CpG motifs: the active ingredient in bacterial extracts? - Nature

© 2003 Nature Publishing Group http://www.nature.com/naturemedicine

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CpG motifs: the active ingredient in bacterial extracts? Arthur M Krieg The use of bacteria and bacterial extracts for immunotherapy has a checkered past. Recent developments in immunology reveal that these nonspecific immune activators actually work by triggering specific receptors that are expressed by subsets of immune cells. Identification of these receptors and the molecular signaling pathways that they activate has enabled a new era of specific targeted immunotherapy using chemically synthesized mimics of pathogen molecules. More than a century has passed since a pioneering surgeon, William B. Coley, discovered that many cancer patients could be cured of advanced metastatic cancer by repeated bacterial infection or injections of bacterial extracts consisting of Streptococcus pyogenes alone or together with Serratia marcescens1,2. This therapy was quite toxic, and there have been numerous attempts to develop more refined approaches. The clinical development of bacterial therapy for cancer was marked by many examples of therapies that worked in mouse models, yet failed in humans. Despite setbacks and inconsistent data, such work led to the approval of bacillus Calmette-Guérin (BCG), which is widely used for the local treatment of bladder cancer. In addition to inducing tumor regression, bacterial extracts can protect against many infectious challenges, and prevent or treat allergic sensitization3. Finally, complete Freund adjuvant (CFA), an emulsion of killed bacteria, is the ‘gold standard’ vaccine adjuvant for immunologists. CFA is highly effective at enhancing vaccine responses in animals but is generally considered to be too toxic for use in humans. This commentary will propose a possible identity for a common bacterial component that may explain the powerful activity of these diverse extracts, and point to ways to achieve better effects with less toxicity. Bacterial extracts are now understood to activate both the innate and adaptive arms of the immune system. One of the most important recent discoveries in the field of immunology is how the innate immune system detects infectious agents and simultaneously distinguishes different classes of pathogens from one another. The innate immune system needs to identify the pathogen class because defending against different pathogens requires radically different types of immune responses, and these must be initiated by innate immune cells. If the invader is an extracellular parasite, then the immune system must mount an appropriate immune response involving the early activation of eosinophils and the subsequent production of inter-

Coley Pharmaceutical Group, Wellesley, Massachusetts 02481, USA. e-mail: [email protected]

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leukin (IL)-4 and IgE, which is termed a T-helper 2 (TH2) response. In contrast, clearing the body of an intracellular viral infection requires a different type of immune response, termed a TH1 response, in which natural killer (NK) cells are activated as quickly as possible to control the infection until specific interferon (IFN)-γ-secreting T cells and cytotoxic T lymphocytes (CTLs) can be produced. Although many of the details remain poorly understood, the immune system is thought to accomplish these related goals in a simple yet elegant manner by using ‘pattern-recognition receptors’ that are expressed on certain innate immune cells, such as dendritic cell (DC) subsets, macrophages, monocytes and neutrophils, and that trigger cell activation when they recognize conserved microbial-specific molecules4. These molecules, including peptidoglycans, zymosan, lipopolysaccharides and unmethylated CpG dinucleotides, used to be thought of as nonspecific immune activators. It is now clear, however, that these molecules are specifically recognized by receptors that are expressed in a cell-specific and compartmentalized manner, and trigger specific signaling pathways. Role of toll-like receptors in detecting pathogens The best-characterized family of pattern-recognition receptors is the Toll-like receptor (TLR) family, of which ten members have been identified in humans4. Different TLRs have distinct patterns and locations of cellular expression. TLR4, for example, is expressed on the surface membrane of human myeloid DCs and monocytes, and is essential for the recognition of lipopolysaccharide derived from Gram-negative bacteria. In contrast, TLR9 seems to be expressed in the endosomal compartment of plasmacytoid DCs and B cells5, and may be essential for the recognition of viral and intracellular bacterial DNA6. Most immune cells express several different TLRs. For example, myeloid DCs express TLR4 and TLR7, whereas plasmacytoid DCs express TLR9 and TLR7 (refs. 4, 7–11). Thus, considering only these DC subsets, a TLR4 ligand will activate myeloid but not plasmacytoid DCs, a TLR9 ligand will activate plasmacytoid but not myeloid DCs, and a TLR7 ligand will activate both. The myeloid and plasmacytoid DC subsets respond quite differently to TLR stimulation, suggesting distinct functions. Activated myeloid DCs produce high levels of IL-12 and proinflammatory cytokines such as IL-6 and tumor necrosis factor-α, but activated plasmacytoid DCs produce very high levels of IFN-α; they produce the majority of the IFN made during a viral infection12. A commonly used TLR7 ligand induces both cytokine patterns11–13 but also stimulates human cells through TLR8, which may cause more complex immune effects14. Thus, because of the distinct cellular patterns of TLR expression, different microbial components

