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Heterosubtypic Immunity to Influenza A Virus in Mice Lacking IgA, All Ig, NKT Cells, or T Cells¹

Kimberly A. Benton^2,*, Julia A. Misplon^*, Chia-Yun Lo^*, Randy R. Brutkiewicz^,, Shiv A. Prasad^3, and Suzanne L. Epstein^*

^* Molecular Immunology Laboratory, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20852; Laboratory of Viral Diseases, Cellular Biology Section and Viral Immunology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and Department of Microbiology and Immunology, Indiana University School of Medicine, Walther Oncology Center, Indianapolis, IN 46202

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mechanisms of broad cross-protection to influenza viruses of different subtypes, termed heterosubtypic immunity, remain incompletely understood. We used knockout mouse strains to examine the potential for heterosubtypic immunity in mice lacking IgA, all Ig and B cells, NKT cells (CD1 knockout mice), or T cells. Mice were immunized with live influenza A virus and compared with controls immunized with unrelated influenza B virus. IgA^-/- mice survived full respiratory tract challenge with heterosubtypic virus that was lethal to controls. IgA^-/- mice also cleared virus from the nasopharynx and lungs following heterosubtypic challenge limited to the upper respiratory tract, where IgA has been shown to play an important role. Ig^-/- mice controlled the replication of heterosubtypic challenge virus in the lungs. Acute depletion of CD4⁺ or CD8⁺ T cell subsets abrogated this clearance of virus, thus indicating that both CD4⁺ and CD8⁺ T cells are required for protection in the absence of Ig. These results in Ig^-/- mice indicate that CD4⁺ T cells can function by mechanisms other than providing help to B cells for the generation of Abs. Like wild-type mice, CD1^-/- mice and ^-/- mice survived lethal heterosubtypic challenge. Acute depletion of CD4⁺ and CD8⁺ cells abrogated heterosubtypic protection in ^-/- mice, but not B6 controls, suggesting a contribution of T cells. Our results demonstrate that the Ab and cellular subsets deficient in these knockout mice are not required for heterosubtypic protection, but each may play a role in a multifaceted response that as a whole is more effective than any of its parts.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Infection with influenza virus of one subtype induces partial immunity against subsequent infection with other subtypes, called heterosubtypic immunity. Rather than completely preventing infection, heterosubtypic immunity promotes viral clearance and recovery in an immunologically specific manner (1, 2, 3). An understanding of the immune mechanisms responsible for this broad, long-lasting (2) cross-protection would be valuable in vaccine development. However, despite a long history of study in animal models, many questions remain about the basis of heterosubtypic immunity.

Multiple studies have examined the potential contribution of Abs to heterosubtypic immunity. Abs to hemagglutinin (HA)⁴and neuraminidase (NA) are predominantly subtype and even strain specific rather than broadly cross-reactive, and thus generally do not contribute to heterosubtypic protection. Abs to the conserved internal proteins nucleoprotein (NP) and matrix protein (M) 1 do not appear to contribute to protection (4, 5). However, Abs to the conserved transmembrane M2 are protective against heterosubtypic challenge (6, 7, 8). Passive transfer of serum generated by live virus immunization has been reported to confer protection against heterosubtypic virus challenge in some (9), but not other (10), studies. In the study in which protection was seen, the specificities of the protective Abs were not identified.

IgA is the predominant isotype in mucosal secretions (11), and is especially prominent in the upper respiratory tract, with the IgA/IgG ratio greater in the upper than lower respiratory secretions (12). Passive Ab studies showed that polymeric IgA is the main, if not the only, isotype protective against homologous influenza challenge in the nose (13). Foster nursing can confer homosubtypic protection, supporting a role for secretory IgA (14). Some reports have suggested that IgA in secretions may be more cross-reactive than IgG (15, 16, 17, 18, 19); such cross-reactivity may be a mechanism through which IgA could contribute to heterosubtypic immunity. Interference with viral replication or assembly during transcytosis (20, 21), as suggested for rotavirus (22), is a potential mechanism that might be mediated by IgA Abs to conserved internal proteins.

