Cooperative Research Centre for Vaccine Technology, Department of Microbiology and Immunology, University of Melbourne, Royal Parade, Parkville, Victoria 3052, Australia
1 Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Parkville, Victoria 3052, Australia
Correspondence to: L. E. Brown
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Abstract |
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Keywords: NF-B, Rel, transcription factors
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Introduction |
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During mouse embryonic development, Rel is first detected in the fetal liver, and later in thymus and spleen (8). In the adult, Rel expression is largely restricted to a number of different haemopoietic cells (911), including B and T lymphocytes, neutrophils (12), and monocytes (13). In B and T cells, the highest levels of nuclear Rel are detected following mitogen stimulation, a finding consistent with its proposed role in the regulation of lymphocyte proliferation (10,14). In haemopoietic cells, Rel is mainly found in association with NF-B1 or RelA (7).
Despite the detailed biochemical and molecular characterization of the different NF-B/Rel complexes, particularly in lymphocytes, little is known about their specific roles in the immune response to infection. The ability to gain further insight in this area has recently been made possible by the generation of mutant mice which lack various NF-
B/Rel subunits (1517). Rel/ and NF-
B1/ mice have normal numbers of haemopoietic cells, indicating that these transcription factors are not essential for haemopoiesis (15,16). However, mature splenic B and T cells from Rel/ mice fail to respond to most mitogenic stimuli in culture (15), and the production of various cytokines by Rel/ T cells and macrophages is impaired (18,19). NF-
B1/ B cells also exhibit a reduced proliferative response to mitogens (16). Consistent with the impaired B cell proliferation, Rel/ and NF-
B1/ mice display a diminished humoral response when challenged with model antigens (15,16). RelB/ mice develop an inflammatory disease that appears to be due to aberrant T cell development (17). While death of RelA/ mice during embryogenesis (20) has prevented a detailed analysis of the role of RelA in immune responses, B and T lymphocytes isolated from RAG-1/ mice reconstituted with RelA/ fetal liver cells exhibit proliferative defects in culture (21).
Although in vitro studies have identified defects in the function of lymphocytes from the various NF-B/Rel-deficient mice, the capacity of these mice to mount an immune response to various pathogens remains unclear. In the case of the Rel/ mice, increased susceptibility to Leishmania major, an intracellular parasite, was linked to reduced cytotoxic killing by Rel/ macrophages (19). NF-
B1/ mice were found to be highly susceptible to staphylococcal infection (16); however, the immunological basis of this phenotype remains to be determined.
Here we examine the ability of Rel/ mice to mount anti-viral immune responses induced by either infection or vaccination. We show that when infected with influenza virus, Rel/ mice can clear the virus from their lungs and have functional cytotoxic T cells. However, they fail to develop the same high titres of serum anti-viral antibody and virus-neutralizing antibody achieved by their Rel+/+ counterparts. In addition, local influenza virus-specific antibody responses are impaired and these animals display a markedly reduced proliferative T cell response to the virus. Despite this, previously infected Rel/ mice have sufficient humoral immunity to allow protection against reinfection with the same virus. Vaccine-induced responses, however, were insufficient to protect Rel/ mice from reinfection.
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Methods |
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Virus
The influenza virus used was Mem 71 (subtype H3N1) virus, a genetic reassortant of A/Memphis/1/71(H3N2)xA/Bellamy/42 (H1N1). The virus was grown for 2 days in the allantoic cavities of 10-day-old embryonated hen's eggs, harvested and stored in aliquots at 70°C. Purified Mem 71 virus was supplied by CSL Limited (Parkville, Victoria, Australia) and was stored at 4°C in PBS with 0.1% sodium azide. Virus titres were determined by the haemagglutination assay (22) and expressed as haemagglutinating units (HAU)/ml. Infectious virus titres, used for quantitation of the intranasal (i.n.) infection dose, were obtained by assay of plaque formation in monolayers of Madin Darby canine kidney (MDCK) cells (23) and expressed as p.f.u./ml.
