The body’s immune system works to combat infection by pathogens in numerous ways. A series of coordinated events take place to eliminate the infection and try and restore the body to the state it was prior to the invasion. The details of the various processes involved differ according to the specifics of the situation. In the case of an infection whereby the pathogen has managed to occupy the space within the cells the immune response will be triggered as would be the case for almost any other form of infection. The first line of defence against the pathogen, the innate immune response requiring the help of NK cells amongst others, will be stimulated. This will be followed by the secondary adaptive immune response to assist with the complete elimination of the pathogen if this has not already happened. In the case of intracellular pathogens, the T cells will be activated to carry out a cell mediated immune response. Although highly effective in the removal of a vast array of pathogens, many have evolved in such a manner that they can avoid such attacks making elimination a challenging process requiring enormous amounts of research to comprehend their behaviour and assist with their removal.
The Humoral Immune Response:
The antibody effectors of this response are well suited to tackling infection; however their highly specific nature is best directed towards antigens lying outside of the cell as they are incapable of reaching pathogens that have integrated their DNA within cells. They are therefore important in helping to contain the spread of the virus if viral material is released from cells into the environment. Total elimination of viruses therefore requires alternative responses.
The cell mediated immune response:
Unlike the antibody response of the humoral branch of immunity, this response incorporates activated macrophages, natural killer cells (NK), and antigen-specific cytotoxic T-lymphocytes (CTL) as well as release of a number of cytokines.
Cytotoxic T lymphocyte response:
One key feature distinguishing effector T cells from their former naïve selves is their ability to express both membrane bound and soluble effector molecules which play an important role in assisting with pathogen elimination. The membrane bound proteins often belong to the tumor necrosis family(TNF) of proteins. This includes the FAS Ligand (FASL) found on the CD8+ cytotoxic T lymphocyte(CTL) involved in the destruction of target cells. TNF-β located on TH1 cells is involved with promoting macrophage activation whilst the CD40 ligand located on TH2 cells is necessary for B cell activation. The soluble effector molecules include various cytotoxins such as perforins and granzymes as well as the cytokines IFNγ and TNF-β released by the cytotoxic T cells. Various other cytokines are also secreted by the helper T cells.
Identification of the target cell involves an initial weak interaction of the T cell receptor and the effector T cell with the antigen-presenting cell, resulting in the production of signals to the T cell if a peptide-MHC complex is recognised. CD2 and LFA-1 are key cell adhesion molecules located on the cell surface of the T cell enabling an extended interaction of approximately 5-10minutes following recognition. CD2 binds to LFA-3 whilst LFA-1 binds intercellular adhesion molecules (ICAMs) both of which are located on the antigen-presenting cells as well as many target cells. This recognition process results in the formation of a cytotoxic T lymphocyte-target-cell conjugate
These cells primarily function through detection of altered self cells and then go on to eliminate these cells. They recognise class I MHC molecules expressed on almost all nucleated cells of the body, as opposed to class II MHC molecules recognised by helper T cells. Activation of these T cells from their naïve state transforms them to a state where they are capable of killing target cells. Following the recognition event described previously costimulatory signals are then sent via the CD28-B7 interaction. IL-2 then interacts with a high-affinity IL-2 receptor resulting in a signal being initiated within the T cell. Proliferation and differentiation of the T cell follows completing the transformation to an effector helper or cytotoxic T cell. Although it is the cytotoxic T cells that eventually kill the target cell the helper T cells are involved in its activation and so indirectly participate in cell destruction as well.
Cytotoxic destruction by T cells:
There are two possible ways in which the initiation of this destructive pathway can occur. They both however have the same final outcome which is the destruction of the infected cell and the pathogen contained within via apoptosis.
Necrosis and apoptosis are two ways in which a cell may die. Necrosis unlike apoptosis involves a highly uncontrolled and toxic response. The cell is made to burst releasing all harmful content inside to its environment. Apoptosis is a much more controlled process where the cell actually works to bring about its own death though an orderly set of steps. Characteristic features of a cell undergoing apoptosis are the decrease in cell volume, modifications to the cytoskeleton, condensation of the chromatin and most importantly the fragmentation of all DNA within the cell. Rather than releasing this material haphazardly into the environment possibly causing more problems for cells in the local environment it releases this material via apoptotic bodies that are finally removed by macrophages.
