Whither regulatory T-cells in MS?

Most data supports that the major culprit in the formation of Multiple Sclerosis is the T-cell, which is a white blood cell that plays a key role in the immune system. These cells are named after the small organ – the thymus gland – where they are produced. In particular, MS is an immune-mediated autoimmune disease caused, in all probability, by a defect in regulatory T-cells: cells which regulate, or maintain order in the immune system, and suppress immune responses either by cell-cell contact or by secreting cytokines (proteins) to regulate the extent of potentially damaging inflammation to the tissues of the body. Unrestrained activation of effector T cells (normally controlled by regulatory T cells) allows them to produce pro-inflammatory cytokines and recruit other inflammatory immune cells to a tissue causing damage such as myelin injury as seen in MS. The consequence of inflammation in MS, in all probability, is a chronic neurodegenerative process called Progressive MS, which is an additional level of complexity that is being intensively investigated.

The medical literature has increasingly begun to vaguely postulate that MS is a disease of mysterious origin; most probably because Neurologists seem to be increasingly unfamiliar with the rapid, widespread and spectacular advances in our knowledge of the workings of the immune system; and how loss of control of immune responses has serious consequences, such as autoimmunity in general and in MS in particular. This is especially puzzling as every study investigating genes which allow susceptibility to MS has demonstrated that the only genes which indicate susceptibility to MS are related to the immune system. The strongest link has been HLA genes (histocompatibility genes – which are used to match tissues for transplantation). These genes are remarkably diverse in their expression in different individuals, resulting in a population able to survive infections because there is an extremely low likelihood that a random individual would not see a protein of a virus or bacterium and thus recognize the organism and trigger a protective immune response. In addition to HLA genes, other immune genes which control signaling responses to antigens (the portion of a molecule, usually a part of a protein) by sensing cytokines (proteins secreted by immune cells to activate or suppress immune responses), have been found in the gene screens (GWAS – gene-wide association studies). While not definitive, there are numerous immune system studies which reflect various forms of immune dysfunction in MS. Further, the standard model for MS in animals is EAE (experimental autoimmune encephalomyelitis), an induced autoimmune disease with resemblance to MS (no other species seems to develop spontaneous autoimmunity against myelin such as in MS so demyelination has to be experimentally induced in animals). Finally, every successful therapy for MS works on the immune system!

In order for the immune system to initiate a tissue-specific autoimmune response, three components of the function of the immune system must occur. First, there must be a heightened immune recognition of a protein/s in that tissue. Second, there must be impaired regulation of the immune response in a setting that, thirdly, allows active recruitment of damaging inflammation directed against that tissue.

The immune system dates back eons in some form or other in animals and plants. Although more advanced in mammals it functions with inherent contradictions. The immune system has to provide protection against a host of infections and environmental challenges and has to do this by recognizing specific tissue molecules on the cells of the body in relation to these noxious materials and without over-reacting to these molecules or tissue cells. Cells that sense and sample the environment are known as antigen presenting cells (APC) and the immune cells that recognize the APC signal (in one instance) are T cells.

The immune system accomplishes this in an ingenious, but potentially hazardous manner. Proteins, (the most common form of antigen), are digested internally in APC, which results in fragments called peptides. The peptides are attached to the HLA molecule which ends up on the cell surface of the APC; or alternatively, the antigen is picked up from the environment of cells and is attached to the HLA proteins which are then placed on the surface of the cell. The HLA molecule sits on the surface of the APC facing outwards where it can be sensed by other cells. The surface view of the HLA molecule is much like looking down on a hot dog where the roll is the HLA molecule, and the hot dog is the antigen which fits into the groove of the roll. Molecular variations in the shape of the HLA roll are unique to each individual and the roll determines how much of the hot dog can fit in and how it lies in relation to the groove (which has a certain degree of flexibility to accomplish this). This complex is then seen by another molecule on the surface of T cells called the T cell receptor (TCR). The genes for the TCR come in countless varieties strung along a particular location in the DNA molecule and by a mix and match process of selecting various bits of TCR DNA we can generate countless millions of different TCR that have myriad ways of recognizing the unique molecular configurations of the HLA/antigen complex). The TCR can align in various configurations above the HLA/antigen hot dog and recognition of antigen attached to the HLA molecule is transmitted to the T cell with different forms of intensity (avidity). Further interactions between additional molecules on the cell surfaces of the APC and T cell as well as cytokines secreted by the APC (and recognized by receptors on the T cell) determine the nature and extent of the immune response to a particular antigen. These recognition events have two consequences. First, there is the recognition of a foreign antigen (say a piece of a virus) which then triggers a protective immune response. Second, and somewhat ironically, immune cells must also recognize antigens from the body itself to be able to effectively carry out this response. Every organ and tissue of the body is recognized and for the most part there is not an overactive response (called immune tolerance). Even more surprising is the fact that to produce an optimal immune response to a foreign antigen we must actually have an underlying current of continuously recognizing our own antigens. So it is not too difficult to imagine that there are a number of possibilities which might exist for some of these critically controlled reactions to go wrong. One of the consequences of the wrong sort of reaction can be heightened activation of the immune system against one of the tissue antigens from the body itself. This is a basis for tissue-specific autoimmunity, such as that against molecules making up myelin.

