Discussion

Discussion

Keran was successively diagnosed with atopic dermatitis, insulin-dependent diabetes mellitus, villous atrophy (flattening and disappearance of the finger-like absorptive processes of thesmall intestine) and autoimmune haemolytic anaemia. Additionally, hyper-IgE and history of a deceased brother at 2-months of age from autoimmune conditions and enteropathy, are evocative of IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy X-linked).

Biological tests confirmed this diagnosis with:

• lack of regulatory T cells (T_regs, defined phenotypically as CD4⁺CD25^hi, CD4⁺CD127^-/lo or CD4⁺FOXP3⁺ T cells),
• a mis-sense hemizygous mutation in the FOXP3 gene on the X chromosome.

IPEX disease is a very rare X-linked poly-autoimmune syndrome due to mutations in the gene encoding for the transcription factor FOXP3, which is essential for the development and function of T_reg, the pillar cells of peripheral immune tolerance. A failure in central or peripheral immune tolerance has severe consequences on homeostasis of the immune system, as illustrated by Keran’s disease.

Definition of tolerance

The immune system maintains integrity of the body by, notably, recognising pathogens and eliminating them or controlling their growth. For this, it has to distinguish dangerous organisms (typically pathogens but also abnormal or cancer cells) from innocuous antigens (typically self, alimentary or harmless environmental antigens). Indeed, the immune system faces many inoffensive foreign antigens throughout life (for eg. aerial antigens in lung, surface antigens on skin, alimentary antigens and microbiota within gut). In a way, the body itself is a universe of self-antigens. An aggressive B or T cell response against such innocuous antigens may lead to an autoimmune or allergic disease.

During their development, B and T cell precursors stochastically generate unique receptors for antigens, i.e. BCRs and TCRs, whose spectrum of recognition covers an extremely broad array of molecules. This is a fascinating aspect of the adaptive immune system which is poised to generate a considerable repertoire of receptor specificities from a limited set of genes, i.e. immunoglobulin and TCR gene loci. During this process, random molecular mechanisms generate clonal specificities directed against self-antigens among many others. It should be kept in mind that self-reactivity is a normal, physiological phenomenon: autoreactive T cells and autoantibodies are naturally present at varied levels within the immune system but are controlled by different safety checkpoints, a process referred to as ‘immunological tolerance’.

Several mechanisms participate in the prevention of undesirable responses of the adaptive immune system.

1.  Negative selection (or central tolerance) eliminates strongly autoreactive B and T cells during their development in the bone marrow and the thymus, respectively. Yet, this process is incomplete and self-reactive B and T cells may escape negative selection and reach the periphery of the immune system. A default in central tolerance may cause autoimmune diseases; e.g. APS-1/APECED syndrome caused by mutation in the AIRE  (autoimmune regulator) gene which is important for negative selection of T cells in the thymus.
2. Ignorance relates to the fact that some self-antigens are inaccessible to recognition by T cells. This is the case for instance when they are physically separated by anatomical barriers, e.g. blood-brain barrier, blood-retinal barrier etc. Nevertheless, in case of breakage of this barrier, autoimmunity may occur. One example is sympathetic ophthalmia (inflammation of the eye), a form of uveitis (inflammation of the middle layer of the eye) occurring after eye injury when ocular self-antigens are released and activate the immune system. Ignorance is not a tolerance mechanism strictly speaking because when antigens are released an immune response occurs, indicating that these T cells are not tolerant. Yet, ignorance participates in the prevention of autoimmunity.
3. Peripheral tolerance prevents activation of autoreactive lymphocytes by intrinsic or extrinsic (regulation) mechanisms. As an example of intrinsic mechanism, a T cell expressing Fas (CD95) on its surface can receive signals from antigen-presenting cells expressing Fas-ligand, leading to apoptosis (peripheral deletion). Hence, a mutation of the Fas gene may cause the ALPS syndrome associating lymphoproliferation and autoimmunity. Other surface molecules including CTLA-4 and PD-1 that inhibit T cell activation also participate in peripheral tolerance. Hence, checkpoint inhibitors such as anti-PD-1 and anti-CTLA4, therapeutic monoclonal antibodies used in cancer therapy, may cause autoimmune side effects. Finally, antigen-presenting cells that are not able to deliver T cell costimulatory signals lead to T cell anergy, i.e. T cells survive but are not fully activated to fulfill their function. Extrinsic mechanisms of peripheral tolerance involve different types of regulatory cells such as T_regs or IL-10 producing T_r1 that exert a dominant negative effect on other immune cells. Among them, T_regs play the major role. They limit the responses of conventional T cells and, notably, prevent activation of autoreactive T cells.

Natural T_regs are generated by the thymus while induced T_regs differentiate from conventional T cells after activation in the periphery. T_regs were initially characterised in the mouse by their constitutive surface expression of CD25, the alpha chain of the interleukin-2 receptor. Yet, expression of CD25 is not T_reg-specific since conventional T cells also transiently express CD25 after activation. In humans, T_regs were initially characterised by a higher level of CD25 expression (CD4⁺ CD25^hi) than conventional CD4⁺ CD25⁺ activated T cells. Another phenotypic characteristic is their low level of CD127 expression (CD4⁺ CD25⁺ CD127^-/lo). Importantly, they specifically express the transcription factor FOXP3 which is essential for their differentiation and function. This is an intracellular molecule whose staining necessitates cell permeabilisation to evaluate the frequency of CD4⁺ CD25⁺ FOXP3⁺ cells.

