Although CD4⁺ Tregs play a vital role in the prevention of autoimmune diseases (see Chapter 20), their role in infections is less clear. CD4⁺ Tregs have a protective role against immune-mediated pathology and their ability to suppress is important in reducing inflammation. For example, lesions in the eye in stromal keratitis are less severe in the presence of CD4⁺CD25⁺ Tregs.

CD4⁺ Tregs can also suppress virus-specific responses. Many pathogens induce high levels of IL-10 and TGFβ, which promote the induction of CD4⁺CD25⁺ Tregs. In chronic viral infections, including HIV, cytomegalovirus (CMV), and herpes simplex virus (HSV) infections, increased numbers of Tregs are responsible for decreased antigen-specific responses by CD4⁺ and CD8⁺ T cells. This can lead to disease development.

CD8⁺ T cells suppress secondary immune responses

CD8⁺ suppressive Tregs can be generated in vitro by stimulating highly differentiated CD8⁺ cells with antigen and IL-10. Many CD8⁺ Tregs work in a cell contact-dependent manner. They cause a reduction of co-stimulatory molecules on dendritic cells and endothelial cells. This induces tolerance because upon the interaction with the TCR and peptide there is insufficient co-stimulation to generate a functional immune response.

In mice the non-classical MHC class Ib molecule, Qa-1, is crucial for suppression. Mice lacking Qa-1 are unable to suppress responses. However when Qa-1 is replaced into these cells their ability to suppress immune responses is restored (Fig. 11.10).

Fig. 11.10 CD4 Qa-1 is essential for CD8⁺ T suppressor function

Qa-1 knockout (KO) mice were generated. Qa-1 KO CD4⁺ cells were mixed with CD8⁺ cells and the level of suppression was assessed. CD8⁺ cells could not suppress CD4 cells lacking Qa-1 expression. Insertion of the Qa-1b allele into CD4⁺ cells resulted in effective suppression of responses.

(Adapted from Hu D, Ikizawa K, Lu L, et al. Nat Immunol 2004;5:516–523.)

CD8⁺ Tregs:

• need to be primed by CD4⁺ cells during a primary response; they then suppress secondary immune responses;

• can target activated effector T cells;

• can kill by direct lysis of target cells;

• can suppress immune responses through the secretion of cytokines such as IL-10.

NK and NKT cells produce immunoregulatory cytokines and chemokines

NK cells make cytokines and chemokines and therefore play an important role in the innate immune response to infections and tumors.

The production of immunoregulatory cytokines and chemokines at early stages in the immune response influences the characteristics of the subsequent adaptive immune reaction and can, therefore, affect the overall outcome of the immune response.

NK cells play a key role in the early immune response to intracellular pathogens, largely through their production of IFNγ, which activates macrophages and facilitates differentiation of TH1 cells.

NK cell activity itself is induced by a variety of cytokines including:

NK cells in turn are negatively regulated by cytokines such as IL-10 and TGFβ.

Q. Which cells will tend to promote the development of NK cells?

A. The TH1 population and macrophages through the production of IL-12, IL-15, and IL-18, whereas the TH2 population will tend to inhibit their development through the production of IL-10.

NK T cells produce cytokines when their TCR engages glycolipids in association with CD1d. It has been suggested that these cells play an immunoregulatory role in the control of autoimmunity, parasite infection, and tumor cell growth.

T cells secreting IFNγ are able to induce NK cell activation, increasing both NK proliferation and cytotoxicity. They are capable of making both TH1-type (IFNγ), TH2-type (IL-4), and TH17 (IL-17A) cytokines depending on the cytokines present in the microenvironment when they are activated (Fig. 11.11). These early sources of cytokine are important in influencing the nature of the T cell response.

Fig. 11.11 NK T cells produce both TH1- and TH2-type cytokines

NK T cells exert effects on many cell types. They can produce TH1, TH2, and TH17 cytokines and are involved in all aspects of the immune response.