A molecular and model diagram of these molecules and their interactions as well as how this triggers stimulation taken from Malissen, B & Bongrand P (Early T cell activation: Integrating biochemical, structural, and biophysical cues. Annual Review of Immunology. 2015. 33:539-561) is shown below.

The process by which T cells and their TCR are educated to recognize specific yet diverse self and non-self-antigens occurs in the thymus during and after fetal life. Taming the extent of the response is known as Central Tolerance. Initially, T cells which either do not respond, or respond excessively to the HLA complex are eliminated (by a process of programmed cell death called apoptosis) in the cortex (outer portion) of the thymus. T cells then move to the inner part of the thymus – the medulla – before exiting to the circulation. While in the thymus T cells see many different self-antigens which are either expressed by thymic medullary cells or dendritic cells (both of which function as APCs). These cells have genes (e.g. AIRE – autoimmune regulator) that turn on segments of other genes which code for parts of molecules normally expressed in different tissues and organs of the body (e.g. thyroid cells or myelin producing cells – oligodendrocytes). The medullary thymic cells may also pass on these antigens to dendritic cells (DC) for antigen presentation. There is a certain amount of molecular degeneracy in the interaction between the HLA molecule and the antigen as well as in the TCR which allows for further increasing the millions of variations in the way that antigens can be seen; and for one TCR to actually see many different antigens (a smart strategy to maximize diversity in this process). In addition, conventional wisdom has it that there is a goldilocks zone controlling the recognition of self-antigens: recognition events which are either too strong or too weak lead to the elimination of T cells, while those in the zone in between survive and are not unduly autoreactive (and regulatory T cells TCR which have a higher affinity for recognition of self-antigens lie at the higher end of this continuum). This process knocks out large numbers of cells which could produce a damaging response to HLA/self-antigens which could then produce autoimmunity. This is known as Central Tolerance. However, some HLA/self-antigen recognizing cells survive this process and exit to the circulation and to the rest of the body. T regulatory cells are then necessary to help control the potential auto-reactivity of these cells (Peripheral Tolerance) and it is not difficult to see how this control mechanism could go wrong leading to autoimmunity. Moreover, it turns out that effector T cells have a certain repertoire of TCR which partially differs from that of regulatory T cells. This allows different parts of a protein (and therefore, different antigens) to be seen by differently functioning T cells. The avidity (strength) of regulatory TCR recognition of HLA/self-antigen is greater than that of effector T cells. Although unconfirmed, it is possible that the alignment of regulatory T cells TCR over the HLA-self-antigen may be spatially different than that of effector T cells which could allow for this). The strength of the regulatory T cell response can also be measured by what is known as the autologous mixed lymphocyte reaction (AMLR) – and this reaction can be increased in some forms of autoimmunity including MS. (More about this in the future as it may be a useful way to investigate what happens in the immune system in MS).

Most of previous research which helped us develop an understanding of the thymic education of T cells relied on the study of collections of many different T cells (called a polyclonal population) as compared with cells specific for just one antigen (monoclonal populations). Recently published studies have enlarged our understanding of more antigen specific processes in the thymus, and are of seminal importance for improving our concept of what might actually happen in MS).