Immunization and challenge of mice
Mice were infected by the i.n. route, under penthrane anaesthesia, with 104.5 p.f.u. of Mem 71 virus in 50 µl PBS or immunized by the s.c. route in the scruff of the neck with 200 µl of virus solution containing 1000 HAU of virus. Proliferative T cell assays were carried out either with spleen cells taken 7 weeks after i.n. infection or with inguinal and popliteal lymph nodes taken 7 days after s.c. inoculation in the hind footpads with 10,000 HAU of purified virus in incomplete Freund's adjuvant (IFA; Sigma, St Louis, MO).
Cell culture medium
T cell culture medium consisted of RPMI 1640 (CSL) supplemented with 10% (v/v) heat-inactivated (56°C, 30 min) FCS, 2 mM L-glutamine, 2 mM sodium pyruvate, 30 µg/ml gentamycin, 100 µg/ml streptomycin, 100 IU/ml penicillin and 0.1 mM 2-mercaptoethanol.
Preparation of lung, spleen and lymph node cells
Lungs were removed after i.n. infection with influenza virus and the cells used as primary effectors in the cytotoxic T cell assay (6 days post-infection) or as a source of B cells in the ELISPOT assay (4, 6, 8 and 10 days post-infection). For the cytotoxic assay, lungs from individual mice were minced and digested with collagenase A (from Clostridium histolyticum; Boehringer, Mannheim, Germany; 4 mg per set of lungs in 2 ml RPMI 1640 supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin and 100 IU/ml penicillin) for 30 min at 37°C. Single-cell suspensions were produced by passing the digest through a wire mesh and the resulting cells were depleted of erythrocytes by treatment with Tris-buffered ammonium chloride (ATC; 0.15 M NH4Cl in 17 mM TrisHCl at pH 7.2). The lymphocytes were purified by centrifugation over Isopaque-Ficoll (24).
For the ELISPOT assay, lungs were sieved through a wire mesh, the resulting cell populations were ATC-treated, and any large clumps of debris that accumulated in these preparations after washing were allowed to settle and then removed prior to addition of cells to the wells. Mediastinal lymph node (MLN) cells used in these assays were also prepared by sieving and ATC treatment.
Spleen cells used in cytotoxic T lymphocyte (CTL) and proliferative T cell assays were similarly sieved and treated with ATC prior to re-stimulation in vitro or addition to nylon wool columns respectively. Inguinal and popliteal lymph nodes, used in the proliferative T cell assay, were sieved through wire mesh, washed and used without further treatment.
ELISA
ELISA was performed on sera from mice bled via the retro-orbital plexus. The assay was carried out as previously described (25) using 96-well polyvinyl chloride microtitre trays (Dynex Technologies Inc., Chantilly, VA) coated with 50 µl of a solution containing 1000 HAU of purified virus/ml. Antibody titres are expressed as the reciprocal of the antibody dilution giving an absorbance of 0.2 U.
ELISA was also used to examine the isotype profiles of influenza virus-specific Ig in the serum samples. Rabbit anti-mouse isotyping reagents (ICN, Costa Mesa, CA) were used for this determination as previously described (26). Antibody titres were expressed as the reciprocal of the dilution of antiserum giving an absorbance of 0.2 U above the background obtained with the relevant isotyping reagent.