T cells have reactivities to conserved viral Ags, and so can contribute to heterosubtypic immunity. A long literature has characterized the CD8⁺ CTL response to conserved internal Ags, predominantly NP (23, 24, 25, 26), and the response of CD4⁺ T cells as measured by proliferation, help for Ab and CTL responses, and cytokine production (27, 28, 29). Th cells specific for NP or M can help for subsequent Ab responses to HA (27), suggesting a mechanism for accelerated responses to a mismatched virus strain at the time of challenge.

Some studies have directly addressed the roles of various T cell populations in heterosubtypic protection in mice primed by virus infection. In the studies of Liang et al. (2), depletion of either CD4⁺ or CD8⁺ T cells during the challenge period led to partial abrogation of heterosubtypic immunity in the nose, and depletion of both CD4⁺ and CD8⁺ cells completely abrogated it. In the lung, depletion of CD8⁺ T cells partially abrogated protection, but depletion of CD4⁺ cells did not and had no further effect when combined with CD8 depletion. We have also observed partial heterosubtypic immunity in the lung despite depletion of both CD4⁺ and CD8⁺ cells (10). These combined results suggest a possible role for some additional effector. One possibility would be CD4/CD8 double-negative T cells, perhaps T cells or NKT cells (NK1.1⁺ cells). Anti-influenza CTL restricted by nonclassical MHC molecules have been reported (30, 31). The in vivo biological role of these cell types has not been established, but one suggested role is the secretion of growth factors promoting tissue repair (32).

Because many questions remain about the contributions of various effectors to heterosubtypic immunity, we reexamined the issue using several different approaches. We tested a requirement for IgA in heterosubtypic immunity by challenge of the full respiratory tract and also challenge restricted to the upper respiratory tract using IgA knockout mice. These mice (33) were previously shown to be capable of protective immune responses against homologous influenza challenge (34), but had not been analyzed for heterosubtypic immunity. We also investigated the potential for heterosubtypic immunity in mice having no Abs at all. Various Ig-deficient mouse strains have been shown by several laboratories to be capable of protective immunity against homologous influenza challenge (3, 29, 35, 36), but little information is available about their potential for heterosubtypic immunity. Additional studies addressed the possible role of double-negative T cells by experiments in CD1 knockout mice lacking NKT cells or TCR knockout mice lacking T cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

The doubly inactivated (DI) mouse strain (J_H/J_H, C/C) that lacks mature B cells and Ig (37, 38, 39) was obtained from A. Jakobovits (Cell Genesys, Foster City, CA) under a Materials Transfer Agreement. The mouse strain lacking IgA (IgA^-/-) (33) was obtained from G. Harriman and the Baylor College of Medicine (Houston, TX) under a Materials Transfer Agreement. DI and IgA^-/- mouse strains were bred in our colony by brother-sister mating, and both sexes were used for these experiments. DI breeders were quality controlled for absence of serum Ig by ELISA. IgA^-/- breeders were quality controlled by PCR. In these mice, a band was observed using primers for the neo insert, but not using primers for the IgA heavy chain. Positive and negative controls for both sets of primers were included in all experiments. In addition, random sampling of IgA^-/- mouse sera for IgA on radial immune diffusion plates (The Binding Site Limited, Birmingham, U.K.) showed no IgA at the lowest level of detection, 2 µg/ml. B6 x 129 F₁ (F₁) mice of both sexes were purchased from Taconic Farms (Germantown, NY). The F₁ hybrid mice served as controls to approximate the genetic mix in the IgA^-/- mice, which contain genetic elements from B6 and 129. The mouse strain lacking CD1 (CD1^-/-) (40) was obtained from L. van Kaer, (Vanderbilt University, Nashville, TN), under a Materials Transfer Agreement, and bred by brother-sister mating. Mice from strains C57BL/6 (B6) and congenic B6.129P2-Tcrd^tm1Mom (^-/-), which lack TCR⁺ T cells (41), were purchased from The Jackson Laboratory (Bar Harbor, ME). The DI mice were housed in sterile caging. The protocols for in vivo experiments were approved by the Center for Biologics Evaluation and Research and National Institute of Allergy and Infectious Diseases Animal Care and Use Committees (Bethesda, MD).

