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Short Communication |
Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK
Correspondence
Geoffrey L. Smith
glsmith{at}imperial.ac.uk
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ABSTRACT |
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T cells, but thereafter, there was a large recruitment of CD4+ and CD8+ T cells. Interestingly, the majority of T cells expressed the natural killer-cell marker DX5. This report is the first to characterize the local immune response sequence to VACV infection and represents a benchmark against which the responses induced by genetically modified VACVs may be compared. |
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; Earl et al., 2004; Xu et al., 2004) and humans (Crotty et al., 2003; Frey et al., 2003; Hammarlund et al., 2003; Amara et al., 2004; Belshe et al., 2004; Combadiere et al., 2004; Greenberg et al., 2005; Pütz et al., 2005; Rock et al., 2005) by modern immunological techniques. However, the immune response at the vaccination site remains poorly understood.
Previously, an intradermal model in the mouse ear pinnae that mimics dermal vaccination with VACV was developed (Tscharke & Smith, 1999). Following vaccination, no signs of systemic illness were observed and there was little or no virus spread from the ear. Subsequently, this model was used to assess virulence of different strains of VACV, other orthopoxviruses and VACV mutants engineered to lack specific virus genes (Tscharke et al., 2002). Fig. 1(a) shows that this vaccination induced protection against challenge with all doses of VACV strain Western Reserve (WR) tested (up to 107 p.f.u.). It was established previously that doses of 105 p.f.u. or greater are lethal in this model (Wilcock et al., 1999; Symons et al., 2002; Fogg et al., 2004; Davies et al., 2005). Another report analysed cells that had infiltrated into VACV-infected ears by separation of the ventral and the dorsal dermal sheets and incubation of these on culture medium at 37 °C for 8–10 h (Reading & Smith, 2003). Non-adherent cells that had migrated from the dermal layers were pooled with the loosely adherent cells recovered by a further incubation (20 min) in PBS containing glucose (2 mg ml–1), but without calcium and magnesium. With this technique, called the ‘migration technique’, only up to 100 000 cells were isolated per mock-infected ear or ear early post-infection (p.i.) (Reading & Smith, 2003) (Fig. 1b), making analysis of the leukocyte population difficult. For example, lymphocyte subsets were able to be characterized only after day 7 p.i. (Reading & Smith, 2003). To increase the number of cells isolated from infected ears in order to permit more extensive lymphocyte characterization, we tried digestion with trypsin, collagenase or liberase and compared cells obtained by these methods with those obtained by migration.
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), but lesions are bigger in C57BL/6 mice (Tscharke et al., 2002) and therefore most of the experiments were undertaken with this strain. For digestions, the ventral and dorsal dermal sheets were separated using forceps and incubated with collagenase (Sigma, crude type XI collagenase; 1 mg ml–1, 75 min at 37 °C) (Belkaid et al., 1996), trypsin (Invitrogen; 0·5–1·0 %, 30 min at 37 °C) (Belkaid et al., 1998) or liberase CI (Sigma; 50 µg ml–1, 40 min at 37 °C) (Hawlisch et al., 2005); cells were then filtered using a 70 µm nylon cell strainer (Becton Dickinson). Cell numbers obtained with trypsin or collagenase were similar and twofold higher than those obtained with liberase (data not shown). However, trypsin treatment removed some cell-surface markers (i.e. CD4 and CD8) that were recovered after 2–4 h incubation at 37 °C (data not shown). Liberase digestion produced fewer contaminating endothelial or epithelial cells, but preferentially isolated lymphocytes and, to a lesser extent, macrophages and granulocytes (data not shown). Therefore, to enable analysis of all leukocyte populations and to avoid the problem of trypsin digestion, we selected collagenase treatment for our study.
Fig. 1(b)
shows the number of cells obtained with migration or collagenase techniques after VACV infection. In mock-infected ears and up to 8 days p.i., there was an increase of >10-fold in cell recovery using collagenase digestion compared with migration and the biggest difference was on day 2 p.i. (>50-fold). Collagenase treatment yielded more than 106 cells per ear for each time point and this was sufficient for phenotypic analysis of the cells. Interestingly, the number of cells recovered by collagenase treatment dropped at day 11 p.i., whereas by using the migration technique, more cells were recovered at this time than earlier (Fig. 1b). Previous results using migration showed an increase in cell number, even at day 15 p.i. (Reading & Smith, 2003), when the lesion size had decreased, and when immunohistochemical detection of VACV with an antibody against the B5R protein was very weak at days 10 and 12 p.i.
