Immunological Tolerance

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Chapter 19 Immunological Tolerance

Summary

Immunological tolerance is the state of unresponsiveness to a particular antigen which is primarily established in T- and B-lymphocytes. The clonal receptors of lymphocytes are generated by random recombination of the many genes that code for the antigen binding regions. This creates the need to sort out dangerous receptors that could recognize and destroy self tissues. The breakdown of immunological tolerance to self-antigens is the cause of autoimmune diseases.

Immunological tolerance is achieved by many different mechanisms operating on different cell types.

Central tolerance refers to the selection processes which T cell precursors undergo in the thymus before they are released as mature naive T cells. Thymic epithelial cells and dendritic cells present self-antigens to the immature T cell precursors. Those T cell precursors that respond strongly to the self-antigens presented in the thymus undergo apoptosis. This is called negative selection. A specialized population of thymic epithelial cells is capable of expressing genes which are expressed in a strictly organ specific manner (e.g. insulin, which is expressed only in the pancreas and the thymus).

Peripheral tolerance refers to the diverse mechanism that enforce and maintain T-cell tolerance outside the thymus. These include the prevention of contact between auto-reactive T-cells and their target antigens (immunological ignorance), the peripheral deletion of auto-reactive T-cells by activation induced cell death or cytokine withdrawal, the incapacity of T cells to mount effector responses upon recognizing their target antigen (anergy), and the suppression of immune responses by regulatory T cells.

B cell tolerance is established by several mechanisms including clonal deletion of autoreactive B cells, mostly in the bone marrow; the rearrangement of autoreactive B cell receptors (receptor editing) or by B cell anergy. In addition B-cell tolerance is maintained by tolerant T cells. The production of high-affinity class-switched antibodies depends on T-cell help. Therefore, if tolerance to a particular antigen is firmly established in the T-cell compartment, B-cells that recognize this antigen will usually remain tolerant.

To establish or re-establish tolerance is a major goal for innovative treatments for autoimmunity, allergy and transplantation. In contrast, to overcome immunological tolerance is one major goal for innovative treatments against cancer.

Generation of autoreactive antigen receptors during lymphocyte development

The specificity of the antigen receptors of T cells and B cells is the result of random shuffling of the many genes that encode the antigen-binding site of these receptors. Theoretically, this process could generate more than 1015 different T-cell receptors, including some that can bind to autoantigens (Fig. 19.1). Similar considerations apply to B-cell receptors. Cells expressing such receptors are often called self-reactive lymphocytes. The immune system has to fulfill two contradictory requirements: on the one hand the repertoire of different antigen receptors needs to be as large as possible to avoid ‘holes in the repertoire’ that could be exploited by pathogens to evade immune detection. On the other hand, the receptor repertoire must be shaped to prevent the immune system from attacking the organism that harbors it. Any disturbance in this delicately balanced system can have pathogenic or even lethal consequences, either from infections or from the unwanted reaction with autoantigens or harmless external antigens as in allergy. This paradox was recognized at the beginning of the last century by Paul Ehrlich who coined the term ‘horror autotoxicus’ for the necessity to avoid immunological reactions against self-antigens. Tolerance is the process that eliminates or neutralizes such autoreactive cells, and a breakdown of this system can cause autoimmunity. To avoid autoreactivity the randomly generated repertoire of T- and B-cell receptors is censored by several different mechanisms. CD4+ TH cells are pivotal for the multitude of immunological mechanisms that induce and maintain immunological tolerance. In this chapter we will discuss immunological tolerance in B cells and conventional αβ TCR expressing T cells.

T cell tolerance

T cell tolerance is established at two levels. Immature thymocytes undergo harsh selection processes in the thymus. This is often called central tolerance and results in the deletion of most T cells with high affinity for self antigens. Mature T cells are also regulated to avoid self-reactivity. The mechanisms that reinforce T cell tolerance outside the thymus are collectively called peripheral tolerance.

Central T-cell tolerance develops in the thymus

The chief mechanism of T-cell tolerance is the deletion of self-reactive T cells in the thymus. Immature T cell precursors migrate from the bone marrow to the thymus. There, they proliferate, differentiate and undergo selection processes before a selected few re-enter the blood stream as mature naive T cells. These differentiation and selection processes depend on interactions with thymic epithelial cells and dendritic cells in specialized microenvironments within the thymus (Fig. 19.2).

Generation of their clonal TCR is the first step in T cell development

In the thymus the T cell precursors – also called thymocytes – start to express the recombinase-activating gene (RAG) products and begin to rearrange their αβ TCR genes. T cell precursors that enter the thymus express neither CD4 nor CD8 and are, therefore, called double negative (DN) thymocytes. The DN precursors actively proliferate, undergoing approximately 20 cell divisions and assemble the TCR β chain. Only those DN cells that have successfully rearranged a TCR β chain will progress to the next stage and express CD4 and CD8 simultaneously. These immature CD4+CD8+ double positive (DP) thymocytes start to reassemble TCR α chains and express T cell receptors (TCR). The randomly rearranged TCRs expressed by the DP thymocytes collectively constitute the organism’s unselected TCR repertoire which is also called the germline repertoire. These thymocytes undergo processes of positive and negative selection. Less than 5% of them survive these selection events and are allowed to exit the thymus as naive mature T cells.

