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CD8+ T Cell Response to Influenza Virus by W MG Daniel Doty HUSETTSIITUTE OF TECHNOLOGY SEP 17 B.S., Molecular and Cellular Biology (2002) University of Arizona ;IRAFRIES_J Submitted to the Department of Biology in partial fulfillment of the degree of Master of Science in Biology at the Massachusetts Institute of Technology September 2005 ©2005 Massachusetts Institute of Technology. All rights reserved. Signature of Author .:,.Department of Biology 2 September, 2005 Certified by Dr. Jianzhu Chen Professor of Biology Thesis advisor Accepted by Stephen P. Bell Professor of Biology Chair, Biology Graduate Committee CD8+ T Cell Response to Influenza Virus by Daniel Doty Submitted to the Department of Biology on 2 September 2005 in partial fulfillment of the requirements for the degree of Master of Science in Biology ABSTRACT The flu is an extremely prevalent and potentially devastating disease, especially dangerous to the very young, the elderly, and to people with compromised immune systems. Influenza has a characteristic course of infection, and is often effectively dispatched by the immune system. The cell-mediated lysis of infected cells is a particularly important step in clearing the infection. Antigen specific CD8+ T lymphocytes are selected and activated in the mediastinal lymph node, proliferate and gain effector function, then migrate to the lungs, where they selectively destroy infected cells. The CD8+ effector population pool undergoes a phase of contraction, when most effector cells die. Those that survive become memory T cells, protecting the body from subsequent influenza infections. The molecular and cellular interactions that comprise the CD8+ cytotoxic T cell response to influenza virus are of particular interest because of their implications for the prevention, treatment, and alleviation of the flu. Thesis advisor: Jianzhu Chen Title: Professor of Biology 9 Introduction Influenza A virus is the cause of one of the most prevalent and harmful human respiratory infections. Typical incarnations of the virus infect 10-20% of the population of the United States, resulting in billions of dollars in lost production and health care costs, as well as up to 40,000 deaths each year (1). Influenza became the agent of the most devastating plague in human history when a particularly virulent strain killed an estimated 40 million people worldwide in the winter of 1918 (2). No disease has ever killed so many in so short a time. Influenza is dangerous because it is highly contagious - the disease spreads readily by aerosol - and because new, highly virulent strains periodically emerge (1). The influenza genome is segmented, and as a result, flu can recombine very easily among different strains. If a cell is infected by two different strains of flu, the progeny that bud out from that cell can have an assortment of genes taken from both viruses. This can have extremely dangerous consequences. Pigs, for instance, can become infected with both avian and human strains of influenza. A new strain of flu virus may arise in concurrently infected pigs that is able to infect humans, but appears completely foreign to any established humoral immunity against ordinary strains of flu, a phenomenon called antigenic shift. The emergence of such a "swine flu" in 1976 prompted the Ford administration to attempt to vaccinate every American, an event that highlighted the looming threat of such emergent strains of influenza (2). Other potential pandemics occasionally arise, but they have thus far been quickly contained by organizations such as the World Health Organization that are constantly vigilant against the emergence of dangerous new flu strains. In addition, the virus can escape from protective immunity by frequent mutation in viral antigens; a phenomenon called antigenic drift. As opposed to chicken pox, for example, where a person who contracts the disease is usually protected for a lifetime from subsequent infection, a person who had the flu or was vaccinated last year is not likely to be protected against the disease in this flu season. Flu vaccines must be formulated every year against the strains that are predicted to be the most prevalent in the coming flu season (1). The constant aggravation and potential threat of influenza make it important to understand the virus and the protective immune response against flu infection. Cytotoxic CD8+ T cells are crucial to the normal clearance of flu infections. Focusing on CD8+ T cells is useful because they provide more general protection against influenza than anti-influenza antibodies. Although antibodies can provide sterilizing protection against the strain for which they are specific, CD8+ T cells can respond to more conserved regions of the influenza proteome, providing greater protection against infection with new strains of flu. It is likely, then, that vaccine strategies that induce both humoral and cellular memory would provide much greater protection against flu infection than the flu shot, which only stimulates an antibody response. Initiation of infection Influenza A is a single-stranded (-)sense RNA virus of the Orthomyxoviridae subfamily. The genome is divided into eight segments, bound with nucleocapsid protein (NP) into ribonucleoprotein complexes inside the capsid. Surrounding the capsid is a lipid membrane envelope, which is studded with two major surface