Deficiencies of NK T cells have also been reported in animal and human autoimmune diseases, highlighting their regulatory roles. For example, non obese diabetic (NOD) mice have a deficit in NK T cells and injection of NK T cells into these mice prevents the spontaneous development of autoimmune diabetes. Human examples where NK T deficiencies may play a role include:

• rheumatoid arthritis;

• psoriasis;

• ulcerative colitis; and

• multiple sclerosis.

Regulation of the immune response by immunoglobulins

Antibody has been shown to exert feedback control on the immune response.

Passive administration of IgM antibody together with an antigen specifically enhances the immune response to that antigen, whereas IgG antibody suppresses the response. This was originally shown with polyclonal antibodies, but has since been confirmed using monoclonal antibodies (Fig. 11.12).

Fig. 11.12 Feedback control by antibody

Mice received a monoclonal IgM anti-SRBC (sheep red blood cells), a IgG anti-SRBC, or a medium alone (control). Two hours later all groups were immunized with SRBC. The antibody response measured over the following 8 days was enhanced by IgM and suppressed by IgG.

The ability of passively administered antibody to enhance or suppress the immune response has certain clinical consequences and applications:

• certain vaccines (e.g. mumps and measles) are not generally given to infants before 1 year of age because levels of maternally derived IgG remain high for at least 6 months after birth and the presence of such passively acquired IgG at the time of vaccination would result in the development of an inadequate immune response in the baby;

• in cases of Rhesus (Rh) incompatibility, the administration of anti-RhD antibody to Rh^– mothers prevents primary sensitization by fetally derived Rh⁺ blood cells, presumably by removing the foreign antigen (fetal erythrocytes) from the maternal circulation (see Chapters 24 and 25).

The mechanisms by which antibody modulates the immune response are not completely defined. In the case of IgM-enhancing plaque-forming cells, there are thought to be two possible interpretations:

• IgM-containing immune complexes are taken up by Fc or C3 receptors on APCs and are processed more efficiently than antigen alone;

• IgM-containing immune complexes stimulate an anti-idiotypic response to the IgM, which amplifies the immune response.

IgG antibody can regulate specific IgG synthesis

IgG can suppress antibody responses in a number of ways.

• Passively administered antibody binds antigen in competition with B cells (antibody blocking) (Fig. 11.13). In this case suppression is highly dependent on the concentration of the antibody, and on its affinity for the antigen compared with the affinity of the B cell receptors. Only high-affinity B cells compete successfully for the antigen. This mechanism is independent of the Fc portion of the antibody.

• Immunoglobulin can inhibit B cell differentiation by cross-linking (receptor cross-linking) the antigen receptor with the Fc receptor (FcγRIIb) on the same cell (see Fig. 11.13). In this case, the suppressive antibody and the B cell’s receptor antibody may recognize different epitopes.

• Doses of IgG that are insufficient to inhibit the production of antibodies completely have the effect of increasing the average antibody affinity because only those B cells with high-affinity receptors can successfully compete with the passively acquired antibody for antigen. For this reason, antibody feedback is believed to be an important factor driving the process of affinity maturation (Fig. 11.14).

Fig. 11.13 Antibody-dependent B cell suppression

Antibody blocking – high doses of soluble immunoglobulin (Ig) block the interaction between an antigenic determinant (epitope) and membrane immunoglobulin on B cells. The B cell is then effectively unable to recognize the antigen. This receptor-blocking mechanism also prevents B cell priming, but only antibodies that bind to the same epitope to which the B cell’s receptors bind can do this. Receptor cross-linking – low doses of antibody allow cross-linking by antigen of a B cell’s Fc receptors and its antigen receptors. The FcγRIIb receptor associates with a tyrosine phosphatase (SHP-1), which interferes with cell activation by tyrosine kinases associated with the antigen receptor. This allows B cell priming, but inhibits antibody synthesis. Antibodies against different epitopes on the antigen can all act by this mechanism.