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PERSPECTIVE will trigger different patterns of immune activation. The most specific TLR pathway for inducing IFN-α expression is TLR9. IFN-α generally controls viral replication, triggering not only innate immune defenses such as NK cells, but also adaptive TH1 T-cell responses15. The possibility of triggering endogenous IFN-α production through deliberate activation of the TLR9 pathway is of considerable interest, given the demonstrated therapeutic activity of IFN-α in human neoplastic and infectious diseases. There are differences and similarities in the signaling pathways induced by various TLRs. All the immune effects of TLR7 and TLR9 are mediated through the adapter protein MyD88, whereas TLR4 activation triggers two distinct signaling pathways, one of which requires MyD88 and/or a different adapter protein, Toll/IL-1 receptor domain–containing adapter protein (TIRAP)16,17. It now seems that TLR4 can also signal through an MyD88-independent pathway using the recently described adapter protein, TIR domain–containing adapter inducing IFN-β (TRIF)18.These differences in the molecular signaling of different TLRs, together with cell-specific and compartmentalized TLR expression, may have evolved to enable the immune system to mount different immune responses against extracellular Gram-negative bacteria and intracellular DNA viruses and retroviruses. Now that specific ligands have been identified for most of the TLRs, it is finally possible for immunotherapy to move away from the nonspecific effects of whole bacterial extracts, and to determine whether the same or even better therapeutic responses may be induced using synthetic TLR ligands. A key question is, which of the TLRs induces the most useful therapeutic effects? In the case of cancer immunotherapy, some data point to TLR9. In 1984, long before the first TLR gene was cloned, Tokunaga et al. showed that the antitumor properties of BCG

could be reproduced with purified bacterial DNA, and that little or no antitumor activity could be detected in bacterial fractions comprising proteins, RNA, lipids or carbohydrates19. More recently, two groups have reported that treatment of bacterial extracts with nuclease severely reduces their immune stimulating activity20,21, further supporting the identification of bacterial DNA as a key active ingredient of bacterial extracts21. In fact, the antitumor activity of purified bacterial DNA was superior to that of whole BCG in several mouse tumor models19. Human clinical trials with a bacterial DNA preparation given by daily peritumoral injection showed substantial promise, with an overall response rate of 43% in 75 patients with a variety of skin malignancies19. Among 17 subjects with either malignant melanoma or squamous-cell carcinoma, there were two complete responses, four partial responses, five “minor remissions” and two with stable disease (the other four patients progressed)19. However, a drug application from these investigators was rejected by the Japanese Ministry of Health and Welfare “because of the difficulty in the production and specification to assure uniform qualities of BCG-DNA as a biologic drug.”19 TLR9 recognizes CpG motifs The molecular structure that the immune system recognizes in bacterial DNA is now known to consist of unmethylated CpG dinucleotides in certain base contexts22,23. This has made it possible to selectively trigger TLR9 using synthetic oligodeoxynucleotides (ODNs) of ∼8–30 bases in length that contain one or more such CpG motifs. Unlike the bacterial DNA preparations of Tokunaga et al.19, ODNs are easy to synthesize and characterize. Because the phosphodiester backbone of native DNA is rapidly digested by serum and cellular nucleases, in vivo applications with CpG ODNs generally use the nuclease-resistant,