glycoproteins, neuraminidase (NA) and haemagglutinin (HA), and lined with matrix protein (MP 1) (3). This membrane makes influenza relatively vulnerable to deactivation by drying, heat, detergents, and solvents. Since NA and HA are exposed to the outside of the viral particle, they are the proteins against which the immune system forms an antibody defense to flu (4). NA and HA are highly mutable, and mutations in these genes can render antibodies that recognize the unmutated form to be useless against the mutant virus. Antibodies are so specific to spatial conformations that small genetic changes can have disproportionately large antigenic consequences. This exerts substantial selective pressure to promote new strains of flu (5). Influenza spreads when mucous droplets containing virus particles are aerosolized through sneezing or coughing of the infected individual and inhaled by a new host, exposing the epithelial cells of the upper airway and lung to virus. The virus attaches to the cell surface by binding HA to mucoproteins containing terminal N-acetyl neuraminic acid groups. Neuraminidase can free the virus from these bonds, preventing the sequestration of the virus by inappropriate cell types that will not promote replication. Virus particles are endocytosed, and are engulfed within endosomes in the cytoplasm. These endosomes are acidified and proteolysis of their contents begins. HA is cleaved by metabolic proteases, causing a conformational change that activates a membrane fusion domain, which then fuses the viral envelope with the membrane of the endosome. MP1 releases the nucleocapsid, spilling it into the cytoplasm (6). Nuclear targeting sequences in the NPs translocate the viral genome into the nucleus (7). In the nucleus, viral RNA is transcribed by the three polymerase polypeptides associated with each genome segment: PBI, PB2, and PA. Viral proteins are then translated by the cell's own ribosomes (3). Initiation of the innate immune response Early in an infection, cytotoxic T cells are not yet activated to destroy infected cells. The immune system must take steps, then, to slow infections until the adaptive immune system is properly armed and deployed. An important early cellular response to replication of many viruses is to produce Type I interferons alpha and beta (IFN-ca and -s). These molecules serve several important functions as a warning signal from infected cells. IFNs-u and -P are quickly produced, likely due to a strong reaction of host cells to double-stranded RNA, which is not normally found in vertebrate cells, but is produced during the influenza infectious life cycle (8 and 9). Type I interferons bind to interferon receptors on nearby cells, as well as on the releasing cell, which induces a Jak-Stat signal transduction pathway that activates the transcription of several genes with antiviral functions (10). One of these genes activates an endoribonuclease that degrades viral RNA. Another IFN-inducible gene phosphorylates eIF-2, a eukaryotic protein synthesis initiation factor, which inhibits translation and thereby slows viral replication. Mx is another interferon-inducible protein, which interferes with influenza virus replication by forming a complex with NP and thereby blocking its activity in viral transcription. Mice that lack the gene for Mx are much more susceptible to influenza infection than genetically normal mice (11). Influenza partially suppresses early Type I IFN signaling with the viral protein NS 1, which represses IFN synthesis by preventing activation of the signaling pathway responsible for inducing transcription of IFN genes, the NF-KB signaling pathway (12 and 8). zI These early defenses are vital to the immune response to flu, but the adaptive immune system is required to clear the infection. The cellular immune response is dependent upon the presentation of viral antigens on host MHC molecules. A CD8+ T cell will recognize its target cell when its T cell receptor (TCR) binds a particular peptide:MHC complex. Proteins within the cytosol, viral and host alike, are regularly broken down by the proteasome and replaced with newly synthesized proteins. The result is a large pool of short peptide sequences, a subset of which are able to bind stably to MHC class I molecules. Peptides are actively transported into the endoplasmic reticulum by a heterodimer consisting of Transporters associated with Antigen Processing-I and -2 (TAP-1 and -2). The TAP transporter preferentially transports peptides that are likely to bind MHC I. Peptides are assembled with MHC I in the endoplasmic reticulum and then the complex is transported to the cell surface (4). Each cell displays on its surface, then, a representation of the proteins produced within that cell; including viral proteins, if present. Antigen specific CD8+ T cells use this display to recognize foreign proteins within host cells. Type I IFNs also aid in fighting viral infection by increasing the likelihood that an infected cell will be destroyed, ending its potential as a virus factory. In order to avoid or delay the cellular immune response, many viruses reduce MHC expression. Type I IFNs induce upregulation of MHC class I, leading to more antigens being presented on the cell surface (8 and 4). This increases the probability that an infected cell will be targeted and destroyed by an effector CD8+ cytotoxic T cell, making T cells more efficient at clearing