ACTIVATION OF T LYMPHOCYTES AND CELL MEDIATED IMMUNITY The recognition of specific antigen by naïve T cell induces its own activation and effector phases. T helper cells recognize peptide antigens through its TCR when the antigen is presented to it by an antigen presenting cell along with self-mhc II molecules. This stimulation is strengthened by binding of CD4 molecule of T cell to β2 chain of MHC II protein. Cytotoxic T cells recognize peptide antigen when presented to it with MHC class I proteins. The CD8 molecule of cytotoxic cell binds to α3 domain of class I molecule. The primary stimulation is further enhanced by binding of other accessory molecules; these include CD28 of T cells to B7 of APCs, LFA-1 of T cells to ICAM-1 of APCs, and CD2 of T cells and LFA-3 of APCs. Antigen recognition by effector T cells triggers the effector functions that eliminate the antigen. The outcome of T cell antigen recognition is determined by the duration and affinity of the TCR-antigen interaction. On any APC, approximately only 1000 out of ~10 5 available MHC molecules are likely to display any one peptide at any time. Incomplete signaling may not lead to any response; insufficient signaling may even inactivate the T cell. Dendritic cells and macrophages are efficient APCs because they express high number of MHC molecules and co-receptors than other APCs. Memory and effector T cells have less threshold for activation than naïve T cells and can respond to antigens that are presented by other APCs. Memory and effector T cells can be stimulated in peripheral tissues outside the secondary lymphoid organs. This allows quick response by T cells wherever they encounter the same antigen. If a recently activated T cell is repeatedly exposed to same antigen, some T cells undergo apoptosis. This is called activation-induced cell death and plays a role in self-tolerance. In the absence of co-stimulation, T cells either fail to respond and die by apoptosis or enter a state of unresponsiveness called anergy. Binding of CD28 to B7-1 or B7-2 on APCs delivers signals that enhance T cell response to antigen, prolong cell survival, production of cytokine (IL-2) and differentiation of naïve cells to effector cells. On APCs other than macrophages and dendritic cells, B7 molecules are expressed at low levels but can be induced to increase expression by bacterial endotoxins, IFN-γ, and binding of T cell CD40L to CD40 on APCs. Adjuvants are also known to induce expression of co-stimulators. APCs in normal tissues contribute to maintenance of tolerance to self-antigens. This is because even though such cells are able to process and present self antigens, lack of co-stimulation results in no activation of T cells. In addition to CD28, other T cell receptors like CD2 can deliver co-stimulatory signals. CD-2 binds to its ligand LFA-3 on APC and delivers costimulatory signals even in the absence of CD28. The interaction of CD40L on T cell with CD40 on APC enhances T cell activation by inducing the expression of B7 molecule on APC and inducing the APC to secrete cytokines such as IL-12 that act on T cells. IL-12 provides necessary signal for T cell proliferation.
CD28-B7 interactions and CD2-LFA-3 interactions promote T cell activation by enhancing transcription in T cell, increase the production of cytokine (IL-2), and promote T cell survival. The recognition of antigen initiates a sequence of biochemical signals in T cells that result in activation of certain genes and the entry of cell into cell cycle. The genes that are activated are responsible for production of proteins that mediate effector functions. The cellular response of T cells to antigens consists of three stages: 1. membrane events, which occur within seconds 2. cytoplasmic signal transduction pathways, which occur within minutes 3. new gene transcription, which occur within hours One of the first steps in activation of T cells is the production of IL-2 by the antigen-stimulated T cell. IL-2 functions as growth and differentiation factor for T cells. On stimulation, T cells also increase the expression of receptors for many cytokines. One such activation receptor that is produced by T cell is IL-2 receptor. The activated T cells secrete IL-2, which act on themselves; hence IL-2 is considered an autocrine growth factor. This results in clonal proliferation of the activated T cells. The progeny of antigen-stimulated CD4+ T cells differentiate into effector T cells that produce cytokines. Subsets of effector CD4+ T cells, namely Th1 and Th2, secrete different cytokines and perform different functions. CD8+ T cells differentiate into functional cytotoxic T cells. The differentiation of T cells is associated with production of effector molecules such as cytokines, cytotoxic granule proteins and ligands such as CD40 Fas protein. The major effector function of CD4+ T helper cells is to activate macrophage and other lymphocytes (e.g., B cell) by the production of cytokines whereas the major effector function of CD8+ T cells is to lyse the antigen bearing target cells. The cytokines produced by helper T cells act on themselves, other T cells, B cells, macrophages, granulocytes and vascular endothelium. Thus these cytokines participate in cell mediated 2
