27 Immunotolerance (and immunosuppression)

Dr. M. N. Gupta

1. Objectives

• To understand how innate immunity does not destroy own cells of the animal

• To understand the phenomenon of central tolerance and peripheral tolerance

• To understand how neuroendocrine system controls immune system

2. Concept Map

3. Description

Tolerance is the absence of the immune system to self antigens.

The early observation regarding tolerance were made with experimental animals. These observations helped Medawar and Burnet in giving final shape to clonal selection theory.

One of the early dilemma facing biologists was that an immune system which reacts to a foreign antigen fails to do so for self-antigens present in the animal. As Burnet (in 1976) said “For many years, I have implied that the basic feature of immunity was the capacity to differentiate between self and non-self”.

What made this complex to understand is that this capacity has to be present for both innate and acquired immune responses. Complete acceptance of the clonal selection theory requires that not only it should be able to explain the reaction to non-self, this feature must co-exist or be balanced by lack of reaction to non-self.

As in many other aspects of immunology, biologists were curious to understand whether tolerance is genetic or somatic, that is, it is part of genetic apparatus or it operates, may be at the protein level. As in the case of immune response, tolerance or lack of immune response arises out of both. The cellular mechanisms are genetically controlled.

Biologists who were convinced that genes decide everything were surprised that tolerance can be “learned”. The difficulties of transplanting organs were known quite early. In fact, so was the fact that these difficulties were not encountered at the embryo level in amphibians and chickens.

In 1945, Ray Owen carried out serological studies with blood of non-identical bovine twins. These had mixture of blood types which persisted throughout the lifetime of twins. The puzzle was that if each calf was born separately, each will have single blood group type present in the mixture form of blood of twins. In that case, blood of X calf injected into calf Y would provoke immune response.

In retrospect, this was the first clue that tolerance can be acquired either at the fetus level or very early in life

In rare instances, non-identical twins sharing the placental circulation in the uterus have different blood cells but „tolerate‟ cells from each other and accept mutual skin graft.

In other cases, as shown in the above figure, non-identical twins from different placentas do not acquire this „tolerance‟. The inference was that undifferentiated cells (stem cells or lymphocyte precursors) if mixed, these develop tolerance to each other indefinitely.

In 1957 (this was when we did not know that these are two different kinds of lymphocytes!), Burnet suggested “newly differentiated immunocytes” were either destroyed or were made incapable of proliferation if exposed to an antigen. Some early experiments were carried out by Beatrice Mintz (tetraparental mice), Frank Dixon, Richard Smith, William Weigle (with rabbits), Avrion Mitchison and David Dresser (with mice).

The observations of these investigators provided valuable insight into tolerance and are listed here:

•  Large and repeated doses of antigen can induce unresponsiveness in adults. Perhaps, constant circulation of antigen is the criterion
•  Younger animals require smaller doses. When rabbits were injected BSA at first week after birth, they became unresponsive. If the injection was given 3 weeks after birth, only 27% became unresponsive. Delaying injection to 4 weeks led to no unresponsive rabbits!
•  Rabbits given a single large dose of BSA early in life, slowly regain response but it was not restored completely
•  In adult mice, injection of soluble gamma-globulin (human or bovine) showed that it has a long life and led to unresponsiveness. This persisted even after the antigen had disappeared from circulation. Aggregated gamma-globulin if injected did not lead to unresponsiveness
•  Unresponsiveness could not be transferred to untreated animals by the transfer of serum or cells

C.R.Parish in G.L.Ada‟s lab at Melbourne, Australia were chemically modified flagellin. Flagellin is a constituent of filaments on Salmonella and has been an important experimental antigen in that institution. We have referred to this while discussing clonal selection theory (Module 15). Flagellin was available in pure form both as monomer or as a multimer. We have referred to the use of albumin and gamma-globulin as antigens. These proteins in pure form were available by then. During World War II, blood became an important system and the U.S. Government funded blood protein fractionation research. The result was pioneering.

work of Edsall and Cohn which laid the foundation of protein isolation and purification as we know it. Horse, man and bovine blood were the main systems chosen and albumin and gamma-globulin fractions were available. This knowledge had led to different laboratories purifying proteins of their interest. The main techniques used were limited to the precipitation by organic solvents, salts and metal ions. Chemical modification of proteins had been reported and a systemic study of that area had been initiated by Stein and Moore while trying to sequence bovine pancreatic RNase A (which was the second protein and first enzyme to be sequenced). The choice of RNase A was made because a meat canning industry in Philadelphia was offering samples of RNase A free to the scientists as their contribution to the U.S. war efforts.