Innate Immunity

Adaptive immunity Increased sensitivity to antigen

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Figure 1 CpG cellular mechanism of action. DNA containing one or more CpG motifs is taken up by endocytosis in most cell types, but only activates cells expressing the TLR9 receptor (B cells and plasmacytoid DCs (pDC) in humans). These cells then create a TH1-like cytokine milieu by secreting IFN-α, IFN-β, IL-12, IP-10 and other TH1-promoting cytokines and chemokines. NK cells are activated secondarily, secreting IFN-γ and gaining lytic activity. In addition, the B cells become more sensitive to activation through their antigen receptor, and both B cells and plasmacytoid DCs have enhanced expression of costimulatory molecules, improving their ability to activate T-cell responses. APC, antigen-presenting cell, MHC, major histocompatibility complex.

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PERSPECTIVE phosphorothioate-modified backbone, which improves their cellular uptake and markedly prolongs the in vivo half-life of modified ODNs, though with some increase in non-sequence-specific effects23. These modified CpG ODNs have proven to be superb vaccine adjuvants and are highly effective immunotherapeutics in numerous animal models of infectious disease, cancer, allergy and asthma23. The immune effects of CpG ODNs may be considered in two stages: an early stage of innate immune activation and a later stage of enhancement of adaptive immune responses (Fig. 1). Within minutes of exposure of B cells or plasmacytoid DCs to CpG, the ODNs appear to enter an endosomal compartment where they interact with TLR9, leading to the activation of cell signaling pathways that culminate in the expression of costimulatory molecules, resistance to apoptosis, upregulation of the chemokine receptor CCR7 that causes cell trafficking to the T-cell zone of the lymph nodes, and secretion of TH1-promoting chemokines and cytokines such as macrophage inflammatory protein-1, IFN-inducible protein-10 (IP-10) and other IFN-inducible genes23. Plasmacytoid DCs secrete type I IFN and mature into highly effective antigen-presenting cells24. These CpG-induced type I IFNs, cytokines and chemokines trigger, within hours, a wide range of secondary effects, including NK cell activation and enhanced expression of Fc receptors on effector cells such as polymorphonuclear leukocytes, with a resultant increase in antibodydependent cellular cytotoxicity (ADCC). Polymorphonuclear leukocytes also exhibit increased migration in response to inflammatory signals, as well as enhanced phagocytosis and respiratory burst23. This innate immune activation and plasmacytoid DC maturation is followed by the generation of adaptive immune responses. B cells are strongly costimulated if they bind specific antigen at the same time as TLR9 stimulation, which selectively enhances the development of antigen-specific antibodies22. After CpG stimulation, antigen presentation to T cells occurs in a TH1-like cytokine milieu and can induce primary CTLs even in the absence of T-cell help25,26. Immunotherapy by activating TLR9 with CpG DNA The innate immune activation triggered by CpG ODNs has proven surprisingly effective at protecting mice against infectious challenges. A single treatment of CpG can protect mice against challenge with a broad range of viruses, bacteria, intracellular parasites and even prions27,28. Protection typically begins within 48 h and lasts for several weeks. Repeated weekly or biweekly CpG injections can maintain a state of heightened resistance to pathogen challenge for several months, without obvious toxicity27. Another innate immune activity of CpG ODNs is the induction of ADCC. The efficacy of antitumor antibodies used in clinical therapy, such as Rituxan and Herceptin, appears to result at least in part from the ADCC mechanism. In mouse models, CpG ODNs have been extremely effective at enhancing the effects of antitumor antibodies, and human trials of this approach are currently under way23. A further immunotherapeutic application for CpG ODNs is in the field of allergic disease29. In mouse models, CpG ODNs can both prevent the induction of allergic responses and reverse established immune responses, whether given alone or together with an allergy vaccine29. The mechanism of action was initially hypothesized to involve the induction of a TH1 response that opposes the TH2 allergic response, but recent studies indicate a more complex mechanism, possibly involving activation of regulatory T cells that can downmodulate allergic responses through immune pathways that are not fully understood30. Initial human clinical trials in seasonal rhinitis have shown that patients injected with a conjugate of a CpG ODN and a ragweed allergen tolerated the treatment quite well and showed significant improvements in both immune and clinical measures31.