Although <30 % of total cells recovered from mock-infected ears by collagenase treatment were leukocytes [detected by CD45 mAb–fluorescein isothiocyanate (FITC); Serotec], compared with around 80 % with the migration technique, the total numbers of macrophages and lymphocytes were >50- and >60-fold-increased with enzymic digestion (Fig. 1c). At day 5 p.i., leukocytes (CD45+) represented 70–90 % of total cells recovered with either technique (data not shown). In addition to different numbers of cells being recovered by the two techniques, the cells recovered showed different kinetics of infiltration in the ears. With collagenase treatment, macrophages, which play an important role in the innate response and were detected with F4/80–Tri-Color mAb (Caltag), peaked at day 5 and then declined to levels found in mock-infected ears by day 11. Similarly, effectors of the adaptive immune response, such as T cells [detected with anti-CD3–phycoerythrin (PE)–Cy5 mAb; BD Pharmingen], recovered by collagenase treatment, peaked at day 8 and then declined. In contrast, with migration, both macrophages and T cells were maximal at day 11 (Fig. 1c). Similar results were observed with the BALB/c mouse strain (data not shown). Isolation of leukocytes by collagenase treatment enabled the beginning and end of the immune innate response and the subsequent adaptive response to be followed. Moreover, when cells were isolated by collagenase treatment, the kinetics of cell infiltration followed the kinetics of the innate and adaptive immune response more closely. In contrast, the immune-cell infiltration observed with the migration technique continued even after the lesion had healed (Tscharke et al., 2002). These results suggested that the collagenase technique is more appropriate than the migration technique to study the kinetics of leukocyte populations after viral infection, and the large number of cells obtained enabled functional analyses of these cells.
Leukocyte populations present in the VACV-infected ear and obtained with the collagenase technique were analysed further (Fig. 2). Resident macrophages constituted the major subpopulation of leukocytes in the mock-infected ear and early after VACV infection (Fig. 2a, b), as reported previously by in situ analysis of the mouse ear dermis (Dupasquier et al., 2004). However, later after infection, the percentage of macrophages decreased (Fig. 2a) and their absolute number declined after day 4 p.i. (Fig. 2b). The proportion of granulocytes (detected by high expression of Ly6G–PE; BD Pharmingen) increased slightly by day 2 p.i. (Fig. 2a) and continued to increase up to day 10 p.i., followed by a decline. The percentage of lymphocytes continued to increase up to day 17 p.i. (Fig. 2a), when the lesions had mostly disappeared and most leukocytes were lymphocytes (Fig. 2a, b). To detect antigen-presenting cells (APCs), cells were stained with anti-CD11c–PE and anti-major histocompatibility complex class II mAb–FITC (both from BD Pharmingen), which recognize both dermal dendritic cells and Langerhans cells. The number of these cells increased only slightly after infection and the proportion of APCs was similar to the percentage found in situ in mouse ear (Dupasquier et al., 2004). Overall, these data suggest that collagenase treatment enabled the kinetic response of the different immune populations to be evaluated after viral infection.
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cells (Fig. 2c, d). Among the T cells, TCR cells are abundant in murine skin (Asarnow et al., 1988). These cells are considered a first line of defence and play a role in the control of viral infection (Ninomiya et al., 2000; Wang et al., 2003), including after intraperitoneal infection with VACV (Selin et al., 2001). Up to day 4, these cells (detected with anti-TCR–PE mAb; BD Pharmingen) were the major population of lymphocytes (Fig. 2c, d). Previously, the lymphocyte populations have been studied only after day 7 p.i., due to the limited number of cells obtained, and TCR T cells were a minor population in the VACV-infected ear (Reading & Smith, 2003). The percentage of these cells did not increase after VACV infection (Fig. 2d), but the absolute cell number increased up to 4 days p.i.