The Perforin/Granzyme Pathway
This pathway of target cell destruction is facilitated by monomers of a pore-forming protein known as perforin. Serine proteases known as granzymes also assist with the process and act as fragmentins destroying proteins in the target cell. At present granzyme B is the most well understood fragmentin. Both are present in storage granules that are absent in the cytoplasm of the T cell prior to activation. Following activation however, golgi stacks and storage granules within the cell appear and alter their positioning within the cell, becoming concentrated in the areas closest to the junction with the target cell.
The two cell destructing granzymes and proteases are then released via exocytosis into the junction. The function of the perforin monomers is similar to that of C9 during complement mediated lysis as it tries to create pores within the cell membrane. It does this through the use of an amphipathic domain which it inserts into the cell membrane. This domain is only present after a conformational change occurring as the perforin contacts the target cell membrane. It then polymerises creating cylindrical pores in the membrane to enable the entry of granzyme. Granzyme B enters the cell membrane and commences a set of reactions. The outcome of this is the disintegration of all DNA present within the cell. In this way the infected cell is effectively removed and so is the virus so that infection can no longer spread to other cells.
Fas Dependent Pathway
This pathway usually occurs in the CTLs lacking the perforin and granzymes necessary for the earlier mentioned pathway to occur. It is however possible for both pathways to occur in the same cell. The transmembrane protein Fas on the target cell interacts with its ligand FasL located on the CTL membrane resulting in a series of signals causing cell death.
The final pathway:
Although the two above mentioned pathways begin in different manners they both initiate the same final set of processes. Enzymes belonging to the caspase family of cysteine proteases present in cells as inactive procaspases are activated. Activation can only occur following proteolytic cleavage, causing the activated caspase to go on and cleave other procaspases. Granzymes mediate this cleaving activity activating an initiator caspase in the target cell. Active caspase 3 is then created. Another complex present within the cell formed of DNase may be activated by caspase to become caspase activatable DNase(CAD). This is normally blocked by an the inhibitor I-CAD(Inhibitor of CAD). Active caspase 3 interacts with this complex leading to cleavage of I-CAD and the release of CAD enabling it to enter the nucleus and fragment DNA and initiate the apoptotic pathway.
The interaction of Fas with its natural ligand leads to the activation of an initiator caspase in the target cell. Fas which is associated with FADD(Fas-associated protein with death domain) cross-links procaspase 8 transforming it to activated caspase 8. This can then activate caspase 3. Caspase 3 then acts in the same way as the caspase 3 of the granzyme/perforin pathway to stimulate apoptosis within the infected cell.
Non-specific natural killer cells:
Natural Killer (NK) cells, thought to be a part of the innate immune response due to their early response to virally infected cells, work in much the same way as CTLs. However they differ in their mechanism of activation. They prevent the virus spreading while T cells are still being activated. T cells are able to completely remove the infected cells and viruses which come into activation approximately 7 days after infection as opposed to the 3 days required for activation by NK cells.
Healthy cells will present MHC molecules on their surface which will be recognised as ‘self’ molecules by NK cells. Infected cells may also present MHC molecules at their surface but with viral peptide in their peptide binding groove to be recognised by the T cell receptors of CTLs. The virus may however prevent MHC expression preventing cytotoxic killing of the cell. This recognition of ‘non-self’ by the NK cells leads to destruction of the cell.
Unlike T cells that require MHC-peptide recognition via a T cell receptor, NK cells work with two different categories of receptors. Whilst one category of receptors functions to deliver inhibitory signals to the NK cell the other delivers activation signals. There is a balance between inhibitory and activating signals defining the recognition of healthy from infected cells. Intracellular sequences of the NK receptors differ according to their function. ITAMs (immunoreceptor tyrosine phosphate activation motif) are associated with activating receptors. ITIMs (immunoreceptor tyrosine phosphate inhibitory motif) however are associated with the inhibitory receptors.
Both types of receptor can be divided into two further categories based on structural features. These receptors maybe lectin-like or immunoglobulin-like. The immunoglobulin-like receptors include KIR (killer immunoglobulin-like receptors) such as an inhibitory KIR2D or KIR3D that bind to MHC-I (HLA-A, HLA-B, HLA-C). At present it is not clear to what these KIRs bind to when activating the NK cell. The lectin-like receptors include amongst others CD94:NKG2 that can exist in two forms. CD94:NKG2A which is an inhibitory receptor while CD94:NKG2C delivers activating signals. CD94:NKG2D is another better understood activation receptor. Ligands to which it may bind, are molecules known as MIC-A and MIC-B often produced in response to cells under stress as will be the case when it is infected by pathogens.
If the cell is healthy with normal levels of MHC-I, these inhibitory receptors bind to MHC-I and send signals to prevent killing. However if there are very little to no MHC-I molecules being expressed on a cell, this recognition of non-self by the NK cell will prevent inhibition signals and activating receptors will simultaneously bind to stress ligands expressed on the cell surface.
