# Clonal anergy

Anergy, within the realm of immunology, characterizes the absence of a response from the body's defense mechanisms when confronted with foreign substances. This phenomenon involves the direct induction of peripheral lymphocyte tolerance. When an individual is in a state of anergy, it signifies that their immune system is incapable of mounting a typical response against a specific antigen, typically a self-antigen. The term anergy specifically refers to lymphocytes that exhibit an inability to react to their designated antigen. Notably, anergy constitutes one of the essential processes fostering tolerance within the immune system, alongside clonal deletion and immunoregulation. These processes collectively act to modify the immune response, preventing the inadvertent self-destruction that could result from an overactive immune system.

## Mechanism

This phenomenon was first described in B lymphocytes by Gustav Nossal and termed "clonal anergy." The clones of B lymphocytes in this case can still be found alive in the circulation, but are ineffective at mounting immune responses. Later Ronald Schwartz and Marc Jenkins described a similar process operating in the T lymphocyte. Many viruses (HIV being the most extreme example) seem to exploit the immune system's use of tolerance induction to evade the immune system, though the suppression of specific antigens is done by fewer pathogens (notably Mycobacterium leprae).
At the cellular level, "anergy" is the inability of an immune cell to mount a complete response against its target. In the immune system, circulating cells called lymphocytes form a primary army that defends the body against pathogenic viruses, bacteria and parasites. There are two major kinds of lymphocytes – the T lymphocyte and the B lymphocyte. Among the millions of lymphocytes in the human body, only a few actually are specific for any particular infectious agent. At the time of infection, these few cells must be recruited and allowed to multiply rapidly. This process – called "clonal expansion" – allows the body to quickly mobilise an army of clones, as and when required. Such immune response is anticipatory and its specificity is assured by pre-existing clones of lymphocytes, which expand in response to specific antigen (process called "clonal selection"). This specific clonal army then combats the pathogen until the body is free of the infection. Following clearance of the infection, the clones that are no longer needed die away naturally.
However, a small number of the body's army of lymphocytes are able to react with proteins that are normally present in a healthy body. The clonal expansion of those cells can lead to autoimmune diseases, wherein the body attacks itself. In order to prevent this process, lymphocytes possess an intrinsic quality-control mechanism. This machinery shuts down the lymphocytes' ability to expand, if the trigger for the expansion turns out to be the body's own protein. T-cell anergy can arise when the T-cell does not receive appropriate co-stimulation in the presence of specific antigen recognition. B-cell anergy can be induced by exposure to soluble circulating antigen, and is often marked by a downregulation of surface IgM expression and partial blockade of intracellular signaling pathways.

