Special regulatory T cell review: The resurgence of the concept of contrasuppression in immunoregulation

Abstract

The original concept of contrasuppression (CS) is evident in many immunoregulatory mechanisms. Inhibition of suppressor activity – CS – may be critical in microbial infection and autoimmunity. The major cellular interactions involved in suppression are the CD25⁺ FoxP3⁺ CD4⁺ T regulatory cells, programmed death-1 (PD-1) : PD-L1/L2 and cytotoxic T lymphocyte antigen-4 (CTLA-4) : CD80/86 pathways. These cellular functions are affected by dendritic cells (DC) and a complex array of cytokines of which interleukin (IL)-2, IL-10, IL-6 and transforming growth factor-β (TGF-β) are especially significant. Inhibition of regulatory cells, suppressor pathways or cytokines, is consistent with CS and can be attributed to IL-6, IL-2, PD-1 or PD-L-1 antibodies, blockade of CTLA-4 : CD80/86 pathway, inhibition of CD40–CD40L pathways, and TGF-β, IL-10 antibodies. Contrasuppression may regulate innate immunity by Toll-like receptor expressed not only in non-cognate DC, monocytes, natural killer cells and γδ T cells but also in adaptive T cells. Furthermore, cross-talk between innate and adaptive immunity may be facilitated by contrasuppressor activity.

‘‘What’s in a name?

That which we call a rose by any other name would smell as sweet.’’ From Romeo and Juliet (II, 47–8) W. Shakespeare

Background

The concept of contrasuppression (CS) was introduced by Richard Gershon and his colleagues a quarter century ago as a novel immunoregulatory activity which inhibits suppressor T-cell functions in mice.¹ The experimental approach taken was that the genetic program of immunologically competent cells express unique surface molecules that carry functional information.² The Ly-1⁺ 2^−* phenotype was programmed to induce effector cells, which in turn induce antibodies, delayed hypersensitivity and cytotoxic functions.^2,3 Positive signals produced by most Ly1⁺ cells, however also contained a small subset of I–J^+* cells whose function was to activate suppressor cells, which counteracted the Ly-1⁺ cells⁴. The Ly1⁻ 2^+* cells, functioned predominantly to suppress immune responses, but a small subset of Ly1⁻2⁺ I–J⁺ cells induced acceptor cells that were Ly1⁺ 2⁺I–J⁺. This phenotype distinguished it from T helper cells.

Both the inducer cell or its biologically active mediator and its acceptor cell were required for the expression of CS. As contrasuppressor cells could block the suppressive activity of cell-free mediators released by Ly2 suppressor T cells, the mechanism of CS was either separate from or in addition to the inactivation of suppressor cells themselves. The effect of suppressor cells on both helper and effector T cells was inhibited interfering with cell-mediated functions, such as delayed hypersensitivity.^5,6 CS have been studied in antibody production,^1,7,8 autoimmunity,⁹ oral tolerance,¹⁰ and tumour regression.¹¹ The cells secrete an antigen-specific CS factor which appeared to have comparable functions to those of the cells of origin.¹² Surprisingly for its time, a murine T-cell hybridoma was generated, which constitutively released CS factor specific for the TNP hapten.¹³ Both, suppressor cells and CS cells generated corresponding suppressor and CS-factors; the suppressor factors inhibited the CS effector T cells in an adoptive cell transfer assay, and the CS-factors protected the effector T cells from the suppressor T cells.¹⁴ The lectin Vicia villosa played an important part in these experiments as the lectin binds CS cells and was used to separate these cells.¹⁵

The human counterpart of CS followed a series of cell depletion and reconstitution experiments involving CD4⁺ and CD8⁺ T-cell interactions.^16,17 As in the mouse experiments, human CD8⁺ T cells were separated on VV-coated plates into VV adherent and non-adherent cells. Reconstitution of CD4⁺ T cells with CD8 VV⁺ cells and streptococcal antigen elicited significant helper factors, which was not evident when unfractionated CD8⁺ or CD8 VV⁻ cells were used. Moreover, a dose-dependent contrasuppressor activity was demonstrated with CD8⁺ VV⁺ T cells in the presence of CD8⁺ VV⁻ suppressor T cells, using radioassay for dinitrophenylated (DNP) antibodies when treated with DNP-streptococcal antigen in the presence of B cells.^16,18,19 The CD8⁺ VV⁺ cell function was most effective if the cells were added to the culture of B cells, CD4⁺ T cells and monocytes before the CD8⁺ VV⁻ cells. The target of CD8⁺ VV⁺ cells or their factors were the CD4⁺ T cells. The potential role of CD8 VV⁺ cells in vivo has been explored in patients with active systemic lupus erythematosus, as well as in the synovial fluid of patients with rheumatoid arthritis.^20,21 A significant increase in CD8 VV⁺ cells was found in these patients consistent with CS playing a part in autoimmune diseases.