Viruses

Influenza virus strains used were A/PR/8/34 (H1N1) obtained from J. Yewdell (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD); A/Udorn/307/72 (H3N2) and B/Ann Arbor/1/86 (abbreviated B/AA) obtained from B. Murphy (National Institute of Allergy and Infectious Diseases, National Institutes of Health); A/Philippines/2/82/X-79 (abbreviated X-79; H3N2, described previously; Ref. 42) and A/Johannesburg/82/96 (H1N1) obtained from R. Levandowski (Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD); and A/Japan/305/57 (H2N2) obtained from American Type Culture Collection (Manassas, VA). A/Philippines/2/82/X-79 is a reassortant virus (43) with the HA and NA genes of A/Philippines/2/82 origin, and the NP and M genes of A/PR/8/34 origin (10). Thus, genes for multiple internal components are shared with A/PR/8, but it lacks shared external Ags reactive with neutralizing Abs to A/PR/8. The stock of X-79 used in vivo for challenge was prepared as a pooled homogenate of lungs from C57BL/6 mice infected 4 days previously (15th passage). Other influenza viruses were prepared in the allantoic cavities of 9- to 10-day-old embryonated hen’s eggs. Virus stocks were frozen at -70°C until use.

Immunization and challenge with live influenza virus

Influenza viruses used for priming and challenge were titrated in vivo to determine doses sublethal and lethal to naive mice. For live virus immunization, mice received a sublethal dose of live influenza virus in a volume of 50 µl of PBS, administered intranasally (i.n.) under light anesthesia with methoxyflurane, which permits infection of the full respiratory tract (44). Twenty-four to forty-four percent mortality was observed in IgA^-/- mice following immunization with A/PR/8 in several experiments. The combinations of homologous and heterosubtypic influenza A viruses used for immunization and challenge are indicated in individual experiments. In all experiments, mice immunized with the heterologous influenza B virus (B/AA) were used as specificity controls, in addition to or instead of a naive control group.

At a minimum of 4 wk after virus priming, mice were challenged with live influenza virus i.n. For most studies, virus was administered under light anesthesia with methoxyflurane, as described for priming. Mice were challenged while awake for studies in which challenge virus exposure limited to the upper respiratory tract was desired. By i.n. challenge without anesthesia, infection is initially limited to the upper respiratory tract and then can spread to the lungs (44). ^-/- and B6 mice were anesthetized for challenge using ketamine and xylazine injected i.p.

Mice were monitored for body weight and mortality, or were sacrificed at the indicated day for in vitro virus titration of lung homogenates and nasal washes. Washing of the nasal cavities was performed by the method of Yetter et al. (44). In brief, a 25-gauge 5/8-inch needle was inserted into the posterior opening of the nasopharynx, and 0.5 ml of sucrose-phosphate-glutamate buffer (220 mM sucrose, 5.5 mM K₂HPO₄, 3.6 mM KH₂PO₄, 5.9 mM monosodium glutamate) was flushed through to the external nares. Lung homogenates and nasal washes were stored at -70°C.

Virus quantitation assays

Influenza virus was quantitated by titration on Madin-Darby canine kidney (MDCK) cells using cytopathic effect as the indicator of presence of virus (45), and titer was expressed as the tissue culture 50% infectious dose/ml (TCID₅₀/ml) (46).

Measurement of serum Ab responses to priming virus

Sera obtained from mice 3 wk after immunization with virus were tested for the presence of Ab by ELISA or virus neutralization assay. For ELISA, lysates of MDCK cells infected with A/PR/8, X-79, or B/AA were used to coat wells. To prepare lysates, MDCK monolayers were infected with virus (multiplicity of infection = 1), cells were harvested 24–36 h later, and the cell pellet was resuspended in PBS containing protease inhibitor PMSF (Sigma, St. Louis, MO) or Complete protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany) and subjected to three freeze-thaw cycles. ELISA plates were coated with lysates diluted 1/400–1/800 in PBS by overnight incubation at 4°C. Plates were then blocked with 1% FBS in PBS. Plates were washed with PBS containing 0.05% Tween 20. Serum samples were titrated in ELISA diluent (PBS-Tween BSA-azide), added to wells, and incubated for 2 h. Plates were washed and incubated 1 h with the secondary Ab, goat anti-mouse IgG alkaline phosphatase diluted 1/1000. Plates were then washed again and incubated with the substrate p-nitrophenyl phosphate in 50 mM sodium bicarbonate buffer, 1 mM MgCl₂, pH 9.8. OD was measured with a Multiscan ELISA plate reader at 405 nm. Titers are reported as the highest dilution that yielded an OD (OD₄₀₅) greater than the mean plus 2 SD of control sera from preimmune mice.