immunity, humoral immunity as well as inflammation. Macrophages and B lymphocytes that present antigens to T helper cells, get activated by the same T cell. Cell mediated immunity (CMI) is the effector function of T lymphocytes. CMI can be transferred to another individual passively by transfer of viable T lymphocytes, this is termed adoptive immunity. It is the principal mechanism of defense against those pathogens that survive within macrophages or infect non-phagocytic cells. Defect in CMI leads to increased susceptibility to virus and intracellular bacteria. T cells that are responsible for activation of macrophage are the differentiated Th1 subsets of CD4+ T cells as well as CD8+ T cells, since both these cells are capable of secreting macrophage activating cytokine, IFN-γ. CMI is responsible for rejection of grafts containing cells that exhibit non-self MHC proteins as well as elimination of cells expressing tumor antigens. T cells also produce tumor necrosis factor (TNF) and lymphotoxin, which promote inflammation. T cell mediated macrophage activation and inflammation may lead to tissue injury and this reaction is called delayed type hypersensitivity (DTH). Some chemicals introduced through skin bind to and modify the self-mhc proteins or molecules on the surface of APC, which are then recognized by CD8+ or CD4+ T lymphocytes. This type of DTH is called contact sensitivity. Role of CD4+ T cells: When protein antigens enter an individual, either they are captured by Langerhans cells in the skin (and similar cells in mucosa) and transported to draining lymph node or the antigen is drained to regional lymph node. The antigens are captured by dendritic cells in lymph nodes and presented on their surface with MHC proteins. During their migration to the regional draining lymph node, Langerhans cells express MHC class II molecules and mature into professional APCs, where they are called dendritic cells. Macrophages too engulf the foreign antigen, process it and present on their surfaces with MHC class II proteins. Presences of foreign antigen at the site of infection also induce innate immunity, which stimulates the APCs to express high levels of co-stimulators such as B7 proteins. Native T lymphocytes also migrate to the same peripheral lymphoid organ and encounter the processed antigen. After the naïve T cells encounter the antigen and receive the necessary co-stimulus, they secrete autocrine growth factor, IL-2. The cell then enters the cell cycle and the antigen-specific T cell clonally proliferates. Antigen specific CD8+ T cells also undergo clonal proliferation when they are suitably presented with antigen. Upon antigenic stimulation, T cells starts expressing CD40L in high numbers, which binds to CD40 expressed on the surface of APC. This stimulates the APC to start secreting IL-12. IFN-γ produced by NK cells during innate immune response also act on APC to induce IL-12 secretion. IL-12 acts on the activated T cells and induce the differentiation into Th1 subsets. Th1 effector cells produce IFN-γ, which acts on the APC resulting in increase in its microbicidal functions as well as further increase in IL-12 production. This results in further development of Th1 cells and thus provide an amplification of CMI. Some of the activated T cells develop into memory cells. Both the effector and memory cells leave the lymph and node and enter the circulation. To trigger the effector phase of CMI, effector or memory cells have to come in contact with APC presenting the antigens that initiated the response. If the response is against microbes that are ingested by macrophage in lymphoid organs, the effector T cells can locate the phagocyte in the same organ and activate them to kill the pathogen. If the infection is at some other location, the effector T cells leave circulation and enter the site of infection. Migration of leucocytes to sites of infection is stimulated by cytokines, which induce leucocyte chemotaxis and the expression of adhesion molecules on endothelial cells. Cytokines are produced by macrophages and endothelial cells that are stimulated by microbial products. Important cytokines participating in this process are the TNF and chemokines. TNF activates endothelial cells to express ligands for leucocyte adhesion molecules, resulting in attachment of leucocytes to the endothelium at the site of infection. Chemokines induce transendothelial migration of the attached leucocyte into the extravascular tissue. These cytokines are also produced by NK cells during innate immune reactions. An early inflammation characterized by leucocyte 3