NK cells are another effector population of the innate response that was reported to be involved in the response to VACV infection (Bukowski et al., 1983; Kennedy et al., 2000), but few studies have addressed NK recruitment after VACV infection (Natuk & Welsh, 1987; Prlic et al., 2005). In this study, usually <1 % of lymphocytes in mock-infected ears were NK cells (detected with NK1.1–FITC mAb; BD Pharmingen) (data not shown), but the percentage and absolute number of NK cells increased and peaked at day 7–10 p.i. NK activity against K562 target cells, detected with a 51Cr-release assay, was observed 6 days p.i. (data not shown). The changes in the numbers of TCR and NK cells were not described previously because these lymphocyte populations were followed only after day 7 p.i. (Reading & Smith, 2003), by when these cell populations had already decreased (Fig. 2c, d).
Compared with other lymphocytes, the infiltration of cells from the adaptive immune response, such as CD4+ and CD8+ T cells, was later and more pronounced, especially on days 7–10 p.i. (Fig. 2c, d). The percentage of CD4+ T cells (detected with anti-CD4–Tri-Color mAb; Caltag) still increased at day 17 p.i. (Fig. 2c), whereas the majority of the lesions had resolved at that time. In contrast, the percentage of CD8+ T cells (detected with anti-CD8–PE–Cy5 mAb; BD Pharmingen) decreased after day 7 p.i. (Fig. 2c). The absolute number of both T-cell subsets declined after day 10 p.i. (Fig. 2d). Cytotoxic T-lymphocyte activity against EL4 cells infected at 10 p.f.u. per cell with VACV strain WR was detected by using a 51Cr-release assay by day 7 p.i. (data not shown), as reported with BALB/c mice (Reading & Smith, 2003). Depletion of lymphocyte subsets showed that both CD4+ and CD8+ T cell-mediated immunity contribute to protection against intraperitoneal infection with VACV (Xu et al., 2004).
An expansion of T cells expressing NK-cell markers was reported in the lungs of influenza virus-infected mice (Kambayashi et al., 2001) and in the spleen of lymphocytic choriomeningitis virus-infected mice (Slifka et al., 2000). These cells are usually referred to as ‘NKT cells’. The proportion of T cells expressing NK1.1, used to quantify the NK cells, was very low (<1 %) in mock-infected ears and did not increase after VACV infection (data not shown). The presence of another NK-cell marker, DX5, which is also expressed on all murine NK cells, was detected on only a few CD3+ T cells in mock-infected ears (Fig. 3a) and the majority of these cells co-expressed NK1.1 (data not shown). However, during VACV infection, a population of T cells expressing DX5 (but not NK1.1) emerged (Fig. 3a, b) and underwent enormous expansion (>400-fold) that was the highest among all of the cell populations studied (Fig. 3b). At day 9 p.i., 67±7 % of these DX5+CD3+ cells were CD4+ and 28±7 % were CD8+ (data not shown). After day 10 p.i., the percentage and number of these cells decreased (Fig. 3b) in parallel with the reduction in lesion size (Tscharke et al., 2002). CD3+DX5+ T cells were associated with activation marker (CD69) and subsequent cell death in influenza virus infection (Kambayashi et al., 2001) and the results presented here suggest that NKT cells could play an important role in the control of VACV infection.
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). The intradermal ear-infection model mimics dermal vaccination because only a local lesion develops (Tscharke & Smith, 1999) and immunity against VACV challenge is induced (Fig. 1a). In this report, the different leukocyte populations infiltrating into the VACV-infected ear are described. VACV encodes numerous proteins that interfere with the host response to infection (Smith et al., 1997; Moss & Shisler, 2001) and their study is relevant to the development of recombinant VACV vaccines. The local immune response reported here could be used as a reference to test the effects of these different immunomodulatory proteins. Moreover, this intradermal model could be suitable to study the kinetics of the immune response in other viral infections.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Asarnow, D. M., Kuziel, W. A., Bonyhadi, M., Tigelaar, R. E., Tucker, P. W. & Allison, J. P. (1988). Limited diversity of antigen receptor genes of Thy-1+ dendritic epidermal cells. Cell 55, 837–847.[CrossRef][Medline]
Belkaid, Y., Jouin, H. & Milon, G. (1996). A method to recover, enumerate and identify lymphomyeloid cells present in an inflammatory dermal site: a study in laboratory mice. J Immunol Methods 199, 5–25.[CrossRef][Medline]
Belkaid, Y., Kamhawi, S., Modi, G., Valenzuela, J., Noben-Trauth, N., Rowton, E., Ribeiro, J. & Sacks, D. L. (1998). Development of a natural model of cutaneous leishmaniasis: powerful effects of vector saliva and saliva preexposure on the long-term outcome of Leishmania major infection in the mouse ear dermis. J Exp Med 188, 1941–1953.