For the virus neutralization assay, 500 TCID₅₀ of virus was added to heat-inactivated serum pools serially diluted in medium containing 0.25 µg/ml trypsin, and incubated for 1 h at 37°C before transfer onto MDCK cell monolayers in 96-well plates. After 3 days, cells were scored visually for cytopathic effect of virus. The highest dilution of serum that inhibited induction of the cytopathic effect of virus on MDCK cells was reported as the virus neutralization titer. For X-79 virus neutralization, sera were pretreated with receptor-destroying enzyme (Denka Seiken, Tokyo, Japan) to reduce the background neutralization repeatedly observed for all sera, including preimmune sera.

In vivo depletion of T cell subsets

The mAbs used for in vivo depletion were: GK1.5 specific for mouse CD4 (47); 2.43 specific for mouse CD8 (48); SFR3-DR5 specific for human leukocyte Ag, used as a negative control; and 30-H12 specific for mouse CD90 (Thy-1.2), a clone that does not activate T cells (49). Ascites fluid was prepared by Harlan Bioproducts for Science (Madison, WI). All mAbs are rat IgG2b Abs and were used as delipified ascites. The concentration of rat IgG2b was measured by radial immunodiffusion, using kits from The Binding Site, and all doses were 1 mg/mouse of each Ab. The acute depletion protocol has been reported previously (10). Flow cytometry was performed, as previously described (50), to confirm completeness of in vivo T cell depletion.

Statistical analysis

Differences in virus titers among groups were compared by one-way ANOVA, followed by pair-wise comparisons by Student’s t test, where indicated. Differences in mortality among groups were first compared by ², followed by pair-wise comparisons by Fisher’s exact test. Statistical analysis was performed using SigmaStat version 2.0, Jandel Scientific Software (San Rafael, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Heterosubtypic immunity in IgA^-/- mice: full respiratory challenge and upper respiratory challenge

IgA^-/- mice were shown by Mbawuike et al. (34) to be robust and able to clear primary infection with influenza A/Taiwan/1/86. We found that they could survive and clear doses of X-79 (H3N2) and B/AA used for priming normal mice, although there was some mortality with the A/PR/8 (H1N1) dose needed to immunize (see Materials and Methods). Mice were immunized with H1N1, H3N2, or heterologous B/AA virus as a control for immune specificity. Most mice produced serum IgG against the immunizing virus (Table I). In an in vitro virus neutralization assay, serum Abs neutralized the immunizing virus, but not heterosubtypic or heterologous virus, demonstrating the functional specificity of the Ab response (Table II).

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Protection of IgA-/- mice from lethal heterosubtypic challenge of the full respiratory tract. IgA-/- mice were immunized i.n. under light anesthesia with the following viruses, in doses of TCID50 per mouse: A/PR/8 (H1N1), 102; X-79 (H3N2), 2 x 102; B/AA, 103. Mice were challenged 4 wk later i.n. under light anesthesia with A/PR/8 (H1N1, 5 x 104 TCID50 per mouse) (A) or X-79 (H3N2, 5 x 105 TCID50 per mouse) (B). The H3N2 and B/AA immunization groups contained eight to nine mice. The H1N1-primed groups initially contained eight mice, but due to deaths resulting from the immunization, these groups contained four to five mice at the time of challenge. Differences in mortality among immunization groups were significant for H1N1 challenge, p < 0.001, and H3N2 challenge, p < 0.001 (2). Pair-wise comparison of homologous or heterosubtypic immunization groups to the B/AA group challenged with the same virus: for H1N1 challenge (A), H1N1 immunization, p = 0.007, and H3N2 immunization, p = 0.003; for H3N2 challenge (B), H3N2 immunization, p = 0.021, and H1N1 immunization, p = 0.002 (Fisher’s exact tests).

