19 Cytotoxic cells and cellular immunity

Dr. M. N. Gupta

1. Objectives

• To understand the role of cytotoxicity and cellular immunity in various diseases.
• To learn about how both innate immunity and adaptive immunity deal with intracellular pathogens.
• To learn about NK cells and cytokines in more detail
• To learn about thymic education.

2. Concept Map

3. Description

Some bacteria enter animal cells. Viruses also infect cells. In such cases, cellular immunity is required for protection.

In some fungal diseases as well, cellular immunity plays an important role.

We have already learnt that T-cells are the ones which mediate cellular immunity. Let us look at that in detail and see how these cells work in concert with rest of the immune system.

The discovery of cellular immunity was made when it was realised that some properties could not be transferred between animals through serum.

A delayed hypersensitivity response to tubercle bacillus protein preparation could be transferred to a normal animal by cells from the spleen, lymph node and a sensitized guinea pig.

At one time it is believed that thymus in mammals discharged the functions of both bursa and thymus in chickens. This should help in understanding the excitement which resulted from the work of Jacques Miller with mice subjected to neonatal thymectomy.

Warner and Saltzberg showed the respective function of thymus and bursa in chickens. Graham Mitchel and Jacques Miller soon thereafter showed the role of T-cells in antibody production in mouse.

Cellular Immunity and Diseases

Humoral response is able to deal with extracellular forms of pathogens and the toxic products. Even these, we have already referred to the importance of Th cells which are part of cell mediated immunity.

Some pathogenic bacteria and parasites and all viruses invade host cells. These intracellular invaders are not accessible to antibodies. Hence the need for T-lymphocytes which mediate cellular immunity.

One kind of T-cells (called CD4 T cells) defend against bacterial infections which are intracellular in nature. Mycobacterium tuberculosis and M. leprae grow inside the membrane bound vesicles of macrophages.

These organisms are responsible for causing tuberculosis and leprosy respectively. The vesicles containing these bacteria do not fuse with the lysosome of the macrophages. Th1 cells make this fusion possible and activate macrophages to cause phagocytosis.

A Th1 cell recognizes the infected macrophage, releases cytokines which facilitate the fusion and bactericidal activity of the macrophage. In absence of Th1 activity, these bacteria go on multiplying inside the vesicles.

The other examples of intracellular pathogenic bacteria are Salmonella typhi and Brucella species which also have vesicular residence.

There are other bacteria such as Chlamydia rickettsia and Listeria species which replicate in the cytoplasm of host cells.

So, one way of classifying intracellular pathogens (bacteria, viruses and parasites) is according to their cytoplasmic or vesicular nature.

It should be made be made clear that virus entry into cells at initial stage of infection can be prevented by neutralizing antibodies released by B-cells. The production of these antibodies is facilitated however by Th cells. These neutralizing antibodies are directed against viral protein coat which is highly antigenic.

However, once the virus entry the host cells, these neutralizing antibody cannot reach it.

The role of T-cells in protecting against these pathogens deserves further attention beyond the simple outline which indicates the importance of T-cells. We will now describe immune mechanism and indicate the importance of cell mediated immunity in such cases.

Some bacteria which do not invade cells exert their pathogenicity via epithelial attachment. These generally release toxins inside the cells. Cornybacterium diphtheria, Clostridium tetani, Vibrio cholarae, GpA staphylococci (which cause sore throat) are such pathogens.

The second class is those of intracellular bacteria which already have been identified.

Many bacteria in fact show mixed behaviour. Staphylococcus aureas and Clostridium perifrigens exemplify this behaviour. There is some limited local invasion. In such cases also cell mediated responses are required.

The bacteria which have capability of invading the cells also have to clear nonspecific barriers which have been discussed earlier and may be recalled again in this context. Commensals (organisms occupying specific niche in various parts of the body) play an important role here.

Vagina epithelium secrets glycogen which commensal bacteria metabolise to lactic acid.

Consequently the acidic pH discourages many infections.

Infections by Candida and Clostridium difficile occur more easily when commensals are destroyed/ disturbed by antibiotic treatment. Both prebiotic and probiotics can help in restabilising the protective flora.