influx may occur as part of innate immunity much before the T cell migration. After the T cells enter the site of infection and are activated by antigen, they stimulate much greater leucocyte migration. The effects of inflammation facilitates the migration and subsequent retention of leucocytes in the extravascular tissue. Previously activated effector and memory cells migrate preferentially to peripheral tissue at the site of infection because their migration is mediated by adhesion molecules, integrins and selectins. Activated T cells express ligands for selectins and express high levels of integrins. The endothelium at sites of infection express high levels of selectins and ligands for integrins. Once in the tissues, T cells encounter microbial antigens presented by APC. Once the T cell recognizes the antigen, the affinity of integrins to their ligands also increase. Two such integrins, VLA-4 and VLA-5 bind to fibronectin in extracellular matrix. Another adhesion molecule CD44, which is also expressed at high levels, binds to hyaluronate. As a result, antigen-specific T cells are preferentially retained at the site of infection. T cells not specific to the antigen returns via the lymphatic vessels to the circulation. Once the activated T cells encounter the specific antigen, they are further stimulated to perform their effector functions, which is to stimulate the microbicidal activities of macrophage. Monocytes that leave circulation and enter the site of infection are acted upon by the cytokines produced by effector T cells. This results in the conversion of inactive monocytes into activated macrophages that are able to engulf and kill microbes. The effector T cell that binds to the macrophage presenting the antigen activates it by production of IFN-γ and by binding of CD40L to macrophage s CD40. In response to CD40 signals and IFN-γ, production of several proteins in macrophage is increased. The effector functions of activated macrophages are: 1. Activated macrophages kill phagocytosed and extracellular microbes by producing microbicidal reactive oxygen intermediates, nitric oxide and lysosomal enzymes. 2. Activated macrophages stimulate acute inflammation through secretion of cytokines such as TNF, IL- 1 and chemokines. The result of this inflammation is to bring in neutrophils, which phagocytose and destroy pathogens. 3. Activated macrophages remove dead tissue and facilitate repair after the infection is controlled. They also secrete growth factors such as platelet-derived growth factor that stimulate fibroblast proliferation and transforming growth factor-β (TGF-β) that stimulate collagen synthesis. Tissue injury associated with CMI: Some tissue injury may normally accompany CMI reactions to microbes in the tissue because the microbicidal products released by the activated macrophages and neutrophils are capable of damaging normal tissue, and do not differentiate between microbes and host tissue. Usually, this tissue injury is limited in extent and duration, and it resolves as infection is cleared. If the activated macrophages fail to eradicate the infection, they continue to produce cytokine and growth factors, which progressively modify the local tissue environment. As a result, tissue injury is followed by replacement with connective tissue (fibrosis), and fibrosis is a hallmark of chronic DTH reactions. In chronic DTH reactions, activated macrophages also undergo changes in response to persistent cytokine signals. These macrophages develop increased cytoplasm and cytoplasmic organelles and histologically resemble skin epithelial cells because of which they are called epitheloid cells. Activated macrophages may fuse to form multinucleated giant cells. Clusters of activated macrophages, often surrounding particulate sources of antigen, produce nodules of inflammatory tissues called granulomas. Granulomatous inflammation is characteristic response to some persistent microbes, such as Mycobacterium tuberculosis and some fungi and represents a form of chronic DTH. Granulomatous inflammation is frequently associated with tissue fibrosis. Although fibrosis is normally a healing reaction to injury, it can also interfere with normal tissue function. 4
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Role of CD8+ T cells: Cytotoxic T lymphocytes are effector T cells that recognize and kill target cells expressing foreign antigens in association with MHC proteins. The foreign antigen that is presented on the surface of the APC could be a viral protein, tumor protein or a bacterial protein that has escaped the lysosome and entered the cytosol. Most cytotoxic cells are CD8+ T cells that recognize antigen when presented with class I proteins. There are few CD4+ T cytotoxic cells that recognize antigen when presented with class II proteins. Differentiation of naïve CD8+ T cells to functional cytotoxic cells require the recognition of class I associated peptide antigen and co-stimulators (or cytokines). CD8+ T cells that leave the thymus after maturation and enter circulation or peripheral lymphoid organs are not fully differentiated to lyse target cells. These undifferentiated, functionally inactive CD8+ T cells are often called pre-ctls. First signals in the activation of pre-t cells into effector cytotoxic cells is the binding of TCR to peptide-class I proteins on APCs. Tumor cells or viral infected cells are often engulfed by macrophages, their antigens move from the phagosome into the cytosol and are presented in association with class I proteins. These professional APCs can provide co-stimulation for the T cells by the same they do for CD4+ T cells. The same antigen would also be processed and presented with class II proteins by APC to CD4+ T cells and activate them. Activated CD4+ T cells, if present in the vicinity of CD8+ T cells, provide cytokine signals that stimulate CD8+ T cells. Helper T cells produce IL-2, which stimulate the clonal proliferation of CD8+ T cells. Helper T cells bind to APC via CD40L and induce them to become more efficient at stimulating the differentiation of CD8+ T cells. Activation of CD8+ T cells by antigen along with second signal lead to their proliferation and differentiation into effector T cells capable of cytolytic functions. The most specific feature of cytotoxic cell differentiation is the development of membrane bound cytoplasmic granules that contain proteins, including perforins and granzymes. The differentiated cytotoxic cells also secrete IFN-γ, TNF and lymphotoxin, which activate phagocytes and induce inflammation. The effector cytotoxic cell binds to the target expressing the same 6