infection. Natural killer cells (NK cells) are also activated by Type I interferons, and these provide an important early defense against infection (4). NK cells can differentiate between infected and uninfected cells and kill infected cells using the same molecular tools as cytotoxic T cells, though NK cells are not antigen specific and require a separate set of signals in order to lyse target cells. Killer Inhibitory Receptors on NK cells transmit an inhibitory signal if they encounter MHC class I molecules on the cell surface (4). Those cells that upregulate MHC I in response to Type I IFNs are protected from NK cells, then, while infected cells that downregulate MHC I are more likely to be destroyed by NK cells. NK cells further aid the immune response by secreting cytokine signals such as IFN-a and the Type II interferon y (4). CD8+ T cell activation Up until this point in the infection, the adaptive immune system has not played much of a role. CD8+ T cells are not activated at the site of infection. Nafve T cells are not armed for lysis and will not mount a response against infection without help. CD8+ T cells are first activated in peripheral lymphoid tissues. Dendritic cells take in antigen at the site of infection in the lung and travel to the draining lymph node, which for the lung is the mediastinal lymph node (MLN). Respiratory dendritic cells migrate rapidly to the MLN during the first 24 hours after infection (13). Dendritic cells take up residence in the lymph node and present the antigens they have gathered. T cells migrate into the node through the afferent lymph duct and come in contact with the antigen presenting dendritic cells. When a T cell and a dendritic cell come in contact with one another, binding of adhesion molecules on their surfaces induce changes in each other's cytoskeleton that leads to the formation of an immune synapse (14). This increases the concentration of MHC and TCR molecules at the synapse, making antigen presentation more effective. If a T cell recognizes antigen presented by a dendritic cell, signaling of the T cell receptor (TCR) will create a conformation change in an adhesion molecule on the surface of the T cell, LFA-1, that will allow it to bind more tightly to ICAM adhesion molecules on the dendritic cell (14 and 4). This increases the interactions between these cells and improves the probability that a T cell will mount a response to antigen presented by a dendritic cell. As the T cells flow through the dendritic cells, rare antigen specific T cells bind their TCRs to peptide:MHC complexes and stay in the lymph node, while those cells that are not specific for any of the antigens being presented leave through the efferent lymph duct. This allows the lymph node to select for antigen specific T cells as an affinity chromatography column may select for specific proteins. Non-specific T cells enter the bloodstream and recirculate to other lymph nodes so that they may one day find their antigens and participate in an immune response, should such an antigen be presented. The cellular immune response is relatively slow to react to infection, but also very potent. A large pool of activated nonspecific T cells could be damaging to tissues in the body, and so a balance must be made between aggressive responsiveness and specificity. Nafve cytotoxic CD8+ T cells require more than one signal in order to activate. These signals are provided by dendritic cells in the draining lymph node, and not by other tissues. The first and most obvious signal is the TCR-peptide:MHC interaction. When TCR binds peptide:MHC, receptor-associated kinases phosphorylate tyrosine-based activation motifs (ITAMs) on the intracellular signaling region of the TCR complex, which then allows a protein called Zap 70 to bind to this region (4 and 15). CD8 on the T cell also binds to MHC, which brings CD8 and TCR in close proximity. CD8 associates with a cytoplasmic tyrosine kinase called Lck, and binding of CD8 to MHC class I brings Lck very close to the intracellular signaling region of the TCR complex, where it can activate Zap 70. Zap 70 initiates several signal transduction cascades that lead to transcriptional changes in the T cell that cause it to proliferate and to differentiate into an effector T cell capable of recognizing and killing infected cells (4 and 15). In summary, activation of Zap 70 mediates the transition from naive to effector T cell, but this requires the binding of both TCR and CD8 to MHC on the target dendritic cell. This signal alone is not sufficient for proliferation of a naive T cell, however. A costimulatory signal is required for proliferation. B7, a molecule present on the surface of dendritic cells, provides an important costimulatory signal by binding to CD28 on the surface of the T cell. CD28 signaling results in an increase of proliferation, cytotoxicity, and cytokine secretion (4 and 16). CD28 is necessary for the synthesis and secretion interleukin-2 (IL-2), which is a powerful T cell growth factor. T cells that do not receive a CD28 costimulatory signal do not produce IL-2 or the IL-2 receptor and will become anergic or die (16). The requirement of a costimulatory signal prevents T cells from becoming activated by healthy cells, which present antigen, but lack B7. Other molecules have also been described as costimulatory, either by promoting more efficient engagement of TCR or by providing additional signals