Many components of innate immunity do play a protective role after intracellular infection by the bacteria. These act independent of B-cell and T-cell mediated immune mechanism.

Many organisms (e.g. nonpathogenic Cocci) are eliminated via these mechanisms without adaptive immune mechanisms Competent activation via alternative pathway independently or by facilitating phagocyte infections can destroy the infected cells. Macrophages release cytokines TNF and IL-1 which not only activate phagocytic cells but also promote their entry into tissue at the site of infection. Data with murine cells shows NK cells, in response to microbial components & TNF in turn release IFNᵞ which further activates macrophages. Mice resist Listeria manocytogens infection even if these have SCID (severe combined immune deficiency) which prevents T-cells maturation.

Depending upon a particular infection, its pathogenicity and severity, T-cell mediated responses often become necessary. The cytokines released determine how T-cells direct various effector mechanism. Thus decision is made by subtype of T-cells called Th (T-helper) cells.

The three key effector mechanisms involve:

•  Tc (Cytotoxic T-cells)

•  Antibody + mast cells and eosinophils

•  Macrophage activation and delayed hypersensitivity.

Thus „decision‟ by Th cells is very critical. The third option is protective in Leishmania whereas second option is hurtful

In the murine system, as well as in humans, different forms of T-cells release different cytokines. So, the sequence of decision making involves cytokines at two stages.

Cell mediated cytotoxicity operates through not only Tc cells but other lymphoid and myeloid cells as well.

It may bring some clarity if we rather look at the way these cytotoxic cells bind to the target cells.

Specific antigen derived peptides (along with MHC proteins) are target by Tc cells via their TCR.

Receptors on NK cells recognize some antigenic determinants like on tumour cells.

Ab-Ag complex (could be viral antigen on infected cell) is recognized by Fc recognizing receptor on K cells. This is called antibody dependant cell mediated cytotoxicity (ADCC)

Tc cells have a very complex interaction with the target cell. It involves multiple linkages which firm up the binding between Tc cells & the target cell. This action is called antibody independent cell mediated cytotoxicity to distinguish it with from ADCC. It should be however very clear that Tc needs to see the antigen on the surface of infected cells.

That is a critical design which enables a Tc cell not to start attacking uninfected cells under normal circumstances. Thus, an important role of Tc is in killing virus infected cells so that the host cells of the virus are destroyed and virus multiplication stops and viral infection spread is halted.

Tc cells action is also different from Th cells which co-operate with humoral response of B-cells. That is another reason why Tc cell mechanism is called antibody independent cell mediated cytotoxicity.

Many times during our discussion we have used words like cytokines, lymphokines and mentioned specific interleukins.

This may be a good time to bring clarity about these terms as we will continue using these even in later modules and quite frequently.

Cytokines

The term lymphokines refers to mediators released by lymphpocytes.

Interleukins along with a number assigned to each (e.g. IL-1 or IL-2) was an attempt to create a system of nomenclature for mediators with an implication that these were part of inter leukocytes (white blood cells). With further work, it has been found that interleukins functions cover even a wide range.

Cytokine is a more general term. It includes lymphokines and interleukins. Cytokines are peptides, often glycosylated.

Excessive release of cytokines can be harmful and can result in severe tissue damage, shock and circulatory collapse.

Multiple effects of cytokines

In pre rDNA era, in areas like immunology and molecular biology, several mediators, effectors and “factors” were reputed. Isolation and characterization of these molecules was difficult as these occurred in small amounts and enzymology did not have tools to operate at micro levels. In many cases, later on it was found that different labs were actually often looking at the same molecule. In some cases these studies were being made in different contexts. Thus, „cachectin‟ cytokine reported in the context of weight loss in chronic infections turned out be same as TNFα.

IL-6, was separately identified as hepatocyte stimulating factor, monocyte inducer, β2 interferon, a B-cell stimulatory factor and few other similar descriptions.

Production of cytokines by cloning helped molecular characterization & brought some clarity. However, cytokine functonng invitro should not be straightaway extrapolated to in vivo physiological effects.

This is because cytokines can act in synergy, in sequence & even antagonistically.