antigen with MHC class I molecule. TCR on the T cell binds to the peptide and CD8 of T cell binds to α3 chain of class I protein on APC. LFA-1 on T cell binds to ICAM-1 on APC. These signals are sufficient for activation of cytotoxic cell. Co-stimulators and cytokines that are required for the differentiation of pre-ctls into active CTLs are not necessary for triggering their effector functions. The principal mechanism of CTL-mediated cytolysis is the delivery of cytotoxic granule proteins to the target cell recognized by the CTL. The cytoplasmic granules of CTL fuses with the plasma membrane and releases the granules. The two most important granule proteins are perforins and granzymes. Perforin is a pore forming protein that is present as a monomer in the granules. When it is exocytosed fro the granules, the perforin monomer comes into contact with high concentration of calcium and undergoes polymerization. Polymerization of perforin occurs in the plasma membrane of target cell and thereby produce many large channels. The cell dies as a result of influx of water as well as apoptosis brought in by high levels of calcium. Granzymes are serine proteases which cleave proteins. They enter into the target cell through the pores created by perforins. Granzyme B proteolyticaly cleaves and activates cellular enzymes called caspases, which in turn cleave several substrates and induce apoptosis. CTLs use another mechanism of cytolysis that is mediated by interaction of membrane molecules on the CTLs and target cells. CTLs express a membrane protein called Fas ligand (FasL), which binds to its target protein Fas that is expressed in many cell types. This interaction also results in activation of caspases and apoptosis of the target cell. During the target cell cytolysis, the enzymatic cleavage occurs of host as well as microbial proteins and DNA. CTLs is then released from the target cell, which may be due to decrease in affinity of accessory molecules for their ligands. CTLS remain unaffected and usually disengage before the actual cytolysis occur. 7
Roles of Th2 cells in CMI The principal function of Th2 cells in CMI is to suppress the inflammatory reaction by cytokines that inhibit macrophage activation. Th2 cells secrete IL-4 and IL-13 that are antagonists of IFN-γ and thus block T cell mediated macrophage activation. Th2 cells also secrete IL-10, which directly inhibit macrophage activation. Th2 usually appear late in the immune response and the cytokine they produce perhaps play a role in limiting the damage brought about by CMI. Some Th2 cells also produce TGF-β, which suppress T cell proliferation and inhibit macrophages. Th2 cells also play a vital role in immunity to helminthic infections since they induce inflammatory reactions that are dominated by eosinophils and mast cells. Immune response to helminths largely consists of Th2 cells, which secrete IL-4 and IL-5. IL-4 stimulates the production of helminthic-specific IgE antibodies, which opsonize the helminths. IL-5 activates eosinophils, which bind to IgE coated on helminths. Degranulation of their contents leads to death of the helminth. Duration of CMI The functional response of T cells to antigen stimulation last only for few days or weeks, thereafter the response diminishes. Once the foreign antigen is eliminated after successful immune response, the T cell response by the effector cells also decline. The decline in T cell response is due to the death (apoptosis) of antigen-activated T cells. As the antigen is eliminated, the lymphocytes no longer get the survival stimuli that was provided by the antigen, the co-stimulators and the inflammatory cytokines. Some of the progeny of antigen-stimulated T cells survive and develop into antigen-specific memory T cells. Memory T cells survive for long periods even in the absence of continuous antigen exposure. The mechanism of memory cell survival is not known. These memory cells are responsible for enhanced and accelerated secondary immune response. Memory cells accumulate with age and it is believed that half of all T cells in adults are memory cells. Memory cells express high levels of integrins and CD44, which promote their migration to peripheral sites of infection and inflammation and their retention in those sites. This property enables the memory cells to rapidly locate and eliminate antigen at any site of infection or inflammation. Only upon re-exposure to the same antigen, memory cells proliferate and differentiate into effector cells. 8