One such study was recently published by Malchow S, et al. (Aire enforces Immune Tolerance by directing autoreactive T cells into the regulatory T cell lineage. Immunity. 2016. 44:1102-1113). If a mutation in the AIRE gene occurs then the central thymic expression of tissue antigens needed to induce Central Tolerance is lost. In humans this results in a disease called APECED or APS-1 (autoimmune polyendocrinopathy – candidiasis – ectodermal dystrophy) in which there is autoimmunity against the adrenal glands, parathyroid glands and prostate, testicles and ovaries (with infertility). It can also include Type I diabetes, GI and thyroid autoimmunity. It was believed that the absence of AIRE allowed for the survival of autoreactive T cells which would otherwise be deleted by Central Tolerance in the thymus. It was found, for example, that there was an increase in T cells recognizing a retinal photoreceptor antigen (Rbp3) were increased when AIRE was absent. However, when AIRE is present there are still Rbp3 reactive T cells, so AIRE deficiency only partially ablates these autoreactive cells. So the authors of this study questioned whether AIRE functioned primarily to eliminate autoreactive cells and suggested along with other studies that perhaps it normally functioned to enhance the induction of regulatory T cells instead. They focused on the prostate gland where they had previously identified autoreactive T cells in the gland.

Surprisingly, they found that regulatory T cells, in contrast to their normal absence, were actually present in the prostate gland when AIRE was absent. They also found activated conventional/effector CD4+Tcells (T cells come in two varieties – CD4 and CD8 T cells –depending on molecules expressed on their cell surface) were now also present; which indicated that the regulatory T cells did not function effectively in this situation to control the entry of conventional/effector T cell into the prostate. They then determined that the regulatory T cells lacked certain TCR which would normally be expressed and that these missing TCR were diverted and then became expressed on the conventional/effector T cells allowing them to become significantly autoreactive (because of the higher affinity of regulatory TCR leads to greater cell activation), causing prostate inflammation and damage. In fact, the TCRs normally expressed on regulatory T cells were the ones which were now expressed on the autoreactive effector/conventional T cells. They then drilled down to a more specific approach to an actual prostate reactive cell. They had previously identified a clone of cells called MJ23 which had a TCR which reacted to a prostate self-antigen and which comprised predominantly regulatory T cells when AIRE was active in the thymus. In the absence of AIRE these cells now became a mix of regulatory T cells and conventional/effector T cells which then infiltrated the prostate and were pathogenic due to regulatory T cell dysfunction. So, they reasonably concluded that the normal function of AIRE which promotes the expression of self-antigens in the thymus, is to promote the development of regulatory T cells which prevent autoimmunity while at the same time it promotes the removal of potentially autoreactive effector/conventional T cells which could be damaging. Absent AIRE this process is reversed with dire consequences.

A schematic taken from their paper which illustrates this immune imbalance is below:

While this is not an identical situation to MS, it does show how an imbalance between opposing T cell subsets in which effector/conventional T cells become resistant to regulatory T cells could result in disease.

In a similar vein Kieback E, et al (Thymus-derived regulatory T cells are positively selected on natural self-antigen through cognate interactions of high functional avidity. Immunity 2016. 44:1114-1126) looked at regulatory T cell generation to Myelin Oligodendrocyte glycoprotein (MOG), a component of the myelin membrane. In the right circumstances mice immunized with this protein or peptides derived from it develop the experimental disease EAE. Regulatory T cells normally prevent autoimmunity to MOG but are unable to control the effector/conventional T cell response in this experimental model resulting in a demyelinating disease.

They then created a transgenic mouse where both regulatory and effector T cells expressed the identical TCR recognizing MOG. Here, the effector T cells were found to be in a state of activation and EAE could be triggered easily with MOG. However, by manipulating the regulatory T cells they could be induced to regulate the effector T cells and thus improve the severity of EAE. Further experiments with an array of TCR recognizing MOG but differing between regulatory and effector T cells demonstrated that the TCR from regulatory T cells were more generally more strongly reactive to MOG (higher affinity) than effector T cells; in line with other experimental data. They then knocked out the MOG gene in mice to determine what happened in T cell development in the thymus if this single protein was absent. In this situation, there was a paucity of regulatory T cells specific for MOG (the fact that there are any is explained by TCR degeneracy mentioned above). In striking contrast, effector T cells which reacted to MOG were found even when MOG was knocked out (degeneracy again)! When the investigators then put a regulatory T cell receptor into a conventional effector T cell not only did EAE occur but it was significantly more severe demonstrating that the higher avidity of a regulatory T cell TCR can have grave consequences, much like to previously reviewed study. The converse was also true, that a high affinity TCR placed in a regulatory T cell could enhance its’ suppressive and disease sparing function.