Fig. 11.14 Antibody feedback on affinity maturation

The effect of passive antibody on the affinity and concentration of secreted antibody. One of two rabbits was injected with antibody (passive antibody) on day 1. Both rabbits were immunized with antigen on day 2 and the affinity and concentration of antibody raised to this antigen were assayed at a later time (day n). The antibody assay results show that passive antibody reduces the concentration, but increases the affinity of antibody produced.

Immune complexes may enhance or suppress immune responses

One of the ways in which antibody (either IgM or IgG) might act to modulate the immune response involves an Fc-dependent mechanism and immune complex formation with antigen.

Immune complexes can inhibit or augment the immune response (Fig. 11.15). By activating complement, immune complexes may become localized via interactions with CR2 on follicular dendritic cells (FDCs). This could facilitate the immune response by maintaining a source of antigen.

Fig. 11.15 Regulatory effects of immune complexes

Immune complexes can act either to inhibit or to augment an immune response. Inhibition – when the Fc receptor of the B cell is cross-linked to its antigen receptor by an antigen–antibody complex, a signal is delivered to the B cell, inhibiting it from entering the antibody production phase. Passive IgG may have this effect. Augmentation – antibody encourages presentation of antigen to B cells when it is present on an APC, bound via Fc receptors or, in this case, complement receptors (CR2) on a follicular dendritic cell (FDC). Passive IgM may have this effect.

Q. How does the presence of antigen on FDCs facilitate the immune response?

A. The antigen is available for uptake by B cells and presentation to T cells within the follicle, a process required for class switching and affinity maturation (see Fig. 9.15).

CR2 is also expressed on B cells and, as co-ligation of CR2 with membrane IgM has been shown to activate B cells, immune complex interaction with CR2 of the B cell–co-receptor complex and membrane Ig might lead to an enhanced specific immune response.

Apoptosis in the immune system

Apoptosis is a cellular clearance mechanism through which homeostasis is maintained.

Unlike cell damage-induced death (i.e. necrosis), which can trigger immune responses, apoptosis maintains intracellular structures within the cell. Apoptopic cells undergo nuclear fragmentation and the condensation of cytoplasm, plasma membranes, and organelles into apoptopic bodies. Apoptopic cells are rapidly phagocytosed by macrophages, which prevents the release of toxic cellular components into tissues, so avoiding immune responses to the dead cells.

Apoptosis is:

• involved in clearing cells with a high avidity for antigen in the thymus and is an important mechanism of immunological tolerance (see Chapter 19);

• an important mechanism in maintaining homeostasis in the immune system.

Following resolution of an immune response the majority of antigen-specific cells die by apoptosis. This ensures that no unwanted effector cells remain and also maintains a constant number of cells in the immune system.

A small number of cells are prevented from undergoing apoptosis and enter the memory T cell pool.

Apoptosis is controlled by a number of factors in the cell and depends on expression of the death trigger molecule CD95 (Fas). Deficiencies in the FAS/ FASL pathway can give rise to lymphoproliferative disorders with autoimmune manifestations.

Q. How does ligation of Fas lead to apoptosis?

A. It causes activation of a series of inducer and effector caspases.

Expression of the anti-apoptotic molecule Bcl-2 makes cells more resistant to cell death. Memory T cells generally express high levels of Bcl-2, which may contribute to the rescue of memory populations from apoptosis.

Different cell populations can express both pro- and anti-apoptotic molecules with the balance of these molecules determining whether a cell survives to participate in immune responses. Interestingly, at least in vitro, TH1 cells appear more susceptible to Fas induced apoptosis than TH2 or TH17 cells. The exact significance of this finding, however, remains to be determined.

Immune regulation by selective cell migration

The spatial and temporal production of chemokines by different cell types is an important mechanism of immune regulation. There is good evidence to suggest that the recruitment of TH1, TH2 and TH17 cells is differentially controlled, thereby ensuring the maintenance of locally polarized immune responses.

The expression of different chemokine receptors on TH1 cells (CXCR3 and CCR5), TH2 cells (CCR3, CCR4, CCR8), and TH17 (CCR6) allows chemotactic signals to produce the differential localization of T cell subsets to sites of inflammation (see Fig. 6.10).

