Elsevier

Molecular Immunology

Volume 45, Issue 10, May 2008, Pages 2888-2896
Molecular Immunology

Perturbed thymopoiesis in vitro in the absence of suppressor of cytokine signalling 1 and 3

https://doi.org/10.1016/j.molimm.2008.01.024Get rights and content

Abstract

Cytokine signals are central to the differentiation of thymocytes and their stepwise progression through defined developmental stages. The intensity and duration of cytokine signals are regulated by the suppressor of cytokine signalling (SOCS) proteins. A clear role for SOCS1 during the later stages of thymopoiesis has been established, but little is known about its role during early thymopoiesis, nor the function of its closest relative, SOCS3. Here, we find that both SOCS1 and SOCS3 are expressed during early thymopoiesis, with expression coincident during the double negative (DN)2 and DN3 stages. We examined thymocyte differentiation in vitro by co-culture of SOCS-deficient bone marrow cells with OP9 cells expressing the Notch ligand Delta-like1 (OP9-DL1). Cells lacking SOCS1 were retarded at the DN3:DN4 transition and appeared unable to differentiate into double positive (DP) thymocytes. Cells lacking both SOCS1 and SOCS3 were more severely affected, and displayed an earlier block in T cell differentiation at DN2, the stage at which expression of SOCS1 and SOCS3 coincides. This indicates that, in addition to their specific roles, SOCS1 and SOCS3 share overlapping roles during thymopoiesis. This is the first demonstration of functional redundancy within the SOCS family, and has uncovered a vital role for SOCS1 and SOCS3 during two important checkpoints in early T cell development.

Introduction

The function of the thymus is to generate T lymphocytes expressing T cell receptors of sufficient diversity to combat challenge by every possible microorganism, while eradicating potentially autoreactive T cells. Thymocyte progenitor cells progressively mature in the thymus through a series of well-defined stages that can be distinguished by differential expression of cell surface markers (Godfrey et al., 1993). Immature double negative (CD4CD8, DN) thymocytes can be classified into four stages according to their expression of CD25 and CD44; DN1 (CD25 CD44+), DN2 (CD25+ CD44+), DN3 (CD25+ CD44) and DN4 (CD25 CD44) (Godfrey et al., 1993). Expression of both CD4 and CD8 is then upregulated (double positive, DP), and those thymocytes surviving selection proceed to the mature single positive (SP) stage and enter peripheral lymphoid tissues. Recently, a culture system has been developed for in vitro differentiation of T cells. Co-culture of progenitor cells with OP9 cells expressing the Notch ligand Delta-like1 (OP9-DL1) has been shown to induce differentiation through all stages of thymopoiesis to SP cells, and is therefore a useful model for studying T lymphopoiesis in vitro (Schmitt and Zuniga-Pflucker, 2002).

Cytokines play an essential role in T cell development, particularly during the early stages of thymopoiesis (Yarilin and Belyakov, 2004). IL-7, in particular, is critical, as T cell development is severely impaired in mice lacking either IL-7 or the IL-7 receptor (Peschon et al., 1994, von Freeden-Jeffry et al., 1995). Cytokines induce highly potent signals that can disrupt immune homeostasis in the absence of appropriate regulation (Fehniger et al., 2001, Kopf et al., 1994, Sadlack et al., 1993, von Freeden-Jeffry et al., 1995). The suppressor of cytokine signalling (SOCS) proteins are key regulators of cytokine signalling (Alexander and Hilton, 2004). Of the eight members of the SOCS family, SOCS1 and SOCS3 appear to be the most important for the regulation of T cell development and function (Fletcher and Starr, 2005). In addition, SOCS1 and SOCS3 are more closely related to each other than to other family members, and share similar functions in vitro, indicating that they may also have overlapping roles in vivo (Starr et al., 1997).

SOCS1 is highly expressed in the thymus (Chong et al., 2003). Overexpression of SOCS1 in T cells in vivo leads to a profound reduction in thymic cellularity, due to a block in development at the TN2:TN3 transition (Fujimoto et al., 2000). This phenotype is reminiscent of mice lacking the common γ chain receptor subunit shared by IL-7, IL-15 and other cytokines (Cao et al., 1995), suggesting that SOCS1, when overexpressed, regulates thymopoiesis by limiting responses to these cytokines. Similarly, studies of SOCS1-deficient mice have suggested that SOCS1 is critical for regulating both IL-7 and IL-15 signalling in the thymus (Chong et al., 2003, Cornish et al., 2003b, Ilangumaran et al., 2003).

Comparatively little is known about the role of SOCS3 during T cell development. SOCS3 is expressed in peripheral T cells and regulates responses to the gp130 cytokines IL-6 and IL-27 (Brender et al., 2007). Overexpression of SOCS3 in vivo has little impact on thymopoiesis, with thymic subsets represented in the appropriate ratio, but overall thymic cellularity is slightly reduced in these mice (Matsumoto et al., 2003).