All this enabled Parish to use various monomeric flagellin preparations which were acylated to a different degree. Acylation was found to reduce the capacity of the antigen to react with a standard preparation of its antibodies in vitro. Flagellin has also been a useful antigen as it has good antigenicity (even in small doses, it produces enough antibodies) and has a short circulatory life time. Parish found out an acylated flagellin which still retained its capacity to precipitate the antibody in vitro to a high degree. However, it was no longer an immunogen, that is, when injected it produced no antibodies. At the same time, its capacity to result in a delayed hypersensitivity increased. This property in fact remained quite well even in case of another flagellin derivative which has lost both immunogenicity as well as antibody precipitating ability. Parish, in fact, carried out quite extensive studies with flagellin and its chemically modified derivatives. These studies indicated that there is a reciprocal relationship between cell mediated immunity and humoral responses.

It may be interesting to note two interesting facts.

Firstly, these earlier workers (so many of them got the Nobel) had so few tools. Yet, they could infer so much out of their data with clear thinking and proper controls while designing the experiments.

Secondly, many areas, especially at the early phase of their development, greatly profit from developments in other areas. The U.S. Government‟s investment in fractionation of blood proteins impacted so many areas in biochemistry or even biology. Immunology was one of these. People who fruitfully employed the knowledge were biologists. Lack of their mental insularity and openness of scientific communication was very important to the development of immunology.

It may be noted that in the later phase of development of clonal selection theory, many leading workers led by Burnet had shifted focus on understanding tolerance. We will now see how the above experimental observation (and a lot more which followed these) were reconciled with the framework of clonal selection theory.

Tolerance can be viewed as a part of regulation of the immune response

So, let us first discuss regulation within the innate immunity system

Two broad ways it operates are:

•  Lack of recognition of self cells. This is called ignorance. During their life cycle, self cells can change their surface structures. In that case this ignorance vanishes.

•  Presence of inhibitory structures on such non-immune cells

Phagocytes are important component of the innate immune system. So, the first question is how these macrophages and neutrophils do not attempt phagocytosis of healthy self cells.

It may be recollected that phagocytes recognize foreign cells of microbes via their pattern recognition receptors. Self cells either do not have surface antigens recognized by these receptors. In many cases, these are covered up, for example, by sialic acid. In fact, this feature also is a part of how old erythrocytes and other dead cells are recognized by phagocytes. The old erythrocytes shed away sialic acid exposing N-acetyl glucosamine. This is recognized by PRR and phagocytosis commences. Similarly with nucleated cells, death results in many surface molecules becoming more exposed. For example, phosphatidyl serine is a phospholipid which is a part of the biomembranes. Normally, a part of the inner surface, apoptosis results in phosphatidyl serine flip to the surface and recognized by phagocytes.

Natural killer cells kill virus-infected cells. Normal self cells survive because of the balance between killer activation receptors (KAR) and killer inhibitory receptors (KIR) on the NK cells KIR recognize MHC molecules on self cells. In virus infected cells, expression of MHC (Class I) proteins is down regulated. This allows KAR to override and NK cells kill infected cells.

The third key part of the innate immunity system is the complement pathway. The one which is antigen independent is the alternative pathway. You may recollect that C3 in that pathway is activated because some microbial surfaces provide stabilization of the hydrolytic activities. The self cells in fact have inhibitory molecules on their surface membranes.

As these regulatory proteins are expressed by self cells and not by microbes, complement has the ability to distinguish between self and nonself cells. Action of some of these regulatory proteins is shown in the figure.

Now we can move over to the discussion on how adaptive immunity is regulated so that tolerance is inbuilt feature of the acquired immunity. Most of the empirical and semi-empirical observation which we referred to earlier have been fairly clearly understood within the framework of clonal selection theory.

Central Tolerance

Tolerance induced during lymphocyte development is called central tolerance. We have in fact briefly covered this while discussing B-cell and T-cell development. Potential B-cells and T-cells with specificities directed towards self cells/molecules are deleted. This process is called clonal deletion.

You may recollect that precursor T-cells migrate from bone marrow to thymus wherein they develop into immunocompetent T-cells. MHC antigens serve as markers of the self. Those T-cells which display high affinity to MHC Class I and II antigens undergo apoptosis. Even out of these small % (.10%) of T-cells which survive, some still have affinity for self and exit thymus to migrate to peripheral lymphoid tissues.