Chemokines can be induced by cytokines released at sites of inflammation, so providing a mechanism for local reinforcement of particular types of response (Fig. 11.16). Once a response is established the T cells can induce further migration of appropriate effector cells. This is clearly illustrated in TH1 responses where the secondary production of CCL2, CCL3, CXCL10, and CCL5 serves to attract mononuclear phagocytes to the area of inflammation. Production of IL-17A can drive the expression of CCL20, the ligand for CCR6, recruiting more TH17 cells to the site of inflammation. The ability of cytokines such as TGFβ, IL-12, and IL-4 to influence chemokine or chemokine receptor expression provides a further level of control on cell migration or recruitment.

Fig. 11.16 Mechanisms for local reinforcement of different modes of immune response

Activated TH1 cells release IFNγ, which induces the chemokines IP-10 (CXCL10) and Mig (CXCL9). These act on the chemokine receptors CXCR3, which are selectively expressed on TH1 cells, thereby reinforcing this type of response. Macrophage chemotactic protein-1 (MCP-1, CCL2), which attracts macrophages and monocytes, is also induced by IFNγ. Mast cells release eotaxin (CCL11) when activated, and endothelial cells and bronchial epithelium can also synthesize this chemokine in response to IL-4 and IL-13 from TH2 cells. Eotaxin acts on CCR3, which is selectively expressed on TH2 cells, thereby reinforcing the TH2 response. Eosinophils and basophils, which mediate allergic responses in airways, also express CCR3. TH17 cells express the chemokine receptor CCR6. IL-17A production by TH17 cells induces expression of CCL20 which acts on CCR6 thus recruiting TH17 cells to the site of infection. Chemokines can therefore potentiate both the initiation and effector phases of a specific type of immune response.

Immune responses do not normally occur at certain sites in the body such as the anterior chamber of the eye and the testes. These sites are called immune privileged.

The failure to evoke immune responses in these sites is partly due to the presence of inhibitory cytokines such as TGFβ and IL-10, which inhibit inflammatory responses. The presence of migration inhibition factor (MIF) in the anterior chamber of the eye also inhibits NK cell activity.

T cell expression of different molecules can mediate tissue localization

Most studies on the human immune system are performed on blood due to the ethical issues in obtaining tissues. However, only a small proportion of the lymphocyte pool is circulating in the blood, most being resident in lymphoid or effector tissues.

The expression of molecules on T cells can mediate circulation through different tissues. Loss of the lymph node homing molecules CCR7 and CD62L on the surface of T cells prevents cells from circulating through lymphoid tissue – in this way a population of highly differentiated memory cells migrate to non-lymphoid sites where they can exert effector functions.

It has recently been suggested that there are two types of memory cell:

• central memory cells express CCR7, home to lymphoid tissues, and do not have immediate effector function;

• effector memory cells do not express CCR7, migrate to non-lymphoid tissues, and produce effector cytokines – effector cells in non-lymphoid tissues, such as the skin, can proliferate and senesce and this has a direct effect on the level of local immune responses.

Q. Why do cells that express CCR7 migrate to lymphoid tissues?

A. They respond to CCL21 expressed only in secondary lymphoid tissues (see Fig. 6.14).

Neuroendocrine regulation of immune responses

It is now widely accepted that there is extensive cross-talk between the neuroendocrine and immune systems. Both systems share similar ligands and receptors that permit intra and inter-system communication. These networks of communication are deemed essential for normal physiological function and good health. For instance they play important roles in modulating the body’s response to stress, injury, disease and infection. The interconnections of the nervous, endocrine, and immune systems are depicted in (Fig. 11.17).

Fig. 11.17 Neuroendocrine interactions with the immune system

The diagram indicates some of the potential connections between the endocrine, nervous, and immune systems. Blue arrows indicate nervous connections, red arrows indicate hormonal interactions, and white arrows indicate postulated connections for which the effector molecules have not been established.

