T Cell Costimulatory Molecules in Anti-Viral Immunity: Potential Role in Immunotherapeutic Vaccines

STANIER REVIEW

T cell costimulatory molecules in anti-viral immunity: Potential role in immunotherapeutic vaccines

Tania H Watts PhD, Edward M Bertram PhD, Jacob Bukczynski BSc, Tao Wen PhD

TH Watts, EM Bertram, J Bukczynski, T Wen. T cell Les molécules costimulatoires des lymphocytes T costimulatory molecules in anti-viral immunity: Potential role dans l’immunité antivirale : Le rôle potentiel in immunotherapeutic vaccines. Can J Infect Dis 2003;14(4):221-229. des vaccins immunothérapeutiques

T lymphocyte activation is required to eliminate or control intracel- L’activation des lymphocytes T est nécessaire pour éliminer ou contrôler lular viruses. The activation of T cells requires both an antigen les virus intracellulaires. Cette activation exige à la fois un signal specific signal, involving the recognition of a peptide/major histo- antigénique précis, exigeant la reconnaissance d’un multimère complexe compatibility protein complex by the T cell receptor, as well as addi- d’histocompatibilité peptide-majeur par le récepteur des lymphocytes T et tional costimulatory signals. In chronic viral diseases, T cell des signaux costimulatoires supplémentaires. En cas de maladies virales responses, although present, are unable to eliminate the infection. By chroniques, les réponses des lymphocytes T, bien qu’elles soient présentes, providing antigens and costimulatory molecules together, investiga- ne réussissent pas à éliminer l’infection. En fournissant à la fois les tors may be able to increase and broaden the immune response, result- antigènes et les molécules costimulatoires, les chercheurs pourraient ing in better immunological control or even elimination of the accroître et étendre la réponse immunitaire, ce qui assurerait un meilleur infection. Recent progress in understanding the function of contrôle immunologique ou éliminerait même l’infection. Les progrès costimulatory molecules suggests that different costimulatory mole- récents dans la compréhension de la fonction des molécules cules are involved in initial immune responses than are involved in costimulatoires laissent supposer que les molécules costimulatoires qui recall responses. These new developments have important implica- participent aux réponses immunitaires initiales diffèrent de celles utilisées tions for therapeutic vaccine design. In this review the authors dis- en cas de réponses après un rappel. Ces nouveaux développements ont des cuss the function of T cell costimulatory molecules in immune conséquences importantes pour la conception de vaccins thérapeutiques. system activation and their potential for enhancing the efficacy of Dans la présente analyse, les auteurs traitent de la fonction des therapeutic vaccines. molécules costimulatoires des lymphocytes T dans l’activation du système immunitaire et de leur potentiel pour améliorer l’efficacité des Key Words: Immunity; Therapeutic vaccines; Lymphocytes; vaccins thérapeutiques. Vaccination; Viral infection

n order to develop successful vaccines against chronic viral this review, we provide some background on how T cell Idiseases it is widely thought that a cell-mediated immune immunity is initiated, the nature of the costimulatory response is required to eliminate or control intracellular molecules involved in this process, and how this knowledge viruses. For a protective immune response it is important to can be applied to therapeutic vaccines. induce long term immunological memory. The initial activation of T cells requires both an antigen specific signal, T CELLS AND IMMUNITY TO VIRUSES involving the recognition of a peptide/major histocompati- Although antibodies can be effective in neutralizing extracel- bility protein (MHC) complex by the T cell receptor as well lular viruses, CD8+ T cells are important in killing virally as additional costimulatory signals. Only when the T cell rec- infected cells and CD4+ T cells are critical in providing help ognizes both the antigen and a costimulatory signal on the for both antibody-mediated and CD8+ T cell-mediated dendritic cell (DC) (an antigen presenting cell [APC]) is an responses. The initial T cell response to pathogens is depend- immune response initiated. The idea of a therapeutic vaccine ent on the activation of APCs. Figure 1 summarizes the cur- is that the natural immune response, although present, may rent view of how this process occurs. DCs are thought to be be suboptimal. By providing antigens and costimulatory mol- the critical APCs for the initiation of T cell responses (1,2). ecules together, one may be able to provide a strong enough DCs are a diverse group of APCs scattered throughout the response to eliminate, or at least better contain, the virus. In skin and tissues. In their resting state, DCs express receptors