Both IgA^-/- and F₁ mice demonstrated control of virus replication in the upper respiratory tract. Relative to heterologous B/AA virus, immunization with either homologous or heterosubtypic influenza A virus resulted in significant reductions in nasal wash virus titers on days 3 and 6 in IgA^-/- mice (Fig. 2A), and on days 2, 3, and 6 in F₁ mice (Fig. 2B). The virus titers in the lungs were more variable than those observed in the nasal wash samples. The considerable variability in lung titers may reflect differences both in initial nasal virus levels and in spread. In IgA^-/- mice (Fig. 2C), homologous immunization resulted in a significant reduction in lung virus titers at days 3 and 6 in comparison with B/AA immunization. With heterosubtypic immunization, a trend toward reduction in lung virus titers at days 3 and 6 was observed, but was not significant (p = 0.06). In F₁ mice (Fig. 2D), lung virus titers were significantly lower at day 6 in the homologous and heterosubtypic immunization groups than in the B/AA group. Thus, IgA does not appear to be required for protection against heterosubtypic influenza infection, as measured either by prevention of mortality following lethal full respiratory tract challenge or by level of virus replication in a nonlethal upper respiratory tract challenge.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Control of virus replication in IgA-/- and F1 mice after heterosubtypic challenge limited to the upper respiratory tract. Mice were immunized i.n. under light anesthesia with the following viruses, in doses of TCID50 per mouse: A/PR/8 (H1N1), 50; X-79 (H3N2), 102; B/AA, 103. Challenge with X-79 (104.5 TCID50 per mouse) was performed 4 wk later by i.n. administration without anesthesia. The mean virus titers ± SE are shown for nasal washes (A and B) and lungs (C and D) for groups of six mice sacrificed at days 2, 3, 6, and 10 postchallenge. Lung titer values of 0.5 were entered as 0.5 for the purpose of t tests. *, Significant difference in lung titers compared with the B/AA-immunized group on the same day, p < 0.05, Student’s t test.

In previous studies, we and others have demonstrated protection of Ig^-/- mice against homologous challenge (3, 29, 35, 36). Instead of µMT mice that have been reported to produce low levels of Ig (29), we chose to use DI mice that are unable to produce mature B cells and Ig due to the knockout of both the heavy and -chain loci (37, 38, 39). Our previous study in DI mice had used B/AA virus. DI mice are highly susceptible to infection with mouse-tropic influenza A strains, and cannot survive infection with lethal strains even at low doses intended to immunize. For the current study, we identified several influenza A strains that were nonlethal in mice and used reduction in lung virus titers 4 days after challenge (during peak virus replication in naive mice) as the measure of immunity.

As shown in Fig. 3, for several virus strain combinations, DI mice showed enhanced clearance of challenge virus after immunization with heterosubtypic as well as homologous influenza A viruses, but not heterologous B/AA virus. In Fig. 3A, immunization with homologous H1N1 or heterosubtypic H3N2 or H2N2 viruses resulted in significant reductions in lung titers of H1N1 challenge virus in comparison with naive mice. The majority of mice in the H3N2- or H2N2-primed groups, but none of the naive mice, cleared the challenge virus to undetectable levels. In Fig. 3B, immunization with H3N2 or H2N2 viruses resulted in significant reductions in lung titers of the H3N2 challenge virus in comparison with the B/AA-primed or naive groups. All of the mice in the heterosubtypic immunization group, and the majority of those in the homologous group, cleared challenge virus to undetectable levels, whereas none of the B/AA-immunized or naive mice were able to clear challenge virus from the lungs by day 4. Homologous protection would be expected to be better than heterosubtypic protection, and the minor fluctuations were not reproducible (see Fig. 4).

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Mice without Ig or B cells can be protected from influenza challenge by prior exposure to homologous or heterosubtypic influenza A virus. DI mice were immunized under light anesthesia with the following viruses, in doses in TCID50/mouse: B/AA, 104; A/Johannesburg (H1N1), 5 x 102; A/Udorn (H3N2), 104; A/Japan (H2N2), 105. Mice were challenged 4 wk later by the same method with 106 TCID50/mouse A/Johannesburg (A) or 104 TCID50/mouse A/Udorn (B), and lungs were harvested at day 4 postchallenge. The mean lung virus titers ± SE are shown. The values shown in parentheses are the number of mice that cleared lung virus/total mice. Mice with lung titer values below the limit of detection of 0.5 TCID50/ml were considered to have cleared the infection, and values of 0.5 were assigned for the purpose of statistical analysis. The differences in mean values among the groups within the same experiment were significant by one-way ANOVA, p < 0.001. *, Significantly different from naive group, p < 0.05. **, Significantly different from naive and B/AA immunization groups, p < 0.05.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Protection against heterosubtypic challenge in mice without Ig requires CD4+ and CD8+ T cells. DI mice were immunized under light anesthesia with the following viruses, in doses in TCID50/mouse: B/AA, 104; A/Udorn (H3N2), 104; A/Japan (H2N2), 105. Mice were challenged 4 wk later by the same method with 104 TCID50/mouse A/Udorn. To deplete the indicated T cell populations, mice were treated with ascites fluids containing mAbs (see Materials and Methods) at days 3 and 1 before challenge and day 2 after challenge. The mean lung virus titers ± SE are shown for day 4 postchallenge. The number of mice that cleared lung virus/total mice is shown in parentheses for each group. Mice with lung titer values below the limit of detection of 0.5 TCID50/ml were considered to have cleared the infection, and values of 0.5 were assigned for the purpose of statistical analysis. The differences in mean values among the groups within the same experiment were significant by one-way ANOVA, p < 0.001. *, Significantly different from control ascites-treated group, p < 0.05. The data are a compilation of two experiments conducted with identical priming and challenging doses and T cell depletion treatments.