There are several routes by which the central nervous system and immune system can interact:

• most lymphoid tissues (e.g. spleen and lymph nodes) receive direct sympathetic innervation to the blood vessels passing through the tissues and directly to lymphocytes;

• the nervous system directly and indirectly controls the output of various hormones, in particular corticosteroids, growth hormone, prolactin, α-melanocyte-stimulating hormone, thyroxine, and epinephrine (adrenaline);

• immune derived growth factors and cytokines can in turn feed back on the neural and endocrine systems, which probably has an important role in regulating the use of the body’s resources.

Lymphocytes express receptors for many hormones, neurotransmitters, and neuropeptides – expression and responsiveness vary between different lymphocyte and monocyte populations, such that the effect of different transmitters may vary in different circumstances.

Corticosteroids, endorphins, and enkephalins, all of which may be released during stress, are immunosuppressive in vivo. Such hormones can have strong effects on lymphocyte proliferation. In particular stress hormones can bring about the reactivation of latent viral infections. The precise in vitro effects of endorphins vary depending on the system and on the doses used – some levels are suppressive and others enhance immune functions.

It is certain, however, that corticosteroids act as a major feedback control on immune responses. It has been found that lymphocytes themselves can respond to corticotrophin releasing factor to generate their own adrenocorticotrophic hormone (ACTH), which in turn induces corticosteroid release. Corticosteroids:

• inhibit TH1 cytokine production while sparing TH2 responses; and

• induce the production of TGFβ, which in turn may inhibit the immune response.

For example, the low levels of plasma corticosteroids found in Lewis rats are believed to contribute to their susceptibility to a variety of induced TH1-type autoimmune conditions.

This interplay between the neuroendocrine system and the immune system is bidirectional. Cytokines, in particular IL-1 and IL-6 produced by T cells, neurons, glial cells and cells in the pituitary and adrenal glands, are potent stimulators of ACTH production through their effect on corticotrophin releasing hormone (CRH).

Q. In what other ways does IL-1 mediate interactions between the immune and nervous systems?

A. It causes an increase in body temperature, suppresses appetite, and enhances the duration of slow-wave sleep (see Chapter 6).

Gender based differences in immune responses also occur. Immune cells have been shown to express receptors for estrogens and androgens, so it is likely that circulating levels of these hormones can affect their function. It is noted that, during reproductive years, females demonstrate more pronounced humoral and cellular immunity than males.

Some autoimmune diseases also show a gender bias. The systemic autoimmune disease systemic lupus erythematosus (SLE) is ten times more common in females than males. Additionally, in animal models of autoimmunity, female NOD mice develop a much higher incidence of diabetes than males (though interestingly this sex bias is not observed in humans) and male BXSB mice have a spontaneously higher incidence of an-SLE like syndrome when compared to females. This provides some evidence for the effects of sex hormones on immune function.

Genetic influences on the immune response

Familial patterns of susceptibility to infectious agents suggest that resistance or susceptibility might be an inherited characteristic. Such patterns of resistance and susceptibility also occur in autoimmune diseases.

Many genes are involved in governing susceptibility or resistance to disease and the disease is said to be under polygenic control. Considerable advances have been made in mapping and identifying the genes governing the response to some diseases as a result of:

• the development of techniques such as microsatellite mapping;

• increased availability of DNA samples; and

• sequencing of the human genome.

In most cases these studies have led to the identification of potential candidate genes, but their real role in disease susceptibility remains to be clarified. In other cases single mutations in genes of known function have been found and the mechanism by which they contribute to disease identified.

MHC haplotypes influence the ability to respond to an antigen

The development of inbred mouse strains conclusively demonstrated that genetic factors have a role in determining immune responsiveness. For example, strains of mice with different MHC haplotypes vary in their ability to mount an antibody response to specific antigens (Fig. 11.18). This function depends on MHC class II molecules (see Chapter 5) and is specific for each antigen – a high responder strain for some antigens may be a low responder strain for others.