ELISPOT assay
Influenza virus-specific antibody-secreting cells (ASC) were enumerated by an ELISPOT assay (27) based on that described by Segwick and Holt (28). Briefly, 200 HAU of purified virus/well were added to 96-well flat-bottomed polyvinyl microtitre plates (Dynex Technologies Inc.) and the plates incubated overnight in a humidified atmosphere at room temperature. Wells were then exposed to PBS containing 10% BSA to block remaining sites. After 1 h incubation, the plates were washed 3 times with PBS containing 0.05% Tween 20 (PBST) and then 50 µl of T cell medium was added to each well. Aliquots (50 µl) of ATC-treated lung and MLN cell suspensions were added to replicate wells, and six serial 2-fold dilutions performed. The plates were then placed in a vibration-free incubator at 37°C in 5% CO2 overnight. After incubation, the cells were lysed and removed by rinsing the plates twice with distilled water and twice with PBST. Rabbit anti-mouse isotyping reagents (ICN), diluted 1:400 in PBST containing 0.5% BSA, were added in 50 µl/well to duplicate titration series and the plates further incubated for 2 h in a humidified atmosphere. Plates were then washed 3 times with PBST and 50 µl/well of alkaline phosphatase-conjugated sheep anti-rabbit Ig (Silenus Laboratories, Hawthorn, Victoria, Australia; 1:1000 dilution in PBST containing 0.5% BSA) was added and incubated for a further 2 h in a humidified atmosphere. The plates were rinsed with PBST 3 times and once with distilled water. The assay was then developed by adding to each well 50 µl of ELISPOT substrate, which contained 1 mg of 5-bromo-4-chloro-3-indolyl phosphate (5-BCIP) per ml in 2-amino-2-methyl-1-propanol (AMP) buffer (28). After 30 min at room temperature green spots appeared, each spot representing a single ASC. The plates were washed 3 times with distilled water and spots counted using an inverted microscope. Results are expressed as the number of ASC (ELISPOTS)/106 cells.
Plaque assay for infectious virus
Lungs were removed, individually washed in HBSS, and transferred to a bijou bottle containing 1.5 ml HBSS supplemented with 100 IU of penicillin/ml, 100 µg of streptomycin/ml and 30 µg of gentamycin/ml and kept on ice. The tissue was disrupted by passage through a wire mesh sieve. The cells and debris were removed by centrifugation (300 g, 5 min) and the supernatant stored in aliquots at 70°C. Virus titres of the lung supernatants were determined by plaque assay on monolayers of MDCK cells (23).
Neutralization assay
Neutralization of virus infectivity was determined by measuring plaque reduction on MDCK monolayers (27). All sera were heat inactivated (56°C, 1 h) and filtered before use. Serial dilutions of sera in RPMI 1640 containing antibiotics were preincubated with 100150 p.f.u. of influenza virus in a total volume of 500 µl for 30 min at 37°C. One hundred microlitres of the mixtures was applied to duplicate washed MDCK cell monolayers. After absorption of virus for 45 min at 37°C (5% CO2), 3 ml of agarose overlay medium, prewarmed to 45°C, was added. After 3 days incubation (37°C, 5% CO2) the plaques were counted and the neutralization titre was determined as the reciprocal of the mean dilution of serum that resulted in a 50% reduction in the number of plaques obtained with virus alone.
Primary cytotoxic T cell assay
The cells used as targets in the cytotoxic T cell assay were EL4 (H-2b) tumor cells. Virus-infected and uninfected targets were prepared by incubating 2x106 EL4 cells in 500 µl of infectious virus solution (4000 HAU/ml) or serum-free RPMI, respectively. After 1 h incubation at 37°C, cells were washed once and resuspended in 200 µl of T cell medium containing 200 µCi 51Cr (Amersham). After 2 h incubation at 37°C the cells was washed 3 times and their concentration adjusted to 105 cells/ml. One hundred microlitre aliquots of target cells were then dispensed into 96-well U-bottom tissue culture plates.
Purified lung lymphocytes were resuspended in T cell medium and added to the wells at various E:T cell ratios, in 100 µl. Cells were gently pelleted by centrifugation of the plates (1000 r.p.m., 30 s). After the target and effector cells had been incubated together at 37°C for 4 h in 5% CO2, 100 µl of supernatant were removed from each well and the amount of radioactivity determined using a -counter. The specific 51Cr release at each E:T ratio is calculated by subtraction of the c.p.m. released spontaneously in wells containing target cells incubated with medium only. These values are then expressed as a percentage of the maximal releasable counts derived from the c.p.m. in samples from wells in which the target cells are incubated in detergent, minus the spontaneous release c.p.m. Spontaneous release usually ranged from 1 to 10% of the maximum releasable counts. Data are presented as the mean of the values obtained from triplicate cultures.