In DI mice, specific immunity must be due to T cell effectors. To examine the roles of T cell subsets in heterosubtypic immunity in DI mice, we depleted groups of immunized mice of CD4⁺, CD8⁺, or both T cell subsets just before and following heterosubtypic challenge (Fig. 4). As in the experiments presented in Fig. 3, heterosubtypic immunization led to a significant reduction in the titers of challenge virus compared with immunization with B/AA virus. Depletion of either CD4⁺ or CD8⁺ T cells alone abrogated the reduction in virus titers. Thus, both CD4⁺ and CD8⁺ cells are required for optimal protection against heterosubtypic challenge in the absence of Ab.

As another approach to examine the potential of CD8⁺ cells to confer heterosubtypic protection in DI mice, we performed adoptive transfer of CD8⁺ splenocytes from virus-primed and boosted DI mice into CD4-depleted naive DI mice challenged with A/Udorn. The results were inconclusive, as transfer of 1.5 x 10⁶ CD8⁺ cells from homologously primed donors (the maximum cell dose available) did not confer protection on the recipients (data not shown). The lack of homologous protection could be due to an insufficient number of cells transferred, or could be due to the fact the recipients were depleted of CD4⁺ cells, which have been shown to be required for homologous protection against challenge with B/AA (3).

Heterosubtypic immunity in mice lacking NKT cells or T cells

Residual protection in normal mice depleted of CD4⁺ and CD8⁺ cells (2, 10) suggested an additional cellular effector in heterosubtypic immunity. To investigate this possibility, we tested whether various CD4/CD8 double-negative T cell populations were required for heterosubtypic immunity. We first examined CD1^-/- mice that lack NKT cells (40). CD1^-/- mice and B6 controls were immunized with H1N1, H3N2, or B/AA virus and challenged 1 mo later with lethal doses of H1N1 or H3N2 virus. Relative to immunization with B/AA virus, immunization with homologous or heterosubtypic virus conferred protection against mortality from challenge with H1N1 (Fig. 5, A and B) or H3N2 (Fig. 5, C and D) virus. This result demonstrates that NKT cells are not required for protection against heterosubtypic influenza challenge.

View larger version (35K): [in this window] [in a new window]   FIGURE 5. Protection of mice lacking NKT cells (CD1-/- mice) from heterosubtypic challenge. CD1-/- mice (A and C) and B6 mice (B and D) were immunized i.n. under light anesthesia with the following viruses, in doses of TCID50 per mouse: A/PR/8 (H1N1), 25; X-79 (H3N2), 25; B/AA, 100. Four weeks after immunization, challenge was performed i.n. under light anesthesia with 5 x 104 TCID50 per mouse A/PR/8 (H1N1) (A and B) or X-79 (H3N2) (C and D). Differences in mortality among immunization groups were significant: for CD1-/- mice, H1N1 challenge, p = 0.005, and H3N2 challenge, p = 0.001; for B6 mice, H1N1 challenge, p < 0.001, and H3N2 challenge, p = 0.027 (-square). Pair-wise comparison of homologous or heterosubtypic immunization groups to the B/AA group challenged with the same virus: for CD1-/- mice, both H1N1 and H3N2 challenge, and for B6 mice, H1N1 challenge, p = 0.02. For B6 mice, the heterosubtypic immunization group differed significantly from the B/AA immunization group with H1N1 challenge, p = 0.05 (Fisher’s exact test).