In addition to αβ T cells, other lineages including natural killer T (NKT) cells and γδ T cells also develop in the thymus. Although much less is known about thymic selection of γδ T cells there are profound differences in the thymic development of γδ T cells and αβ T cells. Ligand mediated selection events do not seem to be required for the selection of the γδ T cell repertoire. In contrast, instruction for particular effector functions occurs in the thymus for γδ T cells. Given the many current uncertainties about γδ T cell selection and NKT selection in the thymus, this section will deal exclusively with thymic selection of αβ T cells.

Positive selection occurs predominantly in the thymic cortex

Specialized APC, the cortical thymic epithelial cells (cTECs) are pivotal for positive selection. cTECs differ in their antigen processing machinery (cathepsins, proteasome subunits) from hematopoietic antigen presenting cells. Given that the number of different peptides that can be presented by any particular MHC molecule is much smaller than the number of different TCRs that undergo positive selection, each peptide must be involved in positively selecting many different T cells. Accordingly, experiments have demonstrated that the peptide on which a TCR is positively selected does not need to share sequence similarity with the peptides recognized by that same TCR in the periphery.

Note that positive selection depends on the recognition of self-peptides bound to self-MHC. The TCRs that are positively selected based on low-affinity interactions with self-peptide/MHC form the selected repertoire that ultimately recognizes microbial antigens, to protect the organism from infectious diseases. This is one illustration for the flexibility of antigen recognition by T cells. The peptides that mediate positive selection in the thymus are also presented outside the thymus where they support survival of mature T cells and may also act as co-agonists that enhance T cell activation by agonist peptides.

An immediate question is why T cells which were selected based upon self-recognition usually do not cause damage to the organism. One answer is that the threshold for TCR signaling is lower in immature DP T cells in the thymus than in mature T cells in the periphery. Thus, DP T cells can respond to low-affinity interaction with peptide/MHC complexes that would not trigger mature T cells. One important regulator of this TCR signaling threshold is the microRNA miR-181a that regulates the expression of several phosphatases involved in TCR signaling. DP T cells express much higher levels of miR181a than mature SP T cells.

A library of self antigens is presented to developing T cells in the thymus

The induction of central tolerance requires the presence of autoantigens in the thymus. This poses an obvious problem for thymic selection: Some autoantigens, e.g. insulin are expressed in a tissue-specific manner, are frequently called tissue-restricted antigens (TRAs). The question, then, is (how) do TRAs get into the thymus for presentation to developing T cells? Some might be brought into the thymus by immigrating antigen presenting cells but it is highly unlikely that this would yield a reliable representation of the organism’s TRAs. Moreover, developmentally regulated TRAs, e.g. antigens that are only expressed after puberty, would not gain access to the fetal thymus. The answer is that specialized cells in the thymus, the medullary thymic epithelial cells (mTECs) express proteins that are otherwise strictly tissue restricted. This has been called ectopic or promiscuous gene expression (Fig. 19.3).

MTECS express several hundreds or even thousands of functionally and structurally highly diverse antigens that represent almost all tissues in the body. Importantly, mTECs express not only tissue restricted antigens but also developmentally regulated antigens. Thus, gene expression in mTECs is uncoupled from spatial and developmental regulation. The exact mechanisms of this promiscuous gene expression are not yet fully understood. It has become clear that not all mTECs express all TRAs. Any particular TRA is expressed by less than 5% of mTECs.

Whereas mTECs are the only cells known to be capable of promiscuous gene expression, they are not the only cells important for negative selection. Thymic dendritic cells can take up TRAs expressed by mTECs and cross-present these TRAs to T cells. Recent intravital imaging studies have yielded the estimate that a thymocyte makes contact with approximately 500 dendritic cells during its sojourn in the thymic medulla. Although we do not have experimentally based quantitative estimates for thymocyte:mTEC contacts, the purging of self-reactive T cells in the thymic medulla is a remarkable achievement.

Subtle quantitative alterations in the thymic expression of TRAs can be consequential. Murine intrathymic expression levels of autoantigens including insulin and myelin antigens correlate inversely with susceptibility to autoimmune diseases, type I diabetes and experimental autoimmune encephalitis (EAE) respectively. Similarly, in humans genetic variants resulting in low levels of intrathymic insulin-expression are strongly associated with susceptibility to type I diabetes.

Qualitative variations in TRAs expressed in mTECs have also been associated with autoimmune disease models. Differential splicing or the expression of embryonic, rather than mature variants of myelin autoantigens have been associated with strain-specific susceptibility to EAE and may well play a role in the pathogenesis of multiple sclerosis in humans.