The concept of contrasuppression incorporated in immunoregulation

With the onset of cloning over 20 years ago CD4⁺ helper and CD8⁺ T-cell cytotoxic cells were readily cloned but not CD8 suppressor cells. As a consequence all work on suppressor cells and consequently contrasuppressors was discontinued. However, as genetic and phenotypic analysis of the control of immunity has advanced, the mechanism of helper and suppressor immune responses have made significant progress. Immunoregulation is critical in maintaining the balance between immunity and tolerance, which are involved not only in preventing infection, but also in limiting collateral immune-mediated tissue damage. Tolerance to the host tissues has to be safeguarded to prevent autoimmune reactions. The following short account will review the involvement of CS as an essential part of control and fine modulation of immunity and tolerance. Regulation of the inhibitory pathways will determine if immunogenicity will be suppressed, maintained or enhanced.

The re-discovery of suppressor cells – CD25⁺ FoxP3⁺ CD4⁺ Treg cells

The resurgence of suppression as a regulatory mechanism in the control of immunity was the discovery of CD25⁺ CD4⁺ T regulatory (Treg) cells.²² At least two types of regulatory cells have been identified. Natural CD4⁺ Treg cells that express constitutively CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4) and glucocorticoid inducible tumour necrosis factor (GITR). The transcription factor FoxP3 is required for generation of natural Treg cells and is the specific marker of this cell.²³ Natural Treg cells develop in the thymus and are found in the peripheral lymphoid tissue. The mechanism of suppression is mediated by interleukin (IL)-10, transforming growth factor-β (TGF-β) or CTLA-4 and has been extensively studied in mice and a variety of infections.²⁴ Inducible Treg cells develop from conventional CD4⁺ T cells under the influence of cytokines or interference with costimulatory signals; these may result in TR1 or T helper 3 (Th3) regulatory cells. Indeed, high concentrations of TGF-β, IL-10 or IL-2 may induce CD4⁺ CD25- T cells to develop suppressive function²⁵ and TGF-β induces FoxP3 transcription factor.²⁶

Recently, one of the early workers reporting on suppression and CS demonstrated that apoptotic cells induce CD8⁺ suppressor T cells without priming CD4⁺ T cells for immunity.²⁷ The mechanism responsible for suppressing immunity was production of TRAIL (tumour necrosis factor related apoptosis-inducing ligand) by the suppressor CD8⁺ T cells. Necrotic cells normally stimulate CD4⁺ T-cell mediated immunity and do not induce TRAIL by CD8⁺ T cells, unless antigen is presented in the absence of CD4⁺ T cells.²⁷ They raised the possibility that CD8⁺ T suppressor factor secreted by CD8⁺ T suppressor cells^7,28,29 may be accounted for by TRAIL. The difficulties encountered in identifying the CD8⁺ T suppressor cells may have been due to the regulatory activity of conventional CD4⁺ helper and CD8⁺ cytotoxic T cells. Thus, CD8⁺ regulatory cell is a ‘helpless’ CD8⁺ T cell.

Immunoregulation of suppression

The CD25⁺ FoxP3⁺ CD4⁺ T regulatory cells prevent autoimmune responses, as well as over-reaction of the immune system.²² The T regulatory cells suppressing immune responses, however in turn require control to enable immunity and tolerance to be modulated according to the needs of controlling infection and immunity (Table 1). Cytokines modulate most immune responses and IL-6³⁰ and IL-2³¹ can reverse the suppressor activity of Treg cells resulting in CS. Targeting TGF-β or IL-10 by corresponding antibodies can also control immune responses in chronic infection.²⁴ Similarly, blockade of CTLA-4 enhances host immunity.^32,33 GITR ligand or antibodies engage GITR on effector T cells which mediates resistance to suppression by CD4⁺ CD25⁺ Treg cells.³⁴ An alternative mechanism has been postulated for the GITR–GITRL interaction, by boosting responder T cell function and enhancing resistance to Treg cell-mediated suppression.³⁵ This is probably the first time that the term ‘CS’ re-emerges in the title of a paper after 20 years of silence.