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Protection of -/- mice from heterosubtypic challenge requires T cells. -/- mice (A) and B6 mice (B) were immunized i.n. under light anesthesia with the following viruses, in doses of TCID50 per mouse: A/PR/8 (H1N1), 102; B/AA, 103. All mice had measurable serum IgG to the immunizing virus 3 wk after immunization, as measured by ELISA (data not shown). One month after immunization, challenge with X-79 (H3N2) was performed i.n. under ketamine/xylazine anesthesia at virus doses of 105 TCID50 per mouse for -/- mice and 104 TCID50 per mouse for B6 mice. To deplete the indicated T cell populations, mice were treated with ascites fluids containing mAbs (see Materials and Methods) at days 3 and 1 before challenge and days 2, 5, 7, and 11 after challenge. Groups with mice still alive and not yet regaining weight were depleted again on day 14. All groups contained 8–10 mice. Differences in mortality among immunization groups were significant for -/- mice and B6 mice (p < 0.001, -square). Pair-wise comparison of depletion groups to the control ascites group: for -/- mice, CD4 and CD8 depletion, p = 0.002, and CD4, CD8, and CD90 depletion, p < 0.001; for B6 mice, CD4 and CD8 depletion, not significant, and CD4, CD8, and CD90 depletion, p = 0.002 (Fisher’s exact test). For B6 mice, CD4 and CD8 depletion group differed significantly from the CD4, CD8, and CD90 depletion group, p = 0.010.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

A role for IgA had been suggested by some previous findings, as discussed earlier, so we studied heterosubtypic protection in IgA^-/- mice. Live virus immunization protected IgA^-/- mice from a lethal heterosubtypic challenge of the entire respiratory tract, demonstrating that IgA is not required for this type of protection. In addition, in the nasopharynx, where IgA is thought to play a prominent role, heterosubtypic immunity led to control of virus replication and control of spread to the lungs following challenge designed to infect only the upper respiratory tract initially. Thus, IgA is not required for heterosubtypic immunity even under these circumstances. Clearance of virus in the nasopharynx was too rapid to attribute to a de novo Ab response to challenge virus, and so is likely to be mediated by functions other than primed T cell help for a subsequent Ab response. In an in vitro assay, serum Abs to the priming virus did not neutralize the heterosubtypic challenge virus. However, a role for preexisting cross-reactive IgG or IgM Abs, if present locally in the respiratory tract, but not in the serum, cannot be ruled out entirely in these mice.

We next studied heterosubtypic protection by T cells acting alone, in DI mice that lack all Ig and B cells. Although subtype differences in HA and NA may not be as relevant as they are to Ab responses, influenza A subtypes also have differences in other proteins, including NP (54, 55, 56). This variation includes residues within and immediately flanking the epitopes recognized by CTL (57) and Th cells (58, 59) and could affect protection. For example, a single amino acid change within the dominant Th epitope of NP decreased recognition by lymph node cells (59), and a point mutation flanking the dominant CTL epitope ablated presentation of that epitope (60). Therefore, heterosubtypic challenge studies in DI mice extend homologous challenge studies by providing information on the breadth of T cell immunity. The influenza strains used in this study (Fig. 3) were isolated 15–39 years apart, and most likely have accumulated sequence variation in internal components.

Others have studied heterosubtypic immunity in µMT mice, another strain lacking Ig and B cells, using lethal challenge models. Nguyen et al. (61) studied heterosubtypic protection in µMT mice, but under conditions in which even homologous protection was not detected. O’Neill et al. (62) reported an experiment showing a delay in death in heterosubtypically immunized compared with naive µMT mice, but did not characterize the immune mechanisms involved.

In heterosubtypically immunized DI mice, control of virus replication occurred early after challenge, and the majority of mice were able to completely clear the infection. Acute depletion of T cell subsets at the time of challenge demonstrated that both CD4⁺ and CD8⁺ cells are required for heterosubtypic immunity in the absence of Ig. These results agree with our previous study in which full protection of DI mice against homologous secondary challenge with influenza B required both CD4⁺ and CD8⁺ cells (3).

