Department of Pathology and Immunology, Monash Medical School, Commercial Road, Prahran, Victoria 3181, Australia1
Department of Microbiology and Immunology, Louisiana State University Medical Center, School of Medicine in Shreveport, Shreveport, LA 71130, USA2
Immunology Division, The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Parkville, Victoria 3050, Australia3
Authors for correspondence: (i) Francis Carbone. Fax +61 3 9903 0731. e-mail carbone{at}med.monash.edu.au(ii) William Heath. Fax +61 3 9347 0852. e-mail heath@wehi.edu.au
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Abstract |
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Introduction |
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Many of the phenotypic differences between naive and memory CTL reside in differential expression of adhesion molecules (Budd et al., 1987 ; Hamann et al., 1988
; Jung et al., 1988
; Lerner et al., 1989
), which suggests that differential CTL migration, as well as quantitative differences in the strength of the response, could also contribute to the enhanced virus clearance evident on subsequent reinfection. For example, activated T cells rapidly downregulate CD62L (Jung et al., 1988
), limiting their ability to enter lymph nodes, while upregulating adhesion molecules that facilitate entry into peripheral compartments (Butcher & Picker, 1996
; Mackay, 1991
). A large proportion of memory CTL retain this CD62Llo phenotype, at least for some time after infection (Doherty et al., 1996
; Tripp et al., 1995
; Zimmerman et al., 1996
), suggesting that these cells would be activated either within the spleen or, more likely, directly within tissues harbouring replicating virus during reinfection. In either case, the loss of CD62L expression renders these memory CTL incapable of transit through the high endothelial venules and thus may limit their direct access to draining lymph nodes on subsequent infection (Butcher & Picker, 1996
; Mackay, 1991
).
In an attempt to understand the mechanisms involved in lymph node T cell activation upon reinfection, we have examined the primary and secondary CTL response to foot-pad infection with herpes simplex virus type 1 (HSV-1). A variety of studies have shown that CTL play an important role in eliminating this virus from the primary site of infection (Bonneau & Jennings, 1989 ; Nash et al., 1987
; Simmons & Tscharke, 1992
). HSV-1-specific CTL from C57BL/6 mice recognize a Kb-restricted immunodominant determinant from glycoprotein B (gB) (Bonneau et al., 1993
; Hanke et al., 1991
; Witmer et al., 1990
). We have previously shown that T cells express a highly restricted pattern of T cell receptor (TCR) V
and junctional sequence conservation, which can be exploited to track the accumulation of gB-specific CTL in lymph nodes draining the site of infection (Cose et al., 1995
, 1997
). Here we use the dominant V
expression to compare the gB-specific CTL accumulation in this site following primary infection and secondary challenge with HSV-1.
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Methods |
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Virus and cell lines.
The KOS strain of HSV-1 was propagated on Vero cells. The gB peptide with the sequence SSIEFARL (single letter amino acid code) corresponding to residues 498 to 505 in HSV-1 gB was synthesized using an Applied Biosystems model 431A synthesizer (ABI) and kindly provided by J. Fecondo, Swinburne University of Technology, Hawthorn, Victoria, Australia. The H-2b thymoma cell line EL4 was grown in complete DMEM supplemented with 10% FCS.
Injection with HSV-1 or gB peptide.
C57BL/6 mice were injected in each hind foot-pad with 4x105 p.f.u. HSV-1 or with approximately 5 µg gB peptide emulsified either in TiterMax (CytRx Corporation) or complete Freunds adjuvant (CFA; Sigma). After 6 weeks these mice and a group of naive C57BL/6 mice were infected in each hind foot-pad with 4x105 p.f.u. HSV-1 and the draining popliteal lymph nodes and the non-draining mesenteric lymph nodes were removed between 1 and 10 days later. Viable cell counts were determined for all lymph node samples by trypan blue exclusion prior to analysis. Lymph node cells were analysed directly and after 3 days in culture in wells of a 96-well flat-bottom plate at a density of 1x106 cells per well in 250 µl complete RPMI containing 10% FCS without exogenous antigen.
Assessment of gB-specific CTL activity.