Secondary cytotoxic T cell assay
Secondary effector CTL were generated from the spleen cells of individual mice infected 4 weeks previously by the i.n. route (27). Briefly, 4x107 spleen cells, depleted of erythrocytes by treatment with ATC, were cultured in 25 cm3 tissue culture flasks (Corning) containing 15 ml of T cell medium. Of these, 1x107 cells had been incubated at 37°C for 30 min with 3000 HAU of infectious virus in 1 ml of serum-free RPMI and washed once prior to addition to the flask. After 5 days of culture at 37°C in a humidified atmosphere containing 5% CO2 the cells were washed 3 times and used in the 51Cr -release assay described above.
T cell proliferation assay
Spleen cell suspensions were prepared from mice 7 weeks after i.n. infection and draining lymph node cell suspensions were prepared 7 days after s.c. immunization with purified virus. T cells were isolated from these suspensions by passage through nylon wool columns (29). Proliferation assays were set up as previously described (30) in 96-well flat-bottomed tissue culture plates (Nunc, Roskilde, Denmark). Wells contained 3x105 T cells, 3x105 irradiated (2200 rad, 60Co source) spleen cells from Rel/ or Rel+/+ mice as a source of antigen-presenting cells and various amounts of purified influenza virus, in a total volume of 250 µl. Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 4 days with the addition of 1 µCi [3H]thymidine during the final 18 h. Cells were then harvested onto filter paper and the incorporation of radioactivity measured on a Packard Matrix 9600 direct ß-counter.
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Results |
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To investigate which, if any, of the mediators of clearance are compromised in Rel/ mice such that recovery is slightly delayed, we first examined the ability of these mice to mount a CTL response. Spleen cells from individual Rel+/+ and Rel/ mice given infectious virus by the i.n. route 4 weeks previously were re-stimulated in culture and examined for their ability to lyse virus infected targets. All mice showed high levels of specific anti-influenza CTL activity (Fig. 2), the levels of lysis of infected targets above that observed with uninfected targets being similar between the two groups of mice. The activity of CTL effector cells was also assessed directly in lung cell populations taken 6 days after i.n. infection with influenza virus. Figure 3
shows that Rel/ and Rel+/+ mice have comparable levels of influenza-specific pulmonary CTL, indicating that the c-rel gene product is not essential for CD8+ CTL induction, maturation and effector function.
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The local humoral immune response to influenza infection was also examined. The isotype profile of influenza-specific ASC was determined by ELISPOT assay of cells isolated from the lungs and MLN, which drain the lungs, taken at various days after i.n. infection (Fig. 5). In Rel+/+ mice, the ASC reached a maximum on day 8 post-infection in both tissues. In the lungs, cells secreting IgA dominated, whereas in the MLN, although IgA ASC were still the most abundant on day 8, ASC of each other isotype made up a considerable proportion of the total response. In the mice lacking Rel, the numbers of influenza-specific ASC detected in both lungs and MLN were reduced by at least 80% with each of the isotypes being affected.
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Discussion |
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In the present study, we have analyzed the immune response of Rel/ mice to influenza, a model viral infection. A normal immune response evoked by this virus can be considered to occur in two phases. During primary infection of naive mice, effective clearance of influenza from the lungs is largely due to CD8+ cytotoxic T cells but is aided by the developing humoral response. Resistance to secondary infection by the same viral strain is predominantly dependent on the production of high titre virus-neutralizing antibody. Our results show that naive Rel/ mice infected i.n. appeared healthy and were able to completely clear a primary respiratory infection, albeit with a delay of 12 days compared to normal mice. The induction of a normal influenza-specific cytotoxic T cell response in Rel/ mice indicates that Rel is not necessary for CTL induction. This differs from the RelB/ mice in which the normal response to an acute infection with lymphocytic choriomeningitis virus is impaired due to a defective CTL response that arises from a markedly reduced expansion of CD8+ T cells (36). These findings reinforce the notion that different NF-B/Rel proteins may serve specific roles in a cellular immune response.