Department of Immunology, University of Toronto, Toronto, Ontario This review is based on a lecture given at the Stanier Institute Symposium, “Vaccine preparations and vaccination through understanding pathogenesis”, held on November 22, 2002, University of British Columbia. Correspondence and reprints: Dr Tania H Watts, University of Toronto, Department of Immunology, Room 5263 Medical Sciences Building, 1 King's College Circle, Toronto, Ontario M5S 1A8. Telephone 416-978-4551, fax 416-978-1938, e-mail [email protected] Received for publication April 23, 2003. Accepted April 24, 2003

a) Pathogens TOLL LIKE RECEPTORS AND MICROBIAL RECOGNITION TLR activation migration Mycoplasmal dsRNALPS flagellin CpG ODNs lipoprotein activation b) TLR3 TLR4 TLR6 TLR5 TLR9 immature DC TLR2 at site of infection

mature DC

Ag/MHC Costimulatory TCR molecules

c) naïve Gene expression T cell in Inflammatory response Activated draining T cell lymph node Figure 2) Toll-like receptors (TLRs) and microbial recognition. There are at least 10 TLRs and their diversity may be increased by het- effector T cells (tissues) erodimerization. A subset of TLRs and their known ligands are shown here. TLRs are pattern recognition receptors that recognize conserved Figure 1) Pathogen activation of dendritic cells and the initiation of a molecular patterns exhibited by pathogens, including bacterial cell wall T cell response. a) Immature dendritic cells in the tissues express recep- components and bacterial or viral nucleic acids. TLR9 recognizes tors for microbial uptake as well as Toll-like receptors (TLRs), pattern demethylated CpG motifs that are suppressed in mammalian DNA. recognition receptors that relay signals to the antigen presenting cell TLR9 is shown here as a surface receptor but there is evidence that the indicating that an infectious agent is present. b) After recognizing the recognition actually takes place intracellularly. Once engaged by their lig- presence of an infectious agent, dendritic cells (DCs) undergo a matu- ands, TLRs, via signaling intermediates, signal the activation of nuclear ration process, change their chemokine receptor expression and migrate factor-κB in antigen presenting cells, leading to new gene transcription to the draining lymph nodes. Mature dendritic cells upregulate major and upregulation of a program of inflammatory gene expression. histocompatibility complex (MHC) proteins as well as costimulatory dsRNA Double-stranded ribonucleic acid; LPS Lipopolysaccharide; molecules important for the activation of naïve T cells. c) Following ODN Oligodinucleotide. Adapted from (72) activation and clonal expansion in the lymphoid tissues, activated effector T cells home to the site of infection and exert their effector functions (cytokine secretion, killing). Ag Antigen; TCR T cell receptor triggering inflammation and immunity. This activation process renders DCs competent to activate naïve T cells in the lymphoid organs. Naive T cells, via their clonally distributed anti- suitable for pathogen uptake, as well as receptors capable of gen-specific T cell receptors, recognize peptide-MHC sensing infection. Once activated by exposure to pathogens or complexes presented on the activated DCs. At the same time, inflammatory stimuli, DCs undergo a maturation process that T cells need to recognize so-called ‘costimulatory molecules’ in involves their migration to the draining lymph node and order to be activated. Antigen presentation in the lymphoid increased cell surface expression of molecules involved in T organs results in clonal expansion leading to a population of cell activation. activated ‘effector’ T cells (Figure 1). These effector T cells DCs and other APCs express a family of receptors known as then home back to the site of infection, where they carry out the Toll-like receptors (TLRs) (3). These receptors are pattern their functions in eliminating or containing the infection. The recognition receptors that bind conserved features of pathogens. finding that the initiation of the T cell response requires the There are at least 10 such receptors and their diversity is presence of costimulatory molecules induced by microbial infec- increased by heterodimerization (Figure 2). For example, tion explains in part how the immune system can respond vigor- TLR4 is required for the response to bacterial lipopolysaccha- ously to an infection while avoiding recognition of self-tissues. ride. TLR3 recognizes double stranded ribonucleic acid (dsRNA) associated with viral infections and TLR9 recognizes WHAT IS A T CELL COSTIMULATORY demethylated CpG motifs that are enriched in bacterial DNA. MOLECULE? Once triggered by these pathogen associated molecular pat- T cells require two signals for activation: an antigen specific sig- terns, TLRs induce a response in the APC leading to new gene nal and a second ‘costimulatory’ signal. The concept of two sig- transcription and the induction of molecules involved in nals for lymphocyte activation goes back 30 years (4). The