Belshe, R. B., Newman, F. K., Frey, S. E., Couch, R. B., Treanor, J. J., Tacket, C. O. & Yan, L. (2004). Dose-dependent neutralizing-antibody responses to vaccinia. J Infect Dis 189, 493–497.[CrossRef][Medline]
Belyakov, I. M., Earl, P., Dzutsev, A. & 8 other authors (2003). Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses. Proc Natl Acad Sci U S A 100, 9458–9463.
Bukowski, J. F., Woda, B. A., Habu, S., Okumura, K. & Welsh, R. M. (1983). Natural killer cell depletion enhances virus synthesis and virus-induced hepatitis in vivo. J Immunol 131, 1531–1538.[Abstract]
Combadiere, B., Boissonnas, A., Carcelain, G., Lefranc, E., Samri, A., Bricaire, F., Debre, P. & Autran, B. (2004). Distinct time effects of vaccination on long-term proliferative and IFN--producing T cell memory to smallpox in humans. J Exp Med 199, 1585–1593.
Crotty, S., Felgner, P., Davies, H., Glidewell, J., Villarreal, L. & Ahmed, R. (2003). Cutting edge: long-term B cell memory in humans after smallpox vaccination. J Immunol 171, 4969–4973.
Davies, D. H., McCausland, M. M., Valdez, C. & 7 other authors (2005). Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice. J Virol 79, 11724–11733.
Dupasquier, M., Stoitzner, P., Oudenaren, A., Romani, N. & Leenen, P. J. M. (2004). Macrophages and dendritic cells constitute a major subpopulation of cells in the mouse dermis. J Invest Dermatol 123, 876–879.[CrossRef][Medline]
Earl, P. L., Americo, J. L., Wyatt, L. S. & 15 other authors (2004). Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox. Nature 428, 182–185.[CrossRef][Medline]
Fogg, C., Lustig, S., Whitbeck, J. C., Eisenberg, R. J., Cohen, G. H. & Moss, B. (2004). Protective immunity to vaccinia virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions. J Virol 78, 10230–10237.
Frey, S. E., Newman, F. K., Yan, L., Lottenbach, K. R. & Belshe, R. B. (2003). Response to smallpox vaccine in persons immunized in the distant past. JAMA 289, 3295–3299.
Greenberg, R. N., Kennedy, J. S., Clanton, D. J., Plummer, E. A., Hague, L., Cruz, J., Ennis, F. A., Blackwelder, W. C. & Hopkins, R. J. (2005). Safety and immunogenicity of new cell-cultured smallpox vaccine compared with calf-lymph derived vaccine: a blind, single-centre, randomised controlled trial. Lancet 365, 398–409.[Medline]
Hammarlund, E., Lewis, M. W., Hansen, S. G., Strelow, L. I., Nelson, J. A., Sexton, G. J., Hanifin, J. M. & Slifka, M. K. (2003). Duration of antiviral immunity after smallpox vaccination. Nat Med 9, 1131–1137.[CrossRef][Medline]
Hawlisch, H., Belkaid, Y., Baelder, R., Hildeman, D., Gerard, C. & Köhl, J. (2005). C5a negatively regulates Toll-like receptor 4-induced immune responses. Immunity 22, 415–426.[CrossRef][Medline]
Kambayashi, T., Assarsson, E., Chambers, B. J. & Ljunggren, H. G. (2001). Expression of the DX5 antigen on CD8+ T cells is associated with activation and subsequent cell death or memory during influenza virus infection. Eur J Immunol 31, 1523–1530.[CrossRef][Medline]
Kennedy, M. K., Glaccum, M., Brown, S. N. & 13 other authors (2000). Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J Exp Med 191, 771–780.