CpG ODNs are also very effective as vaccine adjuvants to enhance adaptive TH1 humoral and cellular immune responses in mice and primates32–43. In mice, CpG ODNs trigger stronger TH1 responses than any other known adjuvant, even CFA32–34,44. The ability of CpG to activate plasmacytoid DCs to secrete IFN-α, which is thought to be important in mediating optimal TH1 responses, may explain why vaccination of mice with this TLR9 ligand gives a higher level of antigenspecific IFN-γ secretion and CD8+ T cells compared with vaccination with a TLR4 ligand, monophosphoryl lipid A (MPL)32,45. CpG ODNs strongly enhance the number and function of tumor-specific CTLs and IFN-γ secreting T cells45–48. CpG ODNs also enhance therapeutic responses to other immune therapies, such as donor lymphocyte infusions49, and to vaccines, such as DC vaccines50, proteins, irradiated cells with granulocyte-macrophage colony-stimulating factor51,52, and long peptide vaccines45. This has enabled the development of therapeutic vaccines in mouse tumor models where no other approach has shown comparable efficacy, even with 1-cm established tumors35,50. Even without a vaccine, CpG ODNs can induce CD8+ T-cell-mediated regression of established tumors with durable memory responses35–39,41. Repeated injection of CpG ODNs has prevented the development of spontaneous tumors in genetically prone mice transgenic for Her-2/neu53 or the c-myc proto-oncogene driven by a mouse immunoglobulin enhancer54. Nevertheless, CpG ODN treatment was unable to overcome tolerance to a viral-derived dominant tumor antigen epitope in transgenic mice, indicating that this therapy may not induce responses to antigens expressed from birth55. From mice to men Of course, results in mice are not necessarily predictive of results in humans, and this is particularly true in the case of the TLRs and immune activation. In contrast to humans, where the only two cell types currently known to express the TLR9 receptor and respond directly to CpG ODNs are plasmacytoid DCs and B cells8,9, TLR9 expression in mice seems to occur in a broader range of cells, including the mouse myeloid DC equivalent and monocytes/macrophages. In addition, there is some species specificity to the TLR9 recognition of CpG motifs: the mouse TLR9 molecule is preferentially activated by the CpG motif GACGTT, whereas the human TLR9 is optimally triggered by the motif GTCGTT10,56. Despite these interspecies differences, a clinical trial in normal human volunteers showed that addition of a CpG ODN to a commercial hepatitis B vaccine markedly accelerated seroconversion, with most of the subjects achieving protective levels of IgG antibodies in 2 weeks57. The magnitude of the adjuvant effect of CpG in humans seemed to be greater than the comparatively modest effects seen in previous clinical trials of the same vaccine using a TLR4 ligand, MPL58,59. For example, a month after the first boost, the geometric mean titer of hepatitis B antibody in subjects vaccinated with an MPL formulation was approximately 40 mIU/ml58, compared with approximately 630 mIU/ml at the same time point in subjects vaccinated with CpG ODN 7909. There was a trend to increased frequency of CTL responses in the CpG-vaccinated subjects, but the number of subjects in the trial was small, and thus did not reach statistical significance. The adjuvant activity of CpG has also been seen in a clinical trial in immunocompromised HIV-infected patients. This study was designed to evaluate the immunogenicity of Engerix-B vaccine plus CpG 7909, and included 38 HIV-seropositive adults aged 18–55 years, half of whom had no prior hepatitis B vaccination, and half of whom had been vaccinated with at least three doses of regular vaccine prior to the study but had not achieved protective antibody titers. Patients receiving CpG 7909 with their hepatitis B vaccine produced protective anti-