T_regs use many different mechanisms to regulate immunity such as T-cell deprivation of interleukin-2 (by binding to highly-expressed CD25), production of inhibitory cytokines (such as IL-10, TGF-β, IL-35) or other soluble mediators (such as adenosine), down-regulation of dendritic cell functions (such as CTLA-4 mediated blocking of dendritic cell costimulatory ligands, i.e. CD80 and CD86) or even cell killing.

 

To fight against and cope with a versatile and changing antigenic world (even ‘self’ changes with age, puberty, environment), adaptive immune systems have developed unique biological mechanisms for the generation of repertoire diversity (like somatic mutations that override the basic laws of genetics such as the conservative replication of DNA) and subsequent regulatory pathways. Keran’s clinical case illustrates the importance of peripheral tolerance since the lack of T_regs is lethal in the absence of therapy. The absence leads to unleashed activation of autoreactive responses with the contemporaneous occurrence (occurring in the same period of time) of several autoimmune diseases. The IPEX syndrome also illustrates that central deletion is not sufficient to prevent autoimmunity because T_regs are indispensable, in particular in the gut environment where most antigens are exogenous and cannot be tolerated by central mechanisms. Similarly, APS-1/APECED syndrome in which central deletion is severely impaired teaches us that T_regs alone are not sufficient to control autoreactive T cells when central tolerance is defective.

The milestones of T_regs and FOXP3 discovery

The existence of a population of suppressive cells (now known as T_regs) was suspected for a long time, before T_regs could be characterized phenotypically. For instance, when thymectomy (surgical removal of the thymus) is performed before day 3 of life, mice develop several autoimmune disorders, demonstrating that autoreactive T cells are produced at (and even before) birth. In contrast, when thymectomy is performed after day 3, mice grow normally without signs of autoimmunity, demonstrating that suppressive cells are produced from day 3 on. The latter control the activity of the former.

In 1995, Sakaguchi et al. demonstrated that transfer of CD4⁺CD25^– T cells to an alymphocytic host provokes multiple spontaneous autoimmune diseases that can be prevented by addback of CD25⁺ cells. This experiment: (1) confirms that a normal repertoire (the donor has no autoimmune disease) physiologically contains autoreactive T cells; (2) demonstrates that CD4⁺CD25⁺ T cells represent a population of naturally occurring T_regs that control the activity of autoreactive T cells, and (3) suggests that T_reg transfer might be used for therapy of autoimmune diseases (Sakaguchi et al., 1995).

IPEX was described in 1982 in a large family with 19 affected males across five generations, as a lethal X-linked disease (Powell et al., 1982). Then, Chatila et al. identified mutations in two unrelated patients with IPEX syndrome (Chatila et al., 2000). The IPEX syndrome was found to be the human equivalent of the Scurfy phenotype, a mouse model of lymphoproliferation (enlarged spleen and nodes) and autoimmunity. These mice have a 2-base pair insertion in a gene they named Scurfin, resulting in a truncated FOXP3 protein. At the same time, two teams independently established that FOXP3 was the human homolog of the mouse Scurfin gene encoding FOXP3 (Bennett et al., 2001; Wildin et al., 2001). Since it was not known yet that FOXP3 was specifically expressed by T_regs, authors suspected that the genetic defect caused hyperactivation of autoreactive T cells rather than a defect in regulation. To date, more than 70 mutations spanning the whole FOXP3 gene have been identified in IPEX patients.

In 2003, Sakaguchi et al. discovered that FOXP3 is selectively expressed by T_regs, and that its transduction confers regulatory phenotype and properties to conventional (non regulatory) T cells (Hori et al., 2003). The central role of FOXP3 in T_regs was subsequently confirmed by invalidation of the FOXP3 gene in mice (Fontenot et al., 2003) that recapitulated the scurfy phenotype with lymphoproliferation and autoimmunity.

The first demonstration of a role for T_regs in a classical autoimmune disease was brought by Salomon et al. who established that invalidation of the CD28 gene in autoimmune-prone NOD mice, reduced T_reg numbers and accelerated type 1 diabetes (Salomon et al., 2000). Then, involvement of quantitative and/or qualitative T_reg deficiencies were found in patients with many types of inflammatory or autoimmune diseases (Viglietta et al., 2004; Boyer et al., 2004; Cao et al., 2004).

In the 2010’s, T_regs started to move to therapy. Many approaches have been considered among which depletion of T_regs to augment T cell reactivity in malignant haemopathies (Maury et al., 2010), or transfer of T_regs to combat GVHD (Brunstein et al., 2011) or autoimmunity (Bluestone et al., 2015). Since low-dose IL-2 was effective in treating type 1 diabetes in NOD mice (Grinberg-Blayer et al., 2010), an alternative to cell therapy is cytokine-mediated in vivo T_reg mobilisation (Saadoun et al., 2011 ; Koreth et al., 2011).

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