10], systemic lupus erythematosus and inflammatory arthritis [11]. In humans there is increasing evidence of Th17 cells having a key role in the pathogenesis of autoimmune diseases including rheumatoid arthritis [12], [13], multiple sclerosis [14], diabetes [15] and allograft rejection [16].

Human Th17 cells are primarily characterised by the secretion of IL-17A, as well as a range of other cytokines including IL-17F, TNFα, IL-6, IL-22, IL-21, IL-26 and CCL20 [17]. Th17 cells express the transcription factor RORγt which is essential for IL-17A production and overexpression of RORγt induces a Th17 phenotype in naïve CD4+ T cells. However, to induce the Th17 phenotype, RORγt is likely to act in concert with other transcription factors as it alone does not act directly on the IL-17 gene [18].

In healthy humans, Th17 cells constitute 1–2% of all peripheral blood mononuclear cells, making the characterisation and manipulation of these cells difficult. Furthermore, there is no single cell surface marker that distinguishes Th17 cells from other CD4 cell types. Currently, human Th17 cells are most commonly identified by the combined expression of CCR6 and CCR4 together with the intracellular expression of IL-17A and RORγt [19]. CD161 has also been used to define Th17 cells [20] but it too is not exclusively expressed by these cells, limiting its utility in their isolation and characterisation. Thus a major challenge in the field is to define surface molecules that specifically identify Th17 cells.

In this study we have established long-term cultures of Th17 (IL-17A secretion) and Th1 (IFNγ secretion) cells derived from a CD4+ effector memory T cell pool. These cells may provide a viable resource for the discovery of cell surface markers, transcriptional factors and metabolic pathways that are unique to the Th17 cell subset.

Section snippets

Peripheral blood mononuclear cells

Healthy donor peripheral blood mononuclear cells (pBMCs) were isolated from buffy coats obtained from the Australian Red Cross Blood Services (VIC, Australia) using a Ficoll-Paque Plus (GE Healthcare, Australia) gradient. The harvested pBMCs were washed 3 times in DMEM (Gibco, Australia) and then centrifuged at progressively slower speeds (400 × g, 300 × g, 200 × g) to eliminate platelet contamination. The pBMCs were resuspended in fully supplemented DMEM (supplemented with 5% AB negative Human Serum

Establishment of human Th17 and Th1 cell lines

In contrast to other studies which have analysed Th17 cultures derived from naïve CD4 T cells or short term cultures of central or effector memory cells [22], [23], [24] we have established a strategy for the development of long term in vitro cultures of human Th17 and Th1 cells (Fig. 1A). Cells that were positive for chemokine receptors CCR6 and CCR4 (CCR6+CCR4+) or negative for both receptors (CCR6CCR4) were isolated from the pre-committed CD4+ memory T cell pool and expanded in the

Acknowledgements

This work was funded by National Health and Research Council Project grants and the Collaborative Research Centre for Biomarker Translation and the Victorian Operational Infrastructure Scheme. KK is supported by Arthritis Australia. PMH is an NHMRC SPRF. The authors also wish to thank Bruce D. Wines and Janine Stubbs for helpful discussions and comment on the manuscript.

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      The p value for the mean fold difference values between compared samples was calculated with an unpaired two tail t-test, with p value <0.05 indicating statistical significance. We had previously established conditions for the in vitro expansion of human Th17, Th1 and Th17 polyfunctional (Th17pf) cells [32]. In that study, the Th17 and Th1 cells were defined by their commitment to PMA and ionomycin induced expression of intracellular IL-17A and IFNγ, respectively, and in the case of Th17 polyfunctional cells by their capacity to synthesise high levels of both cytokines.

    1

    Burnet Institute, Centre for Immunology, PO Box 2284, Melbourne, VIC 3001, Australia.

    2

    Monash University Central Clinical School, Department of Immunology, Alfred Hospital, Commercial Rd., Melbourne, VIC 3004, Australia.

    3

    Leukocyte Biology Laboratory, Women and Children's Health Research Institute, Women and Children's Hospital, Adelaide, SA 5006, Australia.

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