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PERSPECTIVE bodies more rapidly than patients receiving the vaccine alone (C.L. Cooper et al., personal communication). By 8 weeks, only 42% of patients receiving vaccine alone had protective titers of 10 mIU/ml or higher, compared with 89% of CpG subjects. In addition to these increased rates of seroconversion, patients receiving CpG 7909 also had significantly higher antibody titers. This successful strategy of using CpG 7909 to enhance hepatitis B immune response in a known hyporesponsive population of patients may be duplicated for other purposes. In HIV-seropositive individuals, there is potential application in the development of therapeutic and preventative HIV vaccines. Other vaccine-hyporesponsive populations such as alcoholics and the elderly also may benefit from the use of CpG ODNs in vaccination. Recent studies suggest that CpG 7909 is active in human cancer patients as well. A small phase 1 safety trial of CpG ODN 7909 injected intralesionally in patients with basal cell carcinoma or melanoma showed several local regressions after a few injections (U. Trefzer et al., personal communication). A small phase 1 tumor vaccine trial with the melanoma antigen MAGE-3 showed that out of six patients vaccinated every 3 weeks with a 1-mg dose of CpG, two progressed, two had stable disease and two had partial responses (using the RECIST (Response Evaluation Criteria in Solid Tumors) criteria) that have been durable for at least 1 year60. Although this must be considered anecdotal evidence because of the small patient numbers, the fact that these responses occurred in patients with stage IV melanoma is encouraging. Why should a TLR9 ligand be so effective as a vaccine adjuvant? The explanation may involve the strong and relatively specific stimulation of plasmacytoid DCs that TLR9 activation triggers. Recent studies point to a possible role for mouse plasmacytoid DCs in the maintenance of T-cell self-tolerance61–63. If plasmacytoid DCs also normally promote T-cell tolerance to antigens in humans, then activation of plasmacytoid DCs may be essential for inducing the strongest possible response to an antigen62,63. To design optimized vaccines in humans, it may be desirable to include an adjuvant capable of activating plasmacytoid DCs through the TLR9 receptor. The case for treating autoimmunity by inhibiting TLR9 Along with its beneficial effects for immunotherapy, the TLR9 pathway has been implicated in the pathogenesis of experimental autoimmunity. In this regard, a distinction must be made between synthetic CpG ODNs and immune complexes containing DNA. In the clinical trials of CpG ODNs to date, there has been no indication that they have triggered the development of any autoimmune disease. However, the production of rheumatoid factors and perhaps other autoantibodies, such as those against DNA, can be boosted by immune complexes containing self DNA and autoantigens64. It seems that in B cells strongly activated through the B-cell receptor, the TLR9 pathway can be activated by DNA that would normally be nonstimulatory65–67. This may explain why CpG DNA does not induce autoimmunity if it is not in the form of immune complexes68. The TLR9 pathway may have a role in the pathogenesis of human autoimmune diseases such as systemic lupus erythematosus, in which immune complexes containing DNA are present. Therefore, inhibitors of the TLR9 pathway may become quite useful therapeutic agents. Such inhibitors include antimalarial drugs such as chloroquine and quinacrine, and certain suppressive ODN-containing G-rich motifs64,69–73. The existence of suppressive DNA sequences, which selectively block immune stimulation through TLR9 but not through other pathways such as TLR4, explains the lack of immune activation by isolated vertebrate DNA74. Antimalarial drugs, which have been used in the treatment of lupus for several decades, might actually work through inhibition of the TLR9