222 Can J Infect Dis Vol 14 No 4 July/August 2003 Costimulatory molecules and therapeutic vaccination

TWO SIGNAL MODEL FOR T CELL ACTIVATION

TCR/CD3 CD28

Signal 1 Signal 2

Death or Cell survival unresponsiveness Proliferation

Figure 3) The two signal model for T cell activation. Binding of the T cell receptor (TCR)/CD3 complex to the antigen/major histocompatibility complex (MHC) alone (signal 1) can lead to death or induction of a nonresponsive state in the T cell. Only when the T cell receives both a signal through its TCR as well as a costimulatory signal (signal 2) does it survive and make the cytokines necessary for T cell proliferation. Adapted from a figure kindly provided by Linda Wu Figure 4) Key players in the intiation of T cell activation. Antigen presenting cells degrade pathogens. Peptides from the pathogen are brought to the cell surface as a complex with a major histocompatibility complex (MHC) protein. T cells, via their clonally distributed antigen specific current model is derived from experiments in the late 1980s that receptors bind to MHC peptide complexes. The frequency of T cells showed that although an initial transient response might be specific for a specific MHC-peptide complex prior to immunization is observed if T cells were triggered only through their antigen-spe- estimated to be about one in 107. The interaction between CD28 on cific receptors, they went on to die or become unresponsive. the T cell and B7.1 or B7.2 on a mature dendritic cell provides a crit- However, if a signal was given through both the antigen- ical costimulatory signal for T cell activation. The antigen presenting cell-T cell interaction is also stabilized by adhesion molecules. specific T cell receptor and an additional cell surface receptor ICAM Intercellular adhesion molecule-1; LFA-1 Leukocyte function- known as CD28, then the T cells made high levels of cytokines associated antigen-1; TCR T cell receptor required for T cell proliferation and also upregulated survival factors that prevented programmed cell death (Figure 3) (5). Figure 4 shows the important interactions during initial T cell memory versus effector function, or differentiation into differ- activation by a DC that has been exposed to a pathogen. ent kinds of effector cells. Peptides derived from degradation of the infecting pathogen are Two major families of proteins have received a lot of brought to the DC surface via binding to the MHC proteins attention in the field of T cell costimulation. The CD28 (also known as human leukocyte antigen [HLA] proteins in family (8) and the tumor necrosis factor receptor (TNFR) humans) inside the cell. In addition, B7 molecules present on family (9). As discussed above, the T cell surface receptor activated DCs bind to CD28 molecules on the T cell surface. CD28 provides a critical signal for the initiation of T cell An extensive body of evidence supports the idea that the responses (6). Mice lacking CD28 have very poor T cell binding of the T cell surface receptor CD28 to its ligand, B7.1 or responses in general, although they can respond to some B7.2, allows initial T cell expansion and short term survival (6). pathogens, such as lymphocytic choriomeningitis virus Thus, it is now widely accepted that the binding of B7.1 or (10,11). CD28 is part of a family of related receptors, which are B7.2 on pathogen-activated APCs to CD28 on naïve T cells summarized in Table 1. Two of the family members, CD28 and provides the first costimulatory signal for initiation of T cell inducible costimulator (ICOS), are stimulatory, whereas the mediated immunity. However, in recent years the picture has two other family members, PD-1 and cytotoxic T lymphocyte become more complicated. We now know that following ini- associated antigen-4 (CTLA-4), inhibit T cell activation. tial contact there is further activation in both the APC and CD28 is expressed on resting T cells whereas the other mem- the T cell, resulting in the appearance of new receptor-ligand bers of the family are expressed only after T cell activation. pairs on the interacting cells (7). Some of these molecules are CD28 and ICOS bind to different ligands. ICOS binds to a B7- illustrated in Figure 4. One of the challenges facing immunol- related protein known as ICOS ligand or B7-related protein 1 ogists is to understand the specific roles of this large array of (B7RP1). ICOS is important in enhancing the ability of T immune stimulatory molecules. An emerging idea is that while cells to make cytokines (12). Mice lacking ICOS make very CD28 is important for initial T cell activation, other small germinal centers and have a greatly diminished antibody costimulatory molecules may be important in sustaining class switch, resulting in a predominantly Immunoglobulin M responses, diversifying the response or controlling different (IgM) response (13-15). ICOS knockout (––/ ) mice show aspects of the response, such as initial versus recall responses, decreased immune responses in several infectious models,