Table 1.

Inhibition or modulation of regulatory cells, suppressor pathways and cytokines as a function of contrasuppression

Suppression	Contrasuppression
CD25+ FoxP3+ CD4+ T cells	IL-6
	Inhibiting the CD40–CD40L pathway
	My D88-dependent signalingpathway of TLR2 in miceand TLR8 in humans
	TLR8 ligands-ssRNA and poly-Goligonucleotides
CTLA-4-CD80/CD86	Blockade by single-chain antibodyto CTLA-4
PD-1:PD-L1/L2	IL-2
	PD-1 or PD-L1 antibodies
GITR	GITR ligands or antibodies
TGF-β, IL-10	TGF-β, IL-10 antibodies

Immunoregulation of innate immunity may also involve inhibition of the CD25⁺ FoxP3⁺ CD4⁺ T cells. IL-6 is an acute phase protein induced during inflammation and effectively inhibits generation of Treg cells.³⁰ This is an example of a cytokine produced by activating the innate immune system, suppressing Treg cells and eliciting TH17 cells generating IL-17. Thus, a contrasuppressor effect is combined with pro-inflammatory IL-17 production. Suppression by CD4⁺ CD25⁺ Treg cells of innate immune responses of natural killer (NK) cells, monocytes and neutrophils in Helicobacter infection of mice was dependent on the action of IL-10 and TGF-β.³⁶

PD-1–PD-L1 inhibitory pathway in immunoregulation

Programmed death-1 (PD-1) is a transmembrane protein similar to CTLA-4, but does not bind to CD80 or CD86.³⁷ PD-1 is expressed by activated but not naive CD4⁺ and CD8⁺ T cells, and myeloid cells. PD-1 peaks 3 days after activation and then remains on the cell surface. The ligands to PD-1 are PD-L1 and PD-L2, which are members of the B7 family of receptors. Whereas PD-L1 is constitutively expressed on freshly isolated T and B cells, DC macrophages, and surprisingly on CD4⁺CD25⁺ Treg cells, PD-L2 expression is inducible only on DC and macrophages after cytokine stimulation.³⁸ PD-1–PD-L1 interaction inhibits CD4⁺ T-cell proliferation and cytokine production.^39,40 Indeed, PD-1^−/− mice develop autoimmune diseases, such as glomerulonephritis and lupus-like arthritis.^33,41

Exogenous IL-2 or CD28 monoclonal antibodies (mAb) augmented IL-2 can overcome PD-L1-mediated inhibition.⁴² Release from the inhibitory PD-1– PD-L1 pathway has been consistently achieved by antibodies to PD-1 or PD-L1 in humans and non-human primates infected by human immunodeficiency virus-1 (HIV-1), resulting in enhancement of T-cell immune responses to HIV-1.^43–45 A recent report suggests that blockade of the PD-1– PD-L1 pathway abrogates CD4⁺ CD25⁺ Treg cell activity, which is required for alloreactive suppression of CD4⁺ CD25⁻ T cells, both in vitro and in vivo for skin allograft rejection and graft versus host reaction.⁴⁶

The role of Toll-like receptor (TLR) in immunoregulation

To counteract suppression by Treg cells during primary T-cell responses, MyD88-dependent signalling pathway of TLR is involved.⁴⁷ Inhibition of CD25⁺CD4⁺ Treg cells suppressive activity is mediated by TLR2 expressed by these cells in mice⁴⁸ and TLR8⁴⁹ in humans, enhancing CD4⁺ T cell proliferation. The TLR2-dependent inhibition of Treg cells has been confirmed in TLR2^−/− knock out mice. This finding has therapeutic implications as Pam3 Cys is a synthetic TLR2 ligand which can selectively inhibit Treg cell suppressor activity in vivo.⁵⁰ CD4⁺ CD25⁺ Treg cells in humans were also inhibited by TLR8 ligands, such as ssRNA and poly-G oligonucleotides and confirmed by siRNA knock down of TLR8 in vitro and inhibition of Treg cells in vivo anti-tumour immunity.⁴⁹ Immunoregulation is an essential feature in health and disease and the complexity of the mechanism of fine control between immunity and tolerance underlines the critical nature of this activity.