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pathway. Suppressive ODNs seem to represent a new class of agents that may be useful in the treatment of autoimmunity64,75. Conclusion In summary, the recent discoveries of the TLR pathways and their roles in regulating innate and adaptive immune responses provide a new perspective on the long history of therapy with bacterial extracts such as CFA and BCG. Of the various components in these extracts, CpG DNA acting through TLR9 is especially effective at activating plasmacytoid DCs, and seems to be particularly important in triggering therapeutic TH1 responses. The immune pathways that are triggered through TLR9 are easy to manipulate using synthetic CpG ODNs, which are economical and easy to produce in a highly pure form. CpG ODNs may be uniquely well suited to the systemic immunotherapy of human disease. ACKNOWLEDGMENTS The author thanks G. Hartmann and G. Lipford for helpful comments, and D. Arsenault for assistance in manuscript preparation. 1. Coley, W.B. The treatment of malignant tumors by repeated inoculations of erysipelas: with a report of ten original cases. Am. J. Med. Sci. 105, 487–511 (1893). 2. Wiemann, B. & Starnes, C.O. Coley’s toxins, tumor necrosis factor and cancer research: a historical perspective. Pharmacol. Ther. 64, 529–564 (1994). 3. Zuany-Amorim,C. et al. Suppression of airway eosinophilia by killed Mycobacterium vaccae–induced allergen-specific regulatory T cells. Nat. Med. 8, 625–629 (2002). 4. Janeway, C.A. Jr. & Medzhitov, R. Innate immune recognition. Annu. Rev. Immunol. 20, 197–216 (2002). 5. Ahmad-Nejad, P. et al. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol. 32, 1958–1968 (2002). 6. Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000). 7. Hornung, V. et al. Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168, 4531–4537 (2002). 8. Krug, A. et al. Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12. Eur. J Immunol. 31, 3026–3037 (2001). 9. Kadowaki, N. et al. Subsets of human dendritic cell precursors express different tolllike receptors and respond to different microbial antigens. J Exp. Med. 194, 863–870 (2001). 10. Bauer, S. et al. Human TLR9 confers responsiveness to bacterial DNA via speciesspecific CpG motif recognition. Proc. Natl. Acad. Sci. USA 98, 9237–9242 (2001). 11. Ahonen, C.L. et al. Dendritic cell maturation and subsequent enhanced T-cell stimulation induced with the novel synthetic immune response modifier R-848. Cell Immunol. 197, 62–72 (1999). 12. Vasilakos, J.P. et al. Adjuvant activities of immune response modifier R-848: comparison with CpG ODN. Cell Immunol. 204, 64–74 (2000). 13. Ito, T. et al. Interferon-alpha and interleukin-12 are induced differentially by Toll- like receptor 7 ligands in human blood dendritic cell subsets. J. Exp. Med. 195, 1507–1512 (2002). 14. Jurk, M. et al. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat. Immunol. 3, 499 (2002). 15. Biron, C.A., Nguyen, K.B. & Pien, G.C. Innate immune responses to LCMV infections: natural killer cells and cytokines. Curr. Top. Microbiol. Immunol. 263, 7–27 (2002). 16. Yamamoto, M. et al. Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4. Nature 420, 324–329 (2002). 17. Horng, T., Barton, G.M., Flavell, R.A. & Medzhitov, R. The adaptor molecule TIRAP provides signalling specificity for Toll- like receptors. Nature 420, 329–333 (2002). 18. Yamamoto, M et al. Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signalling. J. Immuno. 169, 6668–6672 (2002). 19. Tokunaga, T., Yamamoto, T. & Yamamoto, S. How BCG led to the discovery of immunostimulatory DNA. Jpn. J. Infect. Dis. 52, 1–11 (1999). 20. Brown, W.C., Estes, D.M., Chantler, S.E., Kegerreis, K.A. & Suarez, C.E. DNA and a CpG oligonucleotide derived from Babesia bovis are mitogenic for bovine B cells. Infect. Immun. 66, 5423–5432 (1998). 21. Ronaghy, A. et al. Immunostimulatory DNA sequences influence the course of adjuvant arthritis. J. Immunol. 168, 51–56 (2002). 22. Krieg, A.M. et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546–549 (1995). 23. Krieg, A.M. CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol. 20, 709–760 (2002). 24. Krug, A. et al. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur. J. Immunol. 31, 2154–2163 (2001). 25. Wild, J., Grusby, M.J., Schirmbeck, R. & Reimann, J. Priming MHC-I-restricted cyto-

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