that augment cell survival, promote cell division, or induce effector functions such as cytokine secretion. These costimulatory signals may reinforce TCR signaling by sharing common pathways and signaling intermediates, or may I; comprise distinct signals that do not overlap. It seems, though, that several costimulatory signals may be required to mount an effective CD8T T cell response (17). Proliferation and Differentiation T cells that are activated in the MLN proliferate quickly between days 3-4 post infection (18). During this time, the population of antigen-specific T cells grows extensively, and these cells also undergo extensive changes in gene expression. The changes made to a CD8+ T cell during this phase transforms it from a rare, resting naive cell into a large pool of effector clones. T cells alter their complement of adhesion molecules in order to migrate properly to the site of infection and target infected cells. Effector molecules lose L-selectin, which naive cells use to home to lymph nodes (18 and 4). Expression of VLA molecules is increased, which binds to V-CAM adhesion molecules expressed on inflamed endothelial cell surfaces, and are required for effector T cell retention in the lungs (19). These changes allow effector T cells to migrate to leave the lymph node and efficiently enter the site of infection. Other adhesion molecules, such as LFA-i, are also upregulated that increase interactions between T cells and potential target cells (20). Effector CD8+ T cells also undergo changes that allow them to kill target cells once they arrive at the site of infection. Effector molecules that contribute directly to killing of target cells, such as perforin and granzyme, are greatly upregulated. Fas ligand is expressed on the cell surface, which allows cytotoxic T cells to induce apoptosis in target cells. CD8+ T cells also lose their requirement for costimulation, allowing them to destroy virtually any target cell that presents the right peptide:MHC class I complex (4). Signaling through the TCR becomes more sensitive, as expression of the tyrosine phosphatase CD45 changes. CD45 de-phosphorylates the inhibitory tyrosine on a Srcfamily kinase that participates in phosphorylating TCR ITAMs: it turns on a protein that promotes TCR signaling. A balance is present where a protein called Csk turns off this kinase and CD45 turns it back on. Effector CD8+ T cells switch to a variant splicing of the CD45 gene that associates with the TCR complex (15). This shifts the balance so that more of the TCR associated tyrosine kinases are in a ready state. This makes it more likely that the kinases required for TCR signaling will be ready when TCR binds peptide:MHC, increasing the sensitivity of T cells for antigen recognition. Effector function Armed effector T cells leave the MLN and migrate to the lung at about day 5 after infection with influenza (18). These T cells are guided to the lung by chemokines and by cell adhesion molecules on the target tissue. By days 6-7, there is significant accumulation of effector T cells in the lung (18). The appearance of armed effector CD8+ T cells in the lungs marks a significant reduction in viral load. Effector CD8+ T cells are highly selective and efficient killers, making viral replication much more difficult. When a CD8+ T cell comes in contact with a target cell, its TCR engages the antigen displayed on class I MHC. CD8 also binds to MHC, bringing together the factors needed for TCR signaling. Due to the changes made in differentiating to effector cells, TCR signaling is much more sensitive than in naive T cells and can respond more readily to lower levels of antigen. One of the results of Zap 70 activation in both naifve and effector CD8+ T cells is the activation of phospholipase C-y and an increase in 7 intracellular Ca2+ due to IP3 signaling (4 and 15). In naYve T cells, this Ca 2+induces activation of transcription factors that affect differentiation. In effector CD8+ T cells, this calcium induces the release of lytic vesicles containing perforin and granzyme. Signals within the T cell direct the release of these vesicles towards the target cell. These vesicles maintain conditions that render these molecules inactive and harmless to the T cell. When released, the effector molecules are exposed to conditions that allow them to destroy the target cell (4). Perforin polymerizes to form pores in the membrane of the target cell. Granzymes are serine proteases that are able to enter into the perforin pores and begin to cleave proteins within the target cell. Accumulated damage from granzyme digestion and compromised membrane integrity kill the target cell. Meanwhile, Fas ligand on the surface of the CD8+ T cell binds to Fas on the target cell, activating the target's powerful apoptotic pathway and inducing cell suicide (15 and 4). Several cytokine signals are secreted by effector CD8+ T cells that help coordinate the immune response. Effector CD8+ T cells produce tumor necrosis factor-c and -P (TNF-a and -) and IFN-y (21). TNFa activates endothelial cells, quickly changing their adhesion properties to allow recruitment of a variety of immune cells (9). IFN-y inhibits viral replication and activates macrophages, as well as induces MHC production in host cells. IFN-y has also been found to modulate the priming, recruitment, and apoptosis of activated T cells (9 and 22). Contraction and memory acquisition Effector