Moss, B. & Shisler, J. L. (2001). Immunology 101 at poxvirus U: immune evasion genes. Semin Immunol 13, 59–66.[CrossRef][Medline]
Natuk, R. J. & Welsh, R. M. (1987). Accumulation and chemotaxis of natural killer/large granular lymphocytes at sites of virus replication. J Immunol 138, 877–883.[Abstract]
Ninomiya, T., Takimoto, H., Matsuzaki, G., Hamano, S., Yoshida, H., Yoshikai, Y., Kimura, G. & Nomoto, K. (2000). V1+ T cells play protective roles at an early phase of murine cytomegalovirus infection through production of interferon-. Immunology 99, 187–194.[CrossRef][Medline]
Prlic, M., Gibbs, J. & Jameson, S. C. (2005). Characteristics of NK cell migration early after vaccinia infection. J Immunol 175, 2152–2157.
Pütz, M. M., Alberini, I., Midgley, C. M., Manini, I., Montomoli, E. & Smith, G. L. (2005). Prevalence of antibodies to Vaccinia virus after smallpox vaccination in Italy. J Gen Virol 86, 2955–2960.
Reading, P. C. & Smith, G. L. (2003). A kinetic analysis of immune mediators in the lungs of mice infected with vaccinia virus and comparison with intradermal infection. J Gen Virol 84, 1973–1983.
Rock, M. T., Yoder, S. M., Wright, P. F., Talbot, T. R., Edwards, K. M. & Crowe, J. E., Jr (2005). Differential regulation of granzyme and perforin in effector and memory T cells following smallpox immunization. J Immunol 174, 3757–3764.
Selin, L. K., Santolucito, P. A., Pinto, A. K., Szomolanyi-Tsuda, E. & Welsh, R. M. (2001). Innate immunity to viruses: control of vaccinia virus infection by T cells. J Immunol 166, 6784–6794.
Slifka, M. K., Pagarigan, R. R. & Whitton, J. L. (2000). NK markers are expressed on a high percentage of virus-specific CD8+ and CD4+ T cells. J Immunol 164, 2009–2015.
Smith, G. L., Mackett, M. & Moss, B. (1983). Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen. Nature 302, 490–495.[CrossRef][Medline]
Smith, G. L., Symons, J. A., Khanna, A., Vanderplasschen, A. & Alcami, A. (1997). Vaccinia virus immune evasion. Immunol Rev 159, 137–154.[CrossRef][Medline]
Symons, J. A., Adams, E., Tscharke, D. C., Reading, P. C., Waldmann, H. & Smith, G. L. (2002). The vaccinia virus C12L protein inhibits mouse IL-18 and promotes virus virulence in the murine intranasal model. J Gen Virol 83, 2833–2844.
Tscharke, D. C. & Smith, G. L. (1999). A model for vaccinia virus pathogenesis and immunity based on intradermal injection of mouse ear pinnae. J Gen Virol 80, 2751–2755.
Tscharke, D. C., Reading, P. C. & Smith, G. L. (2002). Dermal infection with vaccinia virus reveals roles for virus proteins not seen using other inoculation routes. J Gen Virol 83, 1977–1986.
Wang, T., Scully, E., Yin, Z. & 7 other authors (2003). IFN--producing T cells help control murine West Nile virus infection. J Immunol 171, 2524–2531.
Wilcock, D., Duncan, S. A., Traktman, P., Zhang, W.-H. & Smith, G. L. (1999). The vaccinia virus A40R gene product is a nonstructural, type II membrane glycoprotein that is expressed at the cell surface. J Gen Virol 80, 2137–2148.
Xu, R., Johnson, A. J., Liggitt, D. & Bevan, M. J. (2004). Cellular and humoral immunity against vaccinia virus infection of mice. J Immunol 172, 6265–6271.
Received 23 September 2005;
accepted 22 December 2005.
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6 except for mock-infected ear with migration technique, where n=1).