AIRE controls promiscuous expression of genes in the thymus

What enables mTECs to express a broad array of TRAs independently of spatial or developmental regulation? mTECs express the transcriptional regulator Aire (autoimmune regulator). Aire controls the expression of a large number of TRAs in mTECs (see Fig. 19.3).

Similarly, in humans, point mutations in the gene coding for Aire are the cause of the rare monogenic autosomal recessive autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) syndrome. APECED is characterized by high titres of several different autoantibodies that cause disease, mainly in endocrine organs. Together these findings strongly suggest that the autoimmune manifestations are caused by diminished expression of TRAs in thymic mTECs due to the Aire deficiency. Aire associates with a large number of partners to form distinct complexes that impinge on different steps of transcription. The different functions of Aire’s partner proteins include chromatin structure and DNA-damage response, gene transcription, RNA processing, and nuclear transport. It is still unclear, however, how Aire controls promiscuous gene expression. Moreover, experimental evidence strongly suggests that additional yet unidentified transcriptional regulators must also be involved in promiscuous gene expression by mTECs.

Peripheral T-cell tolerance

Despite the intricate mechanisms of central tolerance induction in the thymus, approximately one third of the autoreactive clones are not deleted. Thus, a large number of low-avidity self-reactive T cells escapes into the periphery; thus, autoreactive T cells are part of the normal repertoire. For example, T cells that recognize insulin or myelin basic protein can be isolated from people without diabetes or multiple sclerosis. Despite their low avidity for self-antigen these cells are potentially dangerous and can cause autoimmune tissue destruction. Autoimmune diseases, however, are the exception rather than the norm. It follows that peripheral tolerance mechanisms must exist to prevent these autoreactive T cells from causing harm.

Immunological ignorance occurs if T cells do not encounter their cognate antigen

Immunological ignorance is maintained as long as autoreactive T-lymphocytes do not enter the tissue in which the autoantigen that they recognize is expressed. The autoreactive naive T cells are not tolerized and can be activated upon recognizing their cognate antigen.

The importance of immunological ignorance was demonstrated in mice that express a transgene-encoded TCR which recognizes a peptide derived from the lymphocytic choriomeningitis virus (LCMV). These mice were bred with another transgenic strain that expressed the viral peptide on the surface of their pancreatic islet cells. Surprisingly, diabetes did not develop in the offspring even though in vitro their T cells could kill cells that displayed the viral peptide (Fig. 19.4). The T cells in these double-transgenic mice were therefore, not tolerant in vivo, instead they were ignorant of their target cells.

When the mice were infected with LCMV, the transgenic T cells became activated, invaded the pancreas and destroyed the islet cells. Consequently the mice succumbed to diabetes. Importantly, LCMV-infection did not cause diabetes in those mice that expressed the transgenic TCR but not the LCMV-peptide in the pancreas. Once activated by LCMV infection, the hitherto ignorant autoreactive T cells (autoreactive because the LCMV-derived peptide was transgenically expressed in the pancreas) acquired the capacity to migrate into their target tissue where they recognized and destroyed the LCMV-peptide expressing islet cells (see Fig. 19.4).image

Some self antigens are sequestered in immunologically privileged tissues

Self-reactive T cell lines that recognize autoantigens including myelin antigens and pancreatic antigens can easily be cloned from healthy humans. How, then, is autoimmune disease avoided in the presence of potentially pathogenic autoreactive T cells?

One explanation is the sequestration of potentially harmful T cells from the tissues in which their target self-antigens are expressed. Sequestration can be achieved when antigens are physically separated from T cells (e.g. by the blood–brain barrier, see Chapter 12). The blood–brain barrier can be surmounted by activated lymphocytes, however, and many organs do not possess a physical barrier to prevent lymphocytes entering from the bloodstream. Instead, lymphocyte migration is controlled by chemokines, selectins and their receptors.

The amount of released self antigen critically affects sensitization

How does the immune system decide which autoreactive T cells may survive ignorantly and which need to be deleted? Antigen dose (Fig. 19.w1) and TCR avidity play a major role. One key experiment used two different strains of mice that expressed ovalbumin (OVA) specifically in the pancreas. One strain expressed OVA at low levels and the other expressed it at high levels. Only in the high-expressing strain was OVA presented to T cells in the draining lymph nodes, resulting in the deletion of adoptively transferred OVA-specific TC cells. In the low-expressing cells the OVA-specific T cells remained ignorant. Further in vitro assays revealed that the low-level OVA-expression was still sufficient to allow recognition and killing of the OVA-expressing pancreatic β cells by the OVA-specific CTL. Therefore, tissue restricted self-antigens need to be expressed at sufficiently high levels to be presented in the draining lymph nodes. These experiments also clearly showed that self-reactive T cells remain dangerous and poised to wreak havoc even if they are temporarily ignorant. In fact, destruction of pancreatic islet cells can induce the release of sufficient amounts of self-antigen to activate OVA-specific CTL in the OVA-low expressing mice.