IL-2 and IFN-γ together aid cell activation. IL-1 and IL-2 induce different receptors which may be required in a cascade reaction.

IFNγ and IFNα have opposite effects on production of IgE by B-lymphocytes.

The activation of human macrophages by IFNγ is opposed by several cytokines like IL-4 and TGFβ isoforms.

In fact cytokine effects can be inhibited in vivo by three different mechanisms:

The first mechanism is similar to the phenomenon of competitive enzyme inhibition. Cytokine analogues compete with the cytokines for the receptor. Thus the IL-1 inhibitor competes with the IL-1 for the receptor.

Extracelluar domains of cytokine receptors come apart and become free. As these were the components of the receptors with which cytokines were to bind, free complexes of cytokine-receptor fragments are formed which tie up cytokines in interacting with the target cells.

As mentioned and exemplified earlier, two cytokines can oppose each other function. These will have different receptors.

While the few effects of IL-6 were known, work with transgenic animals in which IL-6 was over expressed showed up its role in production of platelates and megakaryocytes.

Cytokines not only part of cell-cell communications, these are important for communication between different organs.

A parallel can be drawn between nervous system and immune system with their common feature of “learning and memory”. Both have different communication systems but with some common features.

Both systems, in fact, also interact. Several cytokines (e.g. IL-1, IL-6, & TNF) have effects on hypothalamus and pituitary.

Let us now continue our discussion of cells other than Tc cells which mediate cytotoxicity. Having learnt about what cytokines are our discussion will continue to include, even in more detail, the role which cytokines play in cytotoxicity.

NK cells

Also called, Null cells/ third population cells constitute about 5-15% of blood mononuclear cells. These do not have receptors like BCR nor TCR but recognise T-cells as well as mononuclear phagocytes. Many of these morphologically appear as large granular lymphocytes (LGL).

NK cells do have many receptor which include CD16 (receptor for Fc), CD2, CD69, lectin like receptors which allow these to bind to wide ranging cells.

KIR (killer immunoglobulin like receptors) are a family of receptors also present on NK cells. These recognise MHC molecules. KIRs occur in two forms. One with ITIH motif and another with ITAM adaptor like motifs in their cytoplasmic tails.

The number of KIRs (CD 158) varies from individuals to individuals. Not all NK cells express all the receptors. Each NK cell has a set of receptors which enable it to monitor change in one group of MHC molecules. Thus NK cells population is able to monitor change in the pattern of MHC expression by the cells of the host animal.

Two mechanisms help NK cells in not killing normal uninfected cells of the animal. This regulation operates via both activation and inhibition mechanism as explained while discussing the role f KIR receptors tracking change in the expression of MHC molecules on animal cell surface.

Lectin-like receptors are calcium binding c-type lectins which can monitor carbohydrate ligands on the animal cells. Intracellular bacterial or viral infections result in changes in expression of the cell surface glycoproteins.

Many viruses try to avoid immune system by downregulating the expression of MHC molecule of the infected cells. (How and why this may work will be clearer only when we discuss MHC in a later molecule). NK cells detect this via KIRs and kill such infected cells. This is so important that even some T-cells may also express some KIRs after activation.

The NK cells activity reflects the balance of signals from ITIM and ITAM which in turn are responding to change in the pattern of MHC of host animal cells.

Lectin-like receptors are a family, each member has two polypeptides NKG2 and CD94. Again these receptors are present on all NK cells and some Tc cells. These receptors recognize leader peptides of MHC molecules. This also serves the purpose of detecting loss of MHC molecule by a cell. These receptors also regulated by their cytoplasmic tails.

Some Toll like receptors on NK cells are also present which aid cytokine production.

NK cells are early defense in some intracellular infections

NK cells provide early defense to intracellular pathogens like herpes virus, protozoan parasite and bacterial pathogen Listeria monocytogens.

NK cells, even if untriggered can kill appropriate cellular targets in vitro. However, cytokines IFN-α, IFN-β and IL-2 produced in early phases of infections increase the NK cytotoxic activity by 20-100 times.