Here, we assess the roles of SOCS1 and SOCS3 during thymopoiesis, both by analysing early thymic differentiation in SOCS-deficient mice in vivo, and using the OP9-DL1 co-culture system in vitro. We find that despite apparently normal thymopoiesis in vivo in the absence of SOCS proteins, progression through the DN3:DN4 and DN:DP stages of thymopoiesis was retarded in SOCS1-deficient cells in vitro. These defects were compounded by the additional removal of SOCS3, leading to an earlier block in differentiation at the DN2:DN3 transition, a stage at which SOCS1 and SOCS3 are co-expressed. This is the first demonstration of shared functions between SOCS family members, and suggests that both SOCS1 and SOCS3 regulate critical checkpoints during early thymopoiesis.

Section snippets

Mice

Socs1ΔLckLck, Socs3ΔLckLck, Socs1+/−, Socs3+/−, Socs1−/−Ifnγ−/− and Ifnγ−/− (obtained from The Jackson Laboratory, Bar Harbor, ME) mice on a C57BL/6 background have previously been described (Alexander et al., 1999, Brender et al., 2007, Chong et al., 2003, Dalton et al., 1993, Roberts et al., 2001, Starr et al., 1998). Mice floxed at both Socs1 and Socs3 loci were generated by breeding Socs1fl/fl mice with Socs3fl/fl mice to produce progeny containing two floxed alleles at each locus (Socs1

Expression of SOCS1 and SOCS3 is coincident during early thymopoiesis

The expression of SOCS1 in the thymus has been well characterised. SOCS1 is expressed throughout thymocyte development, with expression highest in the DP subset (Chong et al., 2003, Trop et al., 2001). Although SOCS3 expression has been studied in peripheral T lymphocytes and during T cell activation (Matsumoto et al., 2003, Yu et al., 2003), little is known of SOCS3 expression during thymopoiesis. We used SOCS1+/− and SOCS3+/− mice to examine the expression of SOCS1 and SOCS3 during early

Discussion

Cytokines play crucial regulatory roles throughout the life of a T lymphocyte, from controlling early events during thymopoiesis to regulating T cell activation and homeostasis in the periphery (Hunter, 2005, Malek et al., 1999, Marsden et al., 2006). These are finely tuned signals that are modulated by a variety of mechanisms, including inhibition of cytokine signalling pathways by the SOCS proteins (Alexander and Hilton, 2004).

Both SOCS1 and SOCS3 appear to play key roles in regulating

Acknowledgements

The authors thank Melanie Rowe and Rebecca Branch for animal husbandry, Catherine Li for cell sorting assistance, Daniela Novembre-Cycon for 5-FU injections, Wei for construction of the MSCV-Cre-IRES-eGFP retrovirus and Drs. Sebastian Carotta and Marnie Blewitt for advice regarding retroviral infection. This work was supported by the Australian National Health and Medical Research Council (Program Grant 461219) and NIH RO1 CA22556; R.S. was supported by a Sylvia and Charles Viertel Senior

References (46)

  • S. Ilangumaran et al.

    Suppressor of cytokine signaling 1 attenuates IL-15 receptor signaling in CD8 + thymocytes

    Blood

    (2003)
  • T.R. Malek et al.

    Multiple gamma c-dependent cytokines regulated T-cell development

    Immunol. Today

    (1999)
  • J.C. Marine et al.

    SOCS3 is essential in the regulation of fetal liver erythropoiesis

    Cell

    (1999)
  • B. Sadlack et al.

    Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene

    Cell

    (1993)
  • T.M. Schmitt et al.

    Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro

    Immunity

    (2002)
  • S. Trop et al.

    Overexpression of suppressor of cytokine signaling-1 impairs pre-T-cell receptor-induced proliferation but not differentiation of immature thymocytes

    Blood

    (2001)
  • L. Van Parijs et al.

    Uncoupling IL-2 signals that regulate T cell proliferation, survival, and Fas-mediated activation-induced cell death

    Immunity

    (1999)
  • U. von Freeden-Jeffry et al.

    The earliest T lineage-committed cells depend on IL-7 for Bcl-2 expression and normal cell cycle progression

    Immunity

    (1997)
  • C.R. Yu et al.

    Suppressor of cytokine signaling 3 regulates proliferation and activation of T-helper cells

    J. Biol. Chem.

    (2003)
  • W.S. Alexander et al.

    The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response

    Annu. Rev. Immunol.

    (2004)
  • G. Balciunaite et al.

    The role of Notch and IL-7 signaling in early thymocyte proliferation and differentiation

    Eur. J. Immunol.

    (2005)
  • J. Buckland et al.

    Co-ordination of the expression of the protein tyrosine kinase p56(lck) with the pre-T cell receptor during thymocyte development

    Eur. J. Immunol.

    (2000)
  • Z. Chen et al.

    Selective regulatory function of Socs3 in the formation of IL-17-secreting T cells

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

    (2006)
  • View full text