Can J Infect Dis Vol 14 No 4 July/August 2003 223 Watts et al

TABLE 1 Costimulatory molecules in T cell activation CD28 superfamily: Receptor Expression Ligands (expression) Function in immune system CD28 Constitutive T cells B7.1 (CD80) Immune stimulation: provides signal 2 for initial survival B7.2 (CD86) and proliferation of naïve T cells Activated APC ICOS Activated T cells B7RP1 also known as LICOS, ICOSL, Immune cell differentiation: enhances T helper cytokine production, Higher expression on activated APC and some antibody class switch, germinal center formation on Th2 cells nonlymphoid tissues CTLA-4 Activated T cells B7.1, B7.2 Inhibitory: downregulates immune response PD-1 Activated T and B cells PD-L1, PD-L2 Lymphoid and Inhibitory: sets threshold for immune activation? non-lymphoid tissues TNFR superfamily: 4-1BB (CD137) Activated CD4+ 4-1BBL (activated APC) CD8 T cell memory (lesser effect on CD4 T cells) and CD8+ T cells Activated dendritic cells Activated NK cells OX40 (CD134) Activated CD4 T cells OX40L (Activated T, B, DC, CD4+ T cell memory vascular endothelial cells) CD40 B cells, dendritic cells CD40L (activated T cells) B cell and dendritic cell activation and macrophages