IL-2 + IFN-α induce NK cells to secret IFN-γ which is critical in immune defense before Tc cells secret IFN-γ. Mice lacking B- and T- cells resist Listeria monocytogen infection initially. Later, NK cells and IFN-α/ IFN-γ levels lowers and mice due to absence of adaptive immune response die.

We have referred to interferons (IFN) several times. Viral infections produce interferons IFN- α and IFN-β. Interferons are glycoproteins and double stranded RNA induce their synthesis both in vivo and in vitro. This RNA is not present in the mammalian cells but is either part of the viral genome or produced during their cycle of infection.

IFN-α and IFN-β bnd to a common interferon receptor on both infected and neighboring cells. This receptor uses Janus-family Tyr kinase mediated signal transduction pathway. STATs (signal transducing activator of transcription) induce synthesis of host proteins which inhibit viral replication.

Figure 22: Perforin released from the lytic granules of cytotoxic T cells can insert into the target cell membrane to form pores

Both NK cells and Tc cells have similar mechanism of cytotoxicity. In both cells, there are lytic granules.

These modified lysosomes have perforins and granzymes which are three serine proteases.

These trigger a cascade. An activated caspase CPP-32 which in turn activates a DNAse.

Degradation of DNA leads to apoptosis.

Development of T-cells

The term T-cells is derived from thymus derived cells. Thymus is necessary for the development and maturation of T-cells.

Thymus, in fact, also provides “thymic education” to T-cells which ensures that T-cells are able to discriminate between host self cells and non-self cells.

Thymus is a bilobed organ situated in front of the heart. Each lobe is organized into lobules.

Each lobule has peripheral cortex and central medulla.

The size of the thymus increases till puberty and thymus gradually atrophies thereafter. The lymphoid tissue is replaced by fat.

Some thymic function continues even in old age. The number of T-cells which matures out of the thymus gradually decreases with age.

With age there is an increase in IL-6 and M-CSF (macrophage colony stimulating factor), decrease in IL-2, IL-10 and IL-13, and no change in IL-7 and IL-15.

As briefly indicated earlier, thymus interacts with the endocrine system. Removal of the thymus results in decrease in levels of pituitary hormones and atrophy of the gonads. Conversely, removal of the pituitary leads to atrophy of the thymus.

Thymosin and thymopoietin are the hormones produced by epithelial cells of the thymus which alongwith cytokines (e.g. IL-7) mature thymocytes into T-cells.

The lymphocytes in the thymus are called thymocytes. It is in the thymus that TCR develops, T-cells reactive to self antigens are destroyed.

Pre T-cells reach thymus from the bone marrow and proliferate in the subcapsular region. Thus CD4-8- cells proliferate into CD4+8+ and reach the cortex region. Cortex contains 85% of the total thymocytes.

By the time these cells reach medulla region, they have functional TCR and are either CD4+ or CD8+.

T-cell precursors spend upto 7 days in thymus in differentiation. About 90-95% thymocytes die during the development phase by apoptosis. Chromatin residue forms resulting apoptotic bodies inside macrophages in the thymic cortex.

Two stage positive and negative selection rescue thymocytes from apoptosis. The positive selection originates through interactions with MHC molecules on the epithelial cells of the thymus. Only CD8+ cells which interact with an affinity within a critical range survive. Those with too high or too low an affinity to MHC molecules undergo apoptosis.

During the negative selections, those binding to self antigens undergo apoptosis. At this stage, one of the surface marker is downregulated and T-cells are either CD4+ or CD8+. The negative selection involves mostly dendritic cells (at the cortex-medullar junctions) and macrophages present in both cortex and medulla With this background, we are now equipped to understand why TCR is so complex and how it develops during thymic education However, before that we will learn about the various types of T-cells and MHC proteins. As we have briefly seen, MHC proteins play an important role in T-cell maturation.

Summary

•  Certain intracellular pathogens require cellular immunity for fighting against them

•  Even these pathogens have to deal with innate immunity

•  Many cells, of which, Tc and NK cells are prominent provide cytotoxicity for the infected cells

•  Cytokines play a key role in these cytotoxic processes

•  Thymus is the site wherein T-cells proliferate and mature