CD8+ T cells quickly route influenza infection, usually eliminating the virus from the respiratory tract by about day 10 after infection (18). With the battle won, the body no longer requires a large pool of armed effector CD8+ T cells. Most of the effector T cells die by apoptosis by about 14 days after infection (18). Only about 5-10% of the peak population remains. The surviving effector CD8+ T cells become long-lived memory T cells (4). This process appears to be pre-programmed, and independent of the magnitude of that peak population. The initial activation of CD8+ T cells seems to drive them through a full program of cell division without the need for addition encounters with antigen (23). The mechanism that mediates this programmed contraction is largely unknown, but evidence suggests a role for IFN-y. Experiments with mice deficient in IFN-y have shown that even though both deficient and wild type groups have similar responses to Listeria through the effector phase, they have diminished contraction of effector T cell populations compared with wild type mice. This leads to a greater number of antigen specific T cells 28 days after Listeria infection, compared with wild type mice (24). Experiments with mice lacking the IFN-y receptor have shown that these mice also experience less apoptosis of effector T cells after infection with lymphocytic choriomeningitis virus (25). IFN-y, then, appears to promote the apoptosis of effector T cells. One of the early determinants of memory appears to be expression of the IL-7 receptor u-chain. IL-7 is known to be an important cytokine for persistence and survival of memory T cells (26). A fraction of CD8+ effector T cells upregulate IL-7Ru earlier in an immune response than other memory markers, and these cells use the IL-7 survival signal to outlast the contraction phase and become memory cells (27). R Memory CD8+ T cells remain in the body in order to mount a more effective response should the individual become infected with the same pathogen. Memory cells have two main advantages over nafve cells when mounting a secondary response. First, there are more memory cells specific to influenza antigens. The memory pool for a particular antigen is approximately 1 02-103 greater than the initial frequency (4). This makes it more likely that a memory cell will recognize antigens from a pathogen that has reinfected the host. Also, memory cells are intrinsically more sensitive to activation than naive cells (28 and 29). This allows for a quicker, more vigorous response from the memory cell. Since memory cells are better suited to fighting reinfection than naifve cells, activation of naYve lymphocytes is suppressed during a secondary response. Two populations of memory CD8 t cells arise from the pool of effector cells that survive contraction. Effector memory cells (EM cells) stay in peripheral tissues and express high levels of integrins and receptors for inflammatory cytokines (29). This seems to allow EM cells to respond quickly at the site of infection. After influenza infection, though, EM cells decline rapidly in the lung. This depletion correlates with a loss of memory response to subsequent influenza infection (30). Central memory cells (CM cells) are recirculated in lymphoid tissues, scanning for the reemergence of viral antigens (31). The pool of antigen specific memory cells appears to remain roughly constant throughout life. Memory cells maintain this population by a low level of proliferation (32). While naive CD8+ T cells require both TCR engagement and cytokine signaling to survive, memory cells have gained the ability to survive without antigen, and require only cytokine signals, such as IL-7 and IL- 15 (32). Experiments have shown that CD4+ helper T cells are important for maintaining the memory population. Mice that lack CD4+ T cells have largely normal CD8+ T cell responses to influenza, but have reduced antigen specific memory pools (33 and 34). The reduced immunological memory of CD4+ T cell deficient mice results in reduced protection against reinfection with influenza. Perspectives on treatment and prevention of influenza The acquisition and maintenance of memory cells is the goal of any vaccination. The traditional influenza vaccine, the flu shot, uses a combination of killed flu virus particles to induce an antibody response. The vaccine must be formulated in advance using three flu strains predicted to be most likely to cause widespread infection (1 and 35). This does not provide protection against other strains, and must be reformulated every year as new strains arise. There is currently a cold adapted live virus vaccine called FluMist available on the market. This is a recombinant live virus that causes an attenuated infection. The virus is cold adapted so that it will replicate in the nasal mucosa, but can not reproduce in warmer areas such as the lungs (36). It is possible to create other attenuated flu viruses, so similar live vaccines may be available in the future. Other treatment options include antiviral drugs that directly inhibit influenza viral replication. These drugs must be taken on a strict schedule to maintain therapeutic levels in the body. Antiviral drugs are generally effective against influenza, but side effects and concerns over patient compliance with taking the drugs as recommended limit their practical widespread use. 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