APC Antigen presenting cell; B7RP1 B7-Related protein 1; CTLA-4 Ctytotoxic lymphocyte associated antigen-4; DC Dendritic cell; ICOS Inducible costimulator; L Ligand; NK cells Natural killer cells including viral and bacterial infections (16-18). However, to cell death pathways. A second group, typified by TNFRII, defects in immune responses in ICOS––/ mice are not as severe lacks death domains and directly binds adaptor proteins called as those in CD28––/ mice (17). CD28 and the inhibitory TNFR associated factors (TRAFs). TRAFs link extracellular homologue CTLA-4, both bind to the same ligands, B7.1 signals through TNFR family members to cell survival and dif- (CD80) and B7.2 (CD86). Initially, T cells express only CD28, ferentiation pathways (26). There are more than 27 members so the effect of binding B7.1 and B7.2 is an increase in the in the TNFR family, a small subset of which are highlighted immune response. However, with time T cells start to express here. The importance of TNFRs and their ligands in the CTLA-4, which has a 20-fold higher affinity for B7.1 and B7.2 immune response is exemplified by the finding that viruses than does CD28, with the net effect that CTLA-4 limits the create soluble homologues of these receptors to subvert their amount of T cell activation (19). Mice lacking CTLA-4 devel- function (27,28). CD40, a member of the TNFR family op a progressive lymphoproliferative disorder, consistent with expressed on DCs and B cells, is highly important in the regu- the role of CTLA-4 in limiting T cell activation (20,21). Mice lation of APCs and B cell function. Upon initial T cell activa- lacking the other inhibitory member of the family, PD-1, tion, T cells upregulate CD40 ligand (CD40L), which can bind develop a late onset lupus-like disease and arthritis, which is to CD40 on DCs to enhance the expression of costimulatory consistent with an immune inhibitory function (22). However, molecules, including B7.1, B7.2, 4-1BB ligand (4-1BBL) and in vitro studies with the ligands for PD-1 have sometimes OX40 ligand (OX40L). In addition, Interleukin 12 (IL-12), a shown inhibition and sometimes stimulation of T cell respons- cytokine important in regulating interferon gamma (IFN-γ) and es, suggesting perhaps that there is another stimulatory recep- T helper 1 (Th1) cell responses, is upregulated by CD40 signal- tor for these ligands. The ligands for PD-1, PD-L1 and PD-L2, ing in DCs (29). Upon activation, T cells also upregulate the are present in both lymphoid and non-lymphoid tissues. It has TNFR family members OX40 and 4-1BB, receptors thought to been proposed that inhibition by PD-1 in the tissues may be important in sustaining T cell responses. 4-1BB is inducible increase the signal threshold required for an immune response on both CD4+ and CD8+ T cells upon activation and is capa- (8). Blockade of CTLA-4’s inhibitory signal is being tested as ble of providing a CD28-independent costimulatory signal to an immune stimulatory regimen in cancer trials (23). CD28– T cells (30,31). OX40 is primarily expressed on acti- However, a caveat of this approach is that it is also likely to vated CD4+ T cells and appears to only work in conjunction increase the risk of autoimmunity and, as such, is unlikely to with a CD28 signal (32). Mice lacking OX40 or its ligand gain widespread support as a means of controlling chronic have impaired secondary CD4+ T cell proliferative responses infectious diseases. (33-36). 4-1BBL––/ mice show decreased secondary CD8 In addition to the focus on the CD28 gene family, several responses to viruses, with no detectable defect in CD4+ T members of the TNFR family have come to prominence in cell responses to virus (37-39). However, in vitro analysis terms of T cell-mediated immunity (9,24,25). The TNFR fam- indicates a role for 4-1BB on both CD4+ and CD8+ T cells ily is a family of receptors involved in the regulation of cell life (40). The finding that 4-1BBL can stimulate CD28–T cells is and death. This family can be broadly divided into two groups. relevant to the human immune response because, in contrast The first group, including Fas and TNFR1, have death to mice, humans accumulate CD28–CD8+ T cells with age domains in their cytoplasmic tails and link extracellular signals (41,42).