CTL lysis was assessed by a 4 h chromium release assay using 51Cr (150 µCi)-labelled EL4 cells in the presence or absence of 1 µg/ml gB peptide. Varying effector-to-target ratios were used for cells analysed directly ex vivo with the results expressed as a percentage of specific lysis. For cells analysed after 3 days of culture without exogenous antigen, the assay was performed using serial dilutions of effectors from one well of culture. Lytic units were determined for 20% specific release and related back to the ex vivo number of cells in each lymph node. Thus, the results are expressed as the number of arbitrary units per lymph node able to specifically lyse 20% of the target cells.
Flow cytometry.
Lymph node cells analysed directly ex vivo or after 3 days culture without exogenous antigen were double-stained with FITC-labelled anti-CD8 (CT-CD8a, Caltag Laboratories) and biotin-labelled anti-V10 (B21.5, Pharmingen) followed by phycoerythrinstreptavidin (Southern Biotechnology Associates). Dead cells were excluded using propidium iodide and the cells were visualized on a Becton Dickinson FACScan. The lymph node cells were separated in small and large (blast) subsets based on forward- and side-scatter parameters for all analyses. Previous analysis using CD44 and CD62L markers showed that the majority of cells falling into the blast cell gate are activated (Cose et al., 1997
). The proportion of CD8+ blasts expressing the V
10 element was determined by flow cytometry as the fraction of CD8+ T cell blasts expressing V
10 receptors and is given as a percentage.
Alternatively, lymph node cells were triple-stained with allophycocyanin-labelled anti-CD8 (53-6.7, Pharmingen), phycoerythrin-labelled anti-CD25 (PC61, Pharmingen) and biotin-labelled anti-V10 (B21.5, Pharmingen) followed by streptavidinFITC (Molecular Probes). Dead cells were excluded using propidium iodide and the cells were visualized on a Becton Dickinson FACScalibur. The proportion of CD8+CD25+ cells expressing the V
10 element was determined by flow cytometry as the fraction of CD8+CD25+ cells expressing V
10 receptors and is given as a percentage.
Determining virus titre.
Hind feet of mice infected with HSV-1 were removed at the ankle and individual feet were ground in a 5 ml homogenizer (Laboratory Supply, Australia) to make a 20% (w/v) suspension in RPMI supplemented with 10% FCS. These suspensions were frozen at -70 °C, then thawed rapidly and centrifuged at 12000 g for 10 min at 4 °C. The supernatant fluid was used immediately in a p.f.u. assay on Vero cells to determine the virus titre. Briefly, serial tenfold dilutions of the supernatant fluids were made in serum-free MEM and added to confluent monolayers of Vero cells in 6-well multiwell dishes (0·9 ml supernatant per well). After 1 h at room temperature with occasional rocking, 3 ml of 1% agarose in MEM supplemented with 2% FCS was added to each well. The plates were incubated at 37 °C for 4 days before being fixed with 10% formalin in phosphate buffer (4 ml per well). After 1 h incubation at room temperature, the formalin and agarose were removed and the cell monolayers stained with 0·01% crystal violet to visualize the plaques. Results are expressed as the number of p.f.u. of virus per foot-pad.
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Results |
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Discussion |
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Given the complexity of the HSV genome it was possible that the secondary response was simply directed to determinants other than gB. This was raised in a previous study which found a reduction in precursor CTL numbers during the peak of the recall response to this virus (Nugent et al., 1994 ). However, analysis of multideterminant lymphocytic choriomeningitis virus (LCMV) and Listeria responses showed no significant changes in the hierarchy of immunodominance when comparing primary and secondary CTL populations (Busch et al., 1998
; Murali-Krishna et al., 1998
). Moreover, while differential specificity selection could occur in convalescent animals infected with HSV-1 this would seem unlikely for the gB peptide-immunized mice. Only gB-specific CTL precursors were primed in this situation and any diminution seen on subsequent infection would be a direct consequence of this initial priming.