Fig. 11.18 Strain differences in the antibody response

Fifteen strains of mice were given a standard dose of the synthetic antigen (TG)-A-L. Antibody responses are expressed as the antigen-binding capacity of the sera. Animals of the H-2^b haplotype are high responders, whereas those of the H-2^d and H-2^k haplotypes are intermediate and low responders, respectively. However, there is some overlap between the levels of response in different haplotypes, indicating that H-2-linked genes are not the only ones controlling the antibody response.

MHC-linked genes control the response to infections

MHC-linked genes (see Chapter 5) are involved in the immune response to infectious agents. In some cases the gene involved is the MHC gene itself, but in others it can be a gene linked to the MHC.

Susceptibility to infection by Trichinella spiralis is affected by the I-E locus in mice

The first observation that genes within the MHC could influence the response to parasites involved the susceptibility to Trichinella spiralis infection. The response of different recombinant mouse strains to infection with T. spiralis is affected by the I-E locus. Mouse strains that express I-E (B10.BR, B10.P) appear to be susceptible whereas those that do not (B10.S, B10.M) are resistant to infection (Fig. 11.19). The response to T. spiralis is also influenced by the MHC-linked gene Ts-2, which maps close to the TNF genes.

Fig. 11.19 Susceptibility to Trichinella spiralis

Association of H-2 haplotype, expression of cell surface I-E molecules, and susceptibility to infection with T. spiralis. The resistance index is measured as the number of parasites present after a constant challenge relative to strains B10.BR (susceptible = 0% resistance) and B10.S (resistant = 100% resistance). B10 shows intermediate resistance.

The I-E locus also influences susceptibility to Leishmania donovani

Using H-2 congenic mice, it was shown that mice expressing the I-E locus were susceptible to visceral leishmaniasis. Parasite clearance was enhanced by anti-I-E antibody, but not by anti-I-A antibody, showing direct involvement of the I-E product. Furthermore, insertion of an I-E transgene into mice lacking the locus prevented effective clearance of parasites from the liver and spleen when compared to the original strain.

Certain HLA haplotypes confer protection from infection

Comparison of HLA haplotypes in humans revealed that certain MHC class I and class II alleles (HLA-B*5301 and DRB1*1302, respectively) are associated with a reduced risk of severe malaria. DRB1*1302 binds different peptides from those bound by DRB1*1301 as a result of a single amino acid difference in the β chain. This change is sufficient to influence the response to the malaria parasite.

HLA-DRB1*1302 has also been associated with an increased clearance of the hepatitis B virus and consequently a decreased risk of chronic liver disease.

The MHC class I type, HLA-A*02, is associated with a reduced risk of disease development following human T-lymphotropic virus-1 (HTLV-1) infection. The viral load was lower in HLA-A*02-positive healthy carriers of HTLV-1 correlating with the presence of high levels of virus-specific CTLs.

In HIV-1 infection a selective advantage against disease has been noted in individuals expressing maximal HLA heterozygosity of class I loci (A, B, and C) and lacking expression of HLA-B*35 and HLA-Cw*04.

Many non-MHC genes also modulate immune responses

Some genes outside the MHC region also govern the immune response. However, these genes are generally less polymorphic than MHC genes and contribute less to variations in disease susceptibility. Nevertheless, their effects are found in autoimmune diseases, allergy, and infection. For example:

• individuals with defects in the complement components C1q, C1r, and C1s (see Chapter 4) are predisposed to develop systemic lupus erythematosus (SLE) and lupus nephritis – the development of SLE-like symptoms in C1q knockout mice parallels the human situation;

• deficiency in C3 leads to an increased susceptibility to bacterial infections and a predisposition to immune complex disease – this is also seen with a deficiency in C2 and C4, both of which are located within the MHC region;

• high IgE production in some allergy-prone families associates with the presence of an ‘atopy gene’ on human chromosome 11q;

• Biozzi generated two lines of mice based on their responsiveness to erythrocyte antigens. The high responder and low responder Biozzi mice make quantitatively different amounts of antibody in response to antigenic challenge and the basis for these differences has in part been attributed to genetic differences in macrophage activity; the high and low responder strains also differ in their ability to respond to parasitic infections, but this does not correlate with the level of antibody produced.