224 Can J Infect Dis Vol 14 No 4 July/August 2003 Costimulatory molecules and therapeutic vaccination

p a. e ep d ti tid p e e p D D M 2 D D E D D E2M Fluorescently tagged

streptavidin

2 E

D D

e e

d p

t t

i p d

e e p

b. Uninfected Influenza infected 7 days previously

Influenza 0.08% 7% NP366/Db tetramer

CD8

Figure 6) Major histocompatibility complex (MHC)/peptide tetramers can be used to monitor antigen-specific T cells. a) Schematic represen- Figure 5) After initial T cell activation, additional costimulatory tation of the structure of an MHC/peptide tetramer. The MHC pro- receptors/ligands are upregulated on the T cell and the antigen pre- teins are engineered in bacterial expression systems and refolded around senting cell (APC). Details are described in the text and in Table 1. a specific peptide (antigen). The tail of the MHC I protein has a target Ag Antigen; ICOS Inducible costimulator; L Ligand; MHC Major sequence designed for specific enzymatic modification with biotin. The histocompatiblity complex; TCR T cell receptor; TNFR Tumour complex is then linked to a fluorescently tagged streptavidin protein, necrosis factor receptor which has four biotin binding sites. b) Representative flow cytometry data showing the proportion of cells binding anti- CD8 or MHCI/peptide tetramers. Cells were stained using anti-CD8 or MHC I-peptide Recent evidence from our laboratory shows that 4-1BBL tetramer attached to different fluorophores. Cells staining with anti- can provide a costimulatory signal to the human CD8 appear on the right quadrants, cells staining with MHC tetramers CD28– T cells, leading to cytokine production, cell appear on the upper quadrants, and cells staining with both reagents survival and the upregulation of molecules associated with appear in the upper right quadrant of the figure cellular cytotoxicity (43). Memory CD8+ T cells specific for chronic viral pathogens (Hepatitis C Virus [HCV], HIV, Cytomegalovirus, Epstein-Barr virus) are found among the – CD28 T cell population in human blood (44,45). The pro- tags that allow one to follow cells of a particular antigen-MHC portion of T cells lacking CD28 is increased in people with specificity using a flow cytometer (53). Using this approach, chronic viral infections or other persistent conditions such as researchers can monitor human blood samples for the presence multiple myeloma (44-49). The observation that 4-1BBL can – of T cells of particular specificities. This has allowed investiga- stimulate CD28 T cells makes it an attractive candidate for tors to analyze T cells in the blood of infected patients without boosting immunity in chronic viral infection, where the lack the need to first expand the specific T cells by restimulation in of CD28 on the memory cells will make this population of cells culture. The resulting new data revealed that past experiments, insensitive to B7 stimulation. As will be discussed below, stim- in which one needed to restimulate and expand T cells in cul- ulatory anti-4-1BB antibodies have been tested in vivo in mouse ture to detect particular specificities, led investigators to great- models. Provision of anti-4-1BB can systemically increase both ly underestimate the number of viral-specific T cells produced anticancer and antiviral immunity (50-52). during acute viral infection. Use of this approach in mouse models and with human blood has revealed a number of impor- MHC TETRAMERS CAN BE USED TO FOLLOW tant insights about the extent and timing of T cell activation CD8 T CELL EXPANSION DURING VIRAL in vivo during acute and chronic infections (54). INFECTIONS Figure 6 describes the structure and application of MHC In the last few years there has been a revolution in our ability tetramers. The tetravalent nature of the MHC-peptide com- to follow the response of T cells to infection without the need plexes allows them to bind in a stable fashion to T cells spe- to culture the T cells. This was made possible by the develop- cific for the MHC-peptide complex making up the tetramer, ment of soluble MHC-peptide tetramers linked to fluorescent so the reagents can be used as antibody-like reagents to detect

Can J Infect Dis Vol 14 No 4 July/August 2003 225 Watts et al

mice about 1% to 2% of the CD8+ T cells in the spleen are still Primary infection Secondary infection specific for the major influenza nucleoprotein (NP) epitope. Hkx31 PR8 Upon subsequent challenge, the response occurs about two 20 8 Wildtype days earlier than the primary response and is of higher magni- 7 4-1BBL-/- tude due to the presence of the expanded memory cell popu- CD28-/- 15 6 n lation that was not present on first exposure to the pathogen.

sio migration/ The kinetics of the primary response to infection do not 5 n a death 10 p appear to be dependent on the infectious dose, but rather 4 x