## Molecular mechanism of anergy induction in T lymphocytes

Understanding the molecular mechanism of anergy induction in T lymphocytes unveils the intricate interplay of signaling pathways governing immune responses. Upon stimulation, the T cell receptor (TCR) in conjunction with co-stimulatory receptors orchestrates a comprehensive activation of all the T-cell’s signaling pathways, collectively termed full T-cell stimulation. Among these pathways, the calcium-dependent arm of lymphocyte signaling is particularly pivotal, triggered by TCR engagement. This initiates a cascade culminating in an elevation of intracellular Ca$^{+II}$ concentration, a critical event in T cell activation. Under such conditions, the calcium-dependent phosphatase calcineurin acts on the transcription factor NFAT, facilitating its translocation to the nucleus, where it regulates gene expression.
Expanding upon this complexity, during full T-cell stimulation the co-stimulatory receptor CD28 activates PI3K and other pathways, augmenting the nuclear levels of key transcription factors such as rel, NF-κB and AP-1 beyond those induced by TCR activation alone. The formation of AP-1, fos/jun heterodimer, further complexes with NFAT, creating a transcriptional complex crucial for the expression of genes associated with T-cell productive responses, including IL-2 and its receptor.
In contrast, TCR signaling in the absence of co-stimulatory receptors predominantly activates the calcium arm of the signaling pathway, leading to NFAT activation alone. However, without the concurrent induction of AP-1 by other pathways, NFAT fails to form the transcriptional complex necessary for a productive T-cell response. Instead, NFAT homodimerizes, functioning as a transcriptional factor that induces anergy in the lymphocyte.
NFAT homodimers play a direct role in the expression of anergy-associated genes, such as the ubiquitin ligase GRAIL and the protease caspase 3. Furthermore, anergized cells exhibit decreased expression levels of IL-2, TNFα, and IFNγ, characteristic of a productive response, while favoring the production of the anti-inflammatory cytokine IL-10. Although three NFAT proteins - NFAT1, NFAT2 and NFAT4 - are preset in T-cells, they demonstrate redundancy to some extent.
In the context of antigen presentation by antigen-presenting cells (APC), T lymphocytes undergo a productive response when the antigen is appropriately presented, activating T cell co-stimulatory receptors. However, encountering antigens not presented by the APCs or weakly presented antigens induces anergic responses in T cells. Notably, strong stimulation through IL-2 or TCR/co-stimulatory receptors can overcome anergy, highlighting the dynamic nature of immune regulation.
Moreover, recent research has illuminated the role of regulatory T cells (Tregs) in modulating T cell responses and maintaining immune tolerance. Tregs, characterized by the expression of the transcription factor Foxp3, exert immunosuppressive effects by inhibiting the activation and function of effector T cells. Importantly, Tregs can directly interact with anergic T cells, further reinforcing their state of unresponsiveness and promoting peripheral tolerance. This interaction involves various mechanisms, including the secretion of inhibitory cytokines such as IL-10 and TGF-β, as well as cell-contact-dependent suppression mediated by molecules like CTLA-4. Understanding the intricate crosstalk between Tregs and anergic T cells provides valuable insights into the maintenance of immune homeostasis and has implications for therapeutic strategies aimed at modulating immune responses in autoimmune diseases and transplantation.

## Clinical significance

Anergy may be taken advantage of for therapeutic uses. The immune response to grafting of transplanted organs and tissues could be minimized without weakening the entire immune system— a side effect of immunosuppressive drugs like cyclosporine. Anergy may also be used to induce activated lymphocytes to become unresponsive with autoimmune diseases like diabetes mellitus, multiple sclerosis and rheumatoid arthritis. Likewise, preventing anergy in response to a tumoral growth may help in anti-tumor responses. It might also be used for immunotherapeutic treatment of allergies.

## Dominant tolerance

Dominant and recessive tolerance are forms of a peripheral tolerance (the other tolerance beside peripheral is a central tolerance). Where so called recessive tolerance is associated with anergized lymphocytes as described above, in the dominant form of tolerance, specialized T-reg cells which actively ablate the immune response are developed from the naive T lymphocyte. Similarly to recessive tolerance, unopposed NFAT signalling is also important for T-reg induction. In this case, the NFAT pathway activates another transcription factor – FOXP3 that is a marker of T-regs and participates in their genetic program.

## Testing

The "Multitest Mérieux" or "CMI Multitest" system (Multitest IMC, Istituto Merieux Italia, Rome, Italy) has been used as a general test of the level of cellular immunity. It is an intradermal test of skin reactivity (similar to tuberculin tests) in which a control (glycerol) is used with seven antigens of bacterial or fungal origin (tetanus toxoid, tuberculin, diphtheria, streptococcus, candida, trichophyton, and proteus). In this test reactions are categorized according to the number of antigens provoking a response and the summed extent of the skin response to all seven antigens. Here anergy is defined as a region of skin reactivity of 0–1 mm, hypoergy as a reaction of 2–9 mm in response to fewer than three antigens, normergic as a reaction of 10–39 mm or to three or more antigens, and hyperergy for a reaction of 40 mm or more.

## Experimental approaches to study anergy

Various chemicals inducing/inhibiting described T cell signalling pathways can be used to study the anergy. The anergy in T cells can be induced by Ionomycin, the ionophore capable of raising intracellular concentration of calcium ions artificially.
Conversely, Ca$^{+II}$ chelators such as EGTA can sequester calcium ions making them unable to cause the anergy. Blocking of the pathway leading to the anergy can be also done by cyclosporin A, which is capable of inhibiting calcineurin – the phosphatase responsible for dephosphorylating of NFAT priming its activation.
PMA, phorbol 12-myristate 13-acetate, along with ionomycin is used to induce full T cells activation by mimicking signals provided naturally by TCR/costimulatory receptors activation.