The double stranded RNA produced by viruses are detected by Toll-like receptors (TLRs) that induce production of IFN-α and IFN-β by the infected cell, macrophages, and monocytes. These can then bind to NK cells inducing their lytic activity. The response is thought to be similar to that of the CTLs whereby granules containing perforin and granzymes are released into the junction to stimulate apoptosis. These granules are always present in the NK cells unlike the granules in T cells which are only present following activation. However the numbers of these granules increase dramatically in NK cells in the presence of TNF-α, IFN-α, IFN-β and IL-12, making them 20-100 times more efficient at killing.
NK cells also produce various cytokines such as IFN-γ which stimulates the participation of macrophages and stimulates them to increase their microbidal activity. It also, more indirectly stimulates the development of TH1 cells through induction of IL-12 by macrophages and dendritic cells. These TH1 cells can then go on to produce IFN-γ activating more macrophages to phagocytose apoptotic bodies. They also release IL-2 and IFN-γ which together help to promote the differentiation of fully developed CTLs.
Antibody-Dependent Cell-mediated Cytotoxicity (ADCC):
Although it is the cell mediated response that is vital in combating viral and pathogenic intracellular infection the humoral response can play an indirect role in eliminating them too. The antibody may bind to the antigen presented on the surface of the cell and the Fc portion of it is available for binding by Fc receptors. These are commonly found on NK cells, macrophages, monocytes, neutrophils and eosinophils. This brings the two cells in closer proximity enabling lysis of the target cell. This method of killing by non specific cells with cytotoxic potential via antigen specific antibodies is known as antibody-dependent cell-mediated cytotoxicity.
The correct response:
The incorrect balance of TH1 and TH2 cells could have detrimental effects on the body as can be seen with the intracellular pathogen causing Leprosy. The cell mediated response stimulated by a TH1-type cell is not actually capable in this case of completely eliminating the virus but can actually localise the pathogen to nodules, known as immune granuloma that wall off the infection, in the airway or gut. This results in tuberculoid leprosy. However if the wrong response were to occur due to a TH2–type response instead, then a more deadly form of lepromatous leprosy would occur. The bacteria will spread as the humoral response which would in this case be stimulated, would be incapable of handling such pathogens.
Viral Evasion of the Immune Response:
Although the immune system has evolved to combat infection, so too have viruses to combat destruction. Like the pathogen causing leprosy, numerous other viruses have evolved to find ways of evading the various immune responses.
Murine Cytomegalovirus (MCMV) has the ability to infect immature dendritic cells (DC) thus preventing their maturation into antigen presenting cells. This functional paralysis of DCs means they become incapable of stimulating an effective T cell response. As such the CTL response is unable to occur.(1) Human cytomegalovirus (HCMV) has the ability to inhibit NK killing as they cause the expression of MHC class-I heavy-chain homologues engaging inhibitory receptors thereby avoiding NK-mediated cytotoxicity. HCMV does so by encoding gpUL18 with a 21% amino acid sequence similarity to human polymorphic MHC class I molecules. (2)(3) These cytomegaloviruses amongst an array of viruses including HIV cause immunosuppression and successfully evade host defence mechanisms.
Therefore it can be seen that there are a number of diverse host mechanisms to respond to the challenge of intracellular infection including cytotoxic T cells, NK cells, macrophages and various cytokines. However, as the host has evolved to protect itself from invasion so too have the invaders and so it is likely that over time these mechanisms may evolve to become more complex and a range of other defences may also come into play, as we become locked in an evolutionary arms race with the pathogens invading our bodies.
(1)Andrews DM, Andoniou CE, Granucci F, Ricciardi-Castagnoli P, Degli-Esposti MA (2001) Infection of dendritic cells by murine cytomegalovirus induces functional paralysis. Nat Immunol 2
(2)Farrell HE, Vally H, Lynch DM, Fleming P, Shellam GR, Scalzo AA, Davis-Poynter NJ (1997) Inhibition of natural killer cells by a cytomegalovirus MHC class I homologue in vivo. Nature 386
(3)Reyburn HT, Mandelboim O, Valés-Gómez M, Davis DM, Pazmany L, Strominger JL (1997) The class I MHC homologue of human cytomegalovirus inhibits attack by natural killer cells. Nature 386
(4)Kindt TJ, Richard AG, Barbara AO (2004) Immunology (Sixth Edition) U.S.A : W.H. Freeman and Company