e 3 memory appear to be preprogrammed (60). Once a T cell is engaged cell survival 5 2 and receives its antigen-MHC and costimulatory signal the 1 cells undergo a series of rapid divisions that are not dependent % o CD8 T cells specific for NP366-374 0 0 on the continued presence of the antigen, resulting in rapid 7 21 38 5 7 expansion of a clone of T cells capable of recognizing infected Days post-infection cells (61-64). The decline of this population also seems to be preprogrammed, and is independent of the disappearance of the pathogen (65). This may be important in chronic infec- Figure 7) CD8+ T cell numbers in influenza infected wildtype (WT), tions because it limits the pathological damage that a sus- CD28 knockout (––/ ) or 4-1BBL––/ mice. Results are plotted as the tained immune response might entail. percentage of CD8+ T cells in the spleen that bind MHC tetramers of The ability to monitor viral-specific responses directly in b D /NP366-374. Note the Y-axis scale is different in the two panels. blood samples using MHC tetramers, combined with sensitive Mice lacking CD28 show very poor initial expansion of influenza spe- methods for detecting which cytokines are produced, has cific T cells following infection, whereas 4-1BBL––/ mice show normal important implications for monitoring vaccine trials. It is now initial expansion and contraction of the response. In contrast, 4-1BBL––/ mice show secondary responses to influenza that are indis- possible to closely monitor CD8+ T cell responses using MHC tinguishable from the primary response, implying a defect in recall or tetramers and intracellular cytokine staining to determine the memory responses. A different recombinant virus is used in primary correlates of protective immunity. For technical reasons, the versus secondary challenge in these experiments. The two viral strains, tools to follow CD4+ T cell responses in the same manner have Influenza A HkX31 and Influenza A PR8 have the same nucleoprotein lagged behind the CD8+ T cell specific reagents; however, this (NP) gene recognized by the CD8+ T cells but differ in the neuro- is currently an area of intense activity. minidase and hemagluttinin genes against which the major neutralizing antibody response is directed. This avoids neutralizing antibodies, pre- ROLE OF COSTIMULATORY MOLECULES CD28 venting viral entry into cells and complicating the interpretation of the AND 4-1BB DURING ACUTE VIRAL CD8+ T cell response. Data from reference 38 INFECTION IN VIVO The use of MHC tetramer technology combined with mouse populations of epitope-specific T cells. In the example shown, models lacking particular costimulatory molecules allows one it can be seen that seven days following infection with to assess the importance of particular ligand-receptor interac- Influenza A, 7% of all CD8+ T cells in the spleen of a C57BL/6 tions in the immune response. Figure 7 shows the impact of the mouse are specific for the major T cell epitope of the influenza removal of CD28 or 4-1BBL on the numbers of CD8+ T cells nucleoprotein NP366-374. This approach can also be com- specific for the immunodominant influenza epitope NP366- bined with the technique of intracellular cytokine staining 374 in C57BL/6 mice (38). Wildtype mice infected intraperi- (55), allowing researchers to determine which cytokines are toneally with influenza A X31 show a rapid expansion of being produced by the activated virus-specific CD8+ T cells. influenza-specific CD8+ T cells in the spleen, peaking at day 7 The use of MHC tetramer technology by the scientific commu- after primary infection at 7% of total CD8+ T cells. This is fol- nity has been accelerated by the creation of a National lowed by a rapid decline of influenza-specific CD8+ T cells in Institutes of Health-supported tetramer facility at Emory the spleen between days 7 and 21, and then a more gradual loss University in the United States. This facility is open to the of these cells over time. Upon subsequent challenge, the international community via an online application process response involves about twice as many T cells as the primary (www.niaid.nih.gov/reposit/tetramer/index.html). response and occurs with slightly enhanced kinetics. However, MHC tetramers have been used to follow the acute response if mice lack CD28 there is a very poor initial expansion of the to infection in several mouse infectious disease models. The T cells and, as a consequence, a very poor secondary response. initial expansion of CD8+ T cells in response to infection By contrast, mice lacking 4-1BBL show little defect in the pri- with influenza virus, lymphocytic choriomeningitis virus or mary response but fail to show an enhancement of CD8+ the bacterium Listeria monocytogenes is very rapid, with the T cell expansion upon secondary challenge. Testing the killing maximum number of antigen-specific CD8+ T cells observed function of the T cells in these mice shows that the ability to in the spleen or lymph node by day 7 or 8 after infection kill target cells coated with viral peptides is proportional to the (56-58). This is followed by a period of contraction in the number of tetramer-binding T cells detected (38). number of T cells in the lymphoid compartment, thought to Further work has shown that the systemic administration of be due to their migration from the lymphoid compartments to stimulatory anti-4-1BB antibodies can correct the defect in the tissues, as well as to programmed cell death of the effector CD28––/ mice when a single dose is provided during the primary cells after they have carried out their functions at the site of influenza infection. This results in full restoration of the mem- infection. As predicted by classical immunology, a proportion of ory T cell response, suggesting that for CD8+ T cells, CD28 is ‘memory’ cells remain behind after this contraction process (59) only required for initial T cell activation and not for recall and approximately three weeks after the influenza infection of responses. Conversely, a single dose of anti-4-1BB antibody