Accordingly, this study demonstrated that a specific self-antigen must be expressed in the thymus for T cells to recognize it and develop mature regulatory or effector function. In the absence of the protein an imbalance between regulatory and effector cells results, with greater activation of effector T cells with the capacity to produce disease. The strength of TCR-induced responses must be greater than that of effector cells in order to effectively mediate their suppressive function; and switching TCR from one cell type to another has significant implications for how effectively a regulatory T cell could function.


And, what about regulatory T cells in MS?

An unfortunate and serious natural experiment occurs when some MS patients who have been treated with Fingolimod (Gilenya) have the drug withdrawn.

In order for a T cell to leave a lymph node and enter the circulation, it must express a receptor called the sphingosine 1 phosphate receptor (S1P1). Fingolimod down regulates the expression of this molecule on the surface of T cells and effectively prevents their entry into the circulation (which is needed to provide immune surveillance as protection against pathogens). This is presumed to additionally prevent the circulation of effector cells responsible for causing inflammation in MS. A further effect of Fingolimod is to enhance the number and function of circulating regulatory T cells.

When Fingolimod is withdrawn in MS about 10% of patients experience an aggressive form of white matter inflammation and demyelination which may be recurrent and is associated with a rapid replenishment of circulating T cells similar to what occurs in the immune reconstitution inflammatory syndrome (IRIS - seen in circumstances where there is rapid repletion of T cells). This occurrence manifests as clinical worsening with lesions on MRI that are inflammatory and tissue destructive. Unfortunately, the destructive inflammation is relatively resistant to Corticosteroid treatment and other MS therapies.

The events which underlie this rebound effect have been examined in the EAE model in mice by Cavone L, et al (Dysregulation of sphingosine 1 phosphate receptor-1 (S1P1) signaling and regulatory lymphocyte-dependent immunosuppression in a model of post-Fingolimod MS rebound. Brain, Behavior, and Immunity. 2015. 50:78-86).

They demonstrate disease recurrence and increased severity when Fingolimod is withdrawn in mice in whom EAE has been induced (below). In the initial part of the graph one sees that Fingolimod treated animals have milder disease (filled squares). However, when Fingolimod is withdrawn as on the right side of the graph, we see a rebound of disease with increased severity not unlike what occurs in MS patients.

When the production of cytokines which reflect inflammation is evaluated, it is clear that there is a particularly brisk production of Interferon gamma, and to a lesser extent Interleukin 17 (black bars).

When the spinal cord of the animals is examined, there is increased inflammation in white matter (compare the left picture with the right. Arrows point out areas of inflammation).

Higher magnification of the Fingolimod withdrawal case to highlight the extent of inflammation.

When Fingolimod is present there is an increase in the number of regulatory cells – Baseline to left, compared with the central bars. Black bars represent Fingolimod treated animals. When Fingolimod is withdrawn there is a brisk reduction in the number of regulatory T cells (Right bars). The consequence of this withdrawal is a reduction in the very cells needed to control inflammation and myelin damage.

Finally the function of regulatory T cells was assessed. If effective, a regulatory T cell will reduce the degree of proliferation of T cells (center bar compared with left bar). When Fingolimod is withdrawn the actual function of regulatory T cells is also impaired (compare the central bar with the one on the right).

The EAE model is very instructive in that is closely mirrors what happens in the MS situation and clearly demonstrates that the rebound in inflammation and demyelination is associated with a defect in number and function of regulatory T cells.



We have seen data on how regulatory T cells are instructed in their development and we have seen examples of what happens when these cells do not function adequately. The task for the future is to further characterize these cells and to learn how to control them in MS. The future direction for us at MSRI is quite clear!