Rather than affecting the bias in determinant immunodominance as a consequence of prior antigen exposure, we favour the notion that the initial priming changed the pattern of gB-specific CTL migration and it was this altered migration that directly, or indirectly, diminished the secondary lymph node response. Naive and memory T cells possess differential migratory properties, with memory lymphocytes in particular preferentially entering infected parenchymal tissue and then returning to the circulation via the afferent lymph (Mackay, 1991 ; Mackay et al., 1990
). This differential migration is combined with a relaxed requirement for co-receptor engagement (Croft et al., 1994
; Luqman & Bottomly, 1992
; McKnight et al., 1994
) and, at least for memory CTL, a capability to kill targets in the absence of any further stimulation (Lalvani et al., 1997
; Zimmerman et al., 1996
). Consequently, on virus challenge memory T cells should be capable of directly dealing with tissue infection without prior activation within the lymphoid organs. Once the peripheral infection is resolved the specific CTL would be expected to either die in situ or return to the circulation via the draining lymph nodes. It is this latter post-infection and post-activation migration that most likely contributes to the transient increase in V
10+ T cells found in the activated CD8+ subset relatively late in both the primary and secondary responses to infection. These are assumed to be gB-specific CTL returning via afferent lymphatics.
Differential migration between naive and memory CTL alone, however, would not explain the diminution of lymph node-specific, gB-specific CTL accumulation during the secondary response. One possibility is that the memory CTL remain in the lymph node for a much shorter period prior to migration to the tissue. Alternatively, it is possible that rapid clearance of virus within the convalescent animals mediated by tissue activated memory CTL, CD4+ T cells or antibody simply limits the available antigen and thus diminishes the resultant CTL activation seen in the draining lymph node. While this seems feasible, it should be noted that more rapid antigen clearance did not appear to suppress other recall responses. Early control of virus and bacteria replication in influenza virus, LCMV and Listeria monocytogenes infections did not limit the massive CTL expansion evident in the recall response to these micro-organisms (Busch et al., 1998 ; Flynn et al., 1998
; Murali-Krishna et al., 1998
). In these studies, CTL numbers rose rapidly after reintroduction of the priming agent and persisted at high levels for some time after infection had been resolved. Unlike the LCMV and Listeria models of infection, HSV does not replicate within the site involved in the actual priming event. Consequently, the rapid and enhanced expansion of splenic CTL seen on LCMV and Listeria challenge could have been due to local proliferation of effector cells in order to deal with micro-organism replication rather than the initial activation of either the naive or memory CTL per se. For this model of HSV infection, priming occurs within the popliteal lymph nodes, while virus replication is confined to the essentially separate peripheral foot-pad compartment (Cook & Stevens, 1973
; Cose et al., 1997
).
The results presented here are consistent with the notion that the initial HSV-specific CTL priming occurs within the lymph nodes draining the site of infection (Carbone et al., 1998 ; Heath et al., 1998
). Presentation would then be mediated by professional antigen presenting cells (APCs), which provide all the co-receptor ligands necessary for effective naive percursor cell priming. Memory CTL have a more relaxed co-receptor and APC requirement and therefore should no longer need the specialized cellular environment found within the draining lymph node. Consistent with peripheral activation for memory CTL, initial T cell priming within primary responses results in the rapid downregulation of CD62L, which promotes extravasation across high endothelial venules (Hamann et al., 1988
; Jung et al., 1988
). We found previously that the lymph node-derived V
10+ blast cells bearing the gB-specific receptors were indeed CD62Llo at the peak of the primary response (Cose et al., 1997
). While some antigen-specific memory CTL regain the CD62L marker (Busch et al., 1998
; Tripp et al., 1995
; Zimmerman et al., 1996
), others maintain their CD62Llo phenotype for considerable periods (Doherty et al., 1996
). The basis for the variable reacquisition of this receptor is unknown. Virus or antigen persistence, especially in peripheral or extra-lymphoid compartments, may favour the retention of a more activated or migratory CD62Llo phenotype. This would appear attractive given the persistent nature of HSV and the fact that one of the major functions of adjuvant emulsions is to form a long-lived antigen depot. If this were the case, then more transient means of antigen delivery might alter the pattern of memory T cell migration to include entry into the lymph nodes and permit subsequent reactivation within this site during the recall response.
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Acknowledgments |
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Received 23 August 1999;
accepted 3 November 1999.