Despite a normal CTL response, titres of serum antiviral antibodies of all isotypes are markedly reduced in the Rel/ mice. This finding is consistent with the impaired response of these mice to haptenated keyhole limpet haemocyanin (a T-dependent antigen), in which the amount of antigen-specific IgG1 and IgG2a is dramatically less than in normal mice (15). Moreover, we have shown that the number of ASC found at the site of infection in the lungs and the MLN is reduced in the infected Rel/ mice. These findings most likely account for the delay in viral clearance observed after infection of Rel/ mice and are compatible with the data derived in mice depleted of CD4+ T cells which also show a slight delay in clearance of virus in the absence of a developing humoral response (37,38). The additional functional consequence of these defects in B cell clonal expansion and antibody secretion in Rel/ mice was that the majority of animals receiving the s.c. vaccine, which is a less potent stimulator of humoral immunity than is infection, remained susceptible to subsequent exposure to virus rather than acquiring the life-long immunity that is normally induced. Infected Rel/ mice, though producing substantially less anti-viral antibody than infected Rel+/+ mice, nevertheless had levels similar to those in vaccinated Rel+/+ mice and were likewise protected against re-infection.
The precise basis for the defective anti-viral humoral response remains unclear. Although a weak antibody response coupled with the reduced proliferative capacity of CD4+ influenza-specific T cells isolated from Rel/ mice when recalled in vitro with influenza virus is consistent with an inefficient CD4+ Th response, an intrinsic defect in the function of Rel/ B cells may also contribute to impaired isotype production in these mice. For example, surface IgM+Rel/ B cells fail to switch to IgG1, IgE and IgG2a in the cultures containing the T cell cytokines IL-4 or IFN- (G. Grigoriadis, R. Grumont and S. Gerondakis, unpublished results), and naïve Rel/ mice show 100-fold less IgG1 and undetectable IgG2a in their serum compared to Rel+/+ mice (15). Furthermore, diminished expansion of antigen-specific B cell clones in vivo is consistent with previous findings showing that Rel is required for B cell proliferation in cultures stimulated with polyclonal mitogens (15). In the future, it should be possible to use adoptive transfer experiments to determine if the restoration of the anti-influenza antibody response requires normal B or T cells, or whether both populations are required.
Of additional interest is the finding that an intact CD8+ T cell response can occur in the presence of an apparently suboptimal CD4+ T cell response. It is well documented that in certain circumstances, CTL induction in vivo requires CD4+ Th cells. For example, the induction of anti-influenza CTL responses in mice primed by immunization with short synthetic peptides representing major CTL determinants of the virus could be completely inhibited by the in vivo depletion of CD4+ T cells (39). However, compatible with our own findings is the notion that induction of effective CTL responses by viruses is much less helper dependent. In studies examining influenza infection of MHC class II-deficient mice, the CTL activity of lung lavage cells was not diminished compared to infected control mice (40).
Influenza infection of Rel/ mice has provided an opportunity to study the functional outcome of a deficit in this transcription factor on both clearance of and protection against viral infection. It now remains to explore issues raised in this study, i.e. the relative contribution of the Th cell deficit to the overall immune deficiency of the Rel/ mice, and to define any differences in the activity of Rel in CD4+ versus CD8+ T cells both at the functional and molecular levels.
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Acknowledgments |
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Abbreviations |
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ASC | antibody-secreting cell |
ATC | Tris-buffered ammonium chloride |
CTL | cytotoxic T lymphocyte |
HAU | haemagglutinating unit |
IFA | incomplete Freund's adjuvant |
i.n. | intranasal |
MDCK | Madin Darby canine kidney |
MLN | mediastinal lymph node |
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Notes |
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Received 29 July 1998, accepted 13 May 1999.
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References |
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