Similarly, clonal deletion of B-cells with potential to attack self molecules/cells takes place through apoptosis. The surviving cells leave fetal liver or bone marrow and reach peripheral or secondary lymphoid organs/tissues.

Also, immature B-cells having receptors specific to self antigens can become unresponsive (tolerant) or anergic. These cells are not eliminated. We had discussed this during B-cell development. These phenomenon involve editing of receptor specificity by light chain rearrangement and receiving incomplete activation signals leading to downregulation of IgM while retaining IgD.

Peripheral Tolerance

This strategy is necessary for achieving tolerance to self antigens which are not present in primary lymphoid organs or reach there by blood stream.

T-cell anergy

Naïve T-cell require signal via TCR as well as from co-stimulatory molecules. We discussed this during T-cell development. CD80 and CD86 are essential co-stimulatory molecules on APCs. CD86 interacts with CD28 on T-cells, APCs also release cytokines. Unless, APCs are not bound to microbes, upreguulation of CD80/CD86 does not take place. So, by default, MHC self antigens interact with naïve T-cells resulting in anergy.

As a failsafe arrangement, if any T-cell activation does take place, these express CTLA-4 whichinteracts with CD80 much more strongly than CD28. Binding of CTLA-4 to CD80 sends a negative signal to T-cell which inhibits their activity.

B-cell anergy

B-cells having specificities towards self antigens require T-cell co-operation to respond to T-cell dependant antigens

Corresponding Th cells have been already eliminated during thymic education (central tolerance). In the absence of this co-operation, B-cells become anergic.

T-cell ignorance

Most of the auto antigens are present at too low level to become target for T-cell recognition Those which generate self peptides at a level high enough to be recognized by effector T-cells but low enough not to induce tolerance, even in such cases, T-cells fail to get activated because of the low affinity. This is called T-cell ignorance.

Idiotypic modulation of the immune responses

During the neonatal period, tolerance is established to self antigens. B-cells and T-cells have receptors which differ in their specificities. For example, B-cell receptors are immunoglobulins. All these have common Fc portion and collectively is present at high concentration to provoke tolerance. The highly variable sequences which define antigen specificity (present in Fab) are determinants is present in too small a concentration to provoke tolerance.

While discussing Jerne‟s network theory, we had learnt that Jerne called those idiotopes and antibodies directed against idiotopes were termed anti-idiotypic antibodies. So, both T-cell receptors and B-cell receptors (immunoglobulin) will generate these anti-idiotypic antibodies.

The administration of anti-idiotypes at neonatal stage has been found to lead to even life long effects

T15 is a major idiotype in the antibody against phosphatidyl choline (a membrane phospholipid). If anti-idiotype T15 antibody is administered to a neonate, long lasting suppression of antibody production against phosphatidyl choline is observed. Significantly, doing the same with adult has limited effect. Hence, while Jerne‟s network theory may not have been successful in explaining all features of the immune system, the concept of idiotypes or idiotopes given by him was valuable in understanding tolerance. The in vitro experiments suggest that idiotypic antibody-idiotype network may operate in inducing tolerance.

Role of neuroendocrine regulation

The main role of the nervous system seems to be dampening down of inflammatory responses and maintaining homeostasis via negative feedback loops.

The hypothalamus-oituitary-adrenal (HPA) axis releases glucocorticoids which are hormones secreted by the adrenal cortex. These suppress inflammation by inhibiting release of cytokines IL-1, IL-2, L-6, IFN-γ, IFN-α (which play mportant role in inflammation) by dendritic cells and macrophages. Antigen presentation and mast cell functions are also inhibited. These multitasker molecules (glucocorticoids) also influence trafficking of the immune cells. Dual mechanism of decreasing the chemokine production and altering the expression of their housing molecules seem to operate in this cell trafficking.

Tolerance and immune suppression are twin phenomenon. We had earlier seen the role played by T-regulator cells in regulation of the immune response. In autoimmunity module we will see how these mechanism fail. In fact, because of these sophisticated tolerance mechanisms, there are fewer autoimmune diseases.

Summary:

• Tolerance is acquired at the fetal or neonate level by animals
• Innate immunity also involves tolerance
• Acquired immunity involves central tolerance and peripheral tolerance
• Immune responses are also subjected to neuroendocrine regulation