226 Can J Infect Dis Vol 14 No 4 July/August 2003 Costimulatory molecules and therapeutic vaccination corrects the defect in the CD8+ T cell recall response in 4- then infused back into the patients and their CD4+ T cell 1BBL––/ mice only when added at the time of viral challenge, counts were found to improve, at least temporarily (71). The arguing that the 4-1BB costimulatory signal is more important advantage of this approach is that the stimulatory agents are during recall responses (unpublished data). Why the immune not delivered systemically, reducing toxicity concerns. This system switches from CD28 to 4-1BB as a costimulatory mole- approach, using antigen-specific activation together with cule during primary versus secondary responses is not clear. costimulatory molecules, such as 4-1BBL, could be used to gen- Interestingly, administration of anti-4-1BB (100µg) during viral erate useful T cells for reinfusion into patients. This approach challenge results in a 2-fold increase in the recall response to could also use a replication-defective viral delivery vector con- influenza virus, even in wild type mice. The physiological levels taining HIV epitopes and costimulatory molecules delivered to of 4-1BBL in mice appear to be very low, as the ligands are dif- the patient’s APCs ex vivo. These would be used to activate ficult to detect except after extensive stimulation of the cells in the patient’s T cells, and the activated T cells are subsequent- vitro (31). This is consistent with the finding that the provision ly reinfused, again avoiding systemic delivery of the virus. of extra 4-1BBL or stimulatory anti-4-1BB antibodies is immune stimulatory even in immunocompetent mice (52,66). CONCLUSIONS These findings suggest that 4-1BBL, rather than B7, may be the Many challenges remain in the development of therapeutic better choice of immune stimulatory agents to use in an vaccines for chronic viral infections. However, great strides immunotherapeutic regimen for antiviral immunity. have been made over the last few years in our understanding of T cell activation and in our ability to precisely follow T cell HOW CAN KNOWLEDGE OF COSTIMULATORY responses during infection. These advances suggest that we MOLECULES BE APPLIED TO THERAPEUTIC will see a number of new therapeutic vaccine approaches VACCINATION? developed over the next few years. With chronic viral diseases such as HIV there may well have been a vigorous initial immune response to the pathogen, but ACKNOWLEDGEMENTS: Funding for our was research provid- the combination of viral escape variants and viral interference ed by the Canadian Network for Vaccines and Immunotherapeutics with the immune system results in the establishment of a (CANVAC) of the Networks of Centres of Excellence Program, the chronic infection. One model for applying a therapeutic vac- Canadian Institutes of Health Research, the National Cancer cine to the problem of HIV is to first use anti-viral therapy to Institute of Canada and by the Arthritis Society. reduce viral load as much as possible. This would be followed by the interruption of anti-viral therapy and provision of HIV CANVAC: Our research in therapeutic vaccines for anti-viral antigens together with costimulatory molecules, in the hope immunity is being conducted under the auspices of the CAN- that the enhanced immune response could eradicate the resid- VAC, the Canadian Vaccines and Immunotherapeutics Network ual virus and prevent further erosion of the immune system. of the Networks of Centres of Excellence program. CANVAC is Again, 4-1BBL is an attractive candidate because of the an interactive group of more than 47 Canadian academics work- evidence that it is important in recall anti-viral responses. ing with private sector biotechnology companies, government and A key issue becomes how to deliver the antigen and nongovernmental partners toward the common goal of rational costimulatory molecules. As has been demonstrated in vaccine design for chronic viral diseases and cancer. Recognizing that the same immunological principles could be applied to cancer mouse models of cancer or acute viral infection, systemic as to chronic viral diseases, CANVAC currently focuses on the administration of stimulatory antibodies against receptors development of vaccines for HIV, HCV as well as prostate cancer. involved in immune triggering is one possible approach. By bringing together expertise in vectors, antigens, costimulatory However, the need to produce large amounts of protein prod- molecules and immune monitoring, as well as expertise in vaccine uct for therapy could be prohibitively expensive. On the other preparedness, epidemiology, ethics, clinical trials and regulatory hand, the recent success of a recombinant protein therapy for issues, CANVAC hopes to tackle the broad number of issues arthritis (soluble TNFR) suggests that such approaches might required to bring new vaccines to the clinic (www.canvacc.org). be feasible (67). An alternative approach that may be cheaper to administer would be to use recombinant replication-defective viruses containing both viral epitopes and the genes REFERENCES 1. Banchereau J, Steinman RM. Dendritic cells and the control of encoding costimulatory molecules as a therapeutic vaccine. immunity. Nature 1998;392:245-52. Replication-defective adenoviruses, modified canary pox, and 2. 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