The mechanism by which CD8⁺ T cells enhance viral clearance is most likely their CTL activity (26). How do CD4⁺ cells contribute? In normal mice, a probable role of CD4⁺ cells is providing help to B cells and thus an accelerated Ab response to challenge virus Ags, as has been demonstrated for adoptively transferred CD4⁺ cells (27). However, in DI mice, this cannot be their role. One possibility is help for responses of CD8⁺ cells, because both subsets are required. The need for CD4 help for CD8 responses was demonstrated in a recent study (63) conducted in µMT mice depleted of CD4⁺ cells, in which virus clearance, clonal expansion of virus-specific CD8⁺ cells in the spleen, and recruitment of virus-specific CD8⁺ cells to the lung were impaired. Class II-restricted cytolysis and production of soluble mediators such as cytokines are also possible roles of CD4⁺ cells and have been reported by others (28, 64, 65).

Despite the apparent requirement for both CD4 and CD8 subsets in DI mice, we observed a suggestion of protection by CD8⁺ or CD4⁺ cells alone in a small number of mice: 3 of 15 mice depleted of CD4⁺ cells, and 1 of 15 mice depleted of CD8⁺ cells, but none of the mice depleted simultaneously of both CD4⁺ and CD8⁺ cells were able to clear virus from the lungs. This trend is in agreement with previous studies in µMT mice, which reported greater contributions by CD8⁺ cells than CD4⁺ cells to protection against primary challenge or secondary homologous challenge through active immunization (29, 36) or adoptive transfer (35). Although CD8⁺ cells may play a greater role in the protective response than do CD4⁺ cells, it has been demonstrated that CD8⁺ cells alone cannot resolve infection/reduce titers in the absence of CD4⁺ cells in µMT mice (63, 66). Nguyen et al. (61) reported that CD4^-/- mice, which had normal class I CTL responses, but did not have measurable influenza-specific Ab after immunization, were not protected against homologous or heterosubtypic challenge, again showing limited function of CD8⁺ cells alone.

In contrast to Ig^-/- mice, heterosubtypic protection in normal mice was mediated by CD8⁺ T cells alone in the study of Liang et al. (2), or by either CD4⁺ or CD8⁺ T cells alone in our previous studies of DNA immunization (50). The need for both T cell subsets in DI mice may reflect the difference in mechanism discussed above, with CD4⁺ cells helping CD8⁺ cell responses, but not B cell responses, or it may reflect that T cell function is suboptimal in DI mice. Suboptimal T cell function in µMT mice was reported by Homann et al. (67), but other studies have shown that Ig^-/- mice have relatively normal total T cell numbers and T cell function by some measures (3, 35, 61, 66, 68).

In normal mice, some heterosubtypic immunity remains, despite simultaneous depletion of CD4⁺ and CD8⁺ cells (2, 10). This protection could be mediated by preexisting Ab, but not by help for a de novo Ab response. Other possibilities include various types of double-negative T cells. We investigated two of the possibilities, NKT cells and T cells. Mouse CD1, a MHC class I-like molecule, is used as a restriction element by NKT cells (69). NKT cells have been proposed to play an immunoregulatory role, in part due to their ability to rapidly secrete cytokines. CD1^-/- mice lack NKT cells (40). Heterosubtypic protection against influenza challenge was observed in CD1^-/- mice; therefore, the activities of cells that recognize CD1 (i.e., NKT cells) may contribute, but are not required.

^-/- mice are more susceptible to some respiratory pathogens (70). T cells accumulate in the lungs of influenza-infected mice 7–10 days after infection (71), and have been proposed to play a role late in infection by facilitating the repair of damaged lung, possibly by the secretion of growth factors (32, 72). Our data showed that knockout mice lacking T cells were protected against heterosubtypic challenge, which indicates that T cells are not required for heterosubtypic immunity. However, a role for T cells was suggested by our observation that heterosubtypic protection was abrogated by depletion of CD4⁺ and CD8⁺ cells in ^-/- mice, but not in B6 mice. Additional depletion of CD90⁺ cells abrogated protection in B6 mice. These data suggest that cells present in the CD4/CD8-depleted group of B6 mice were required for heterosubtypic protection in the absence of CD4⁺ and CD8⁺ effectors. It is possible that cells contribute to heterosubtypic immunity in the presence of these effectors, but that the effect is difficult to isolate and identify in the presence of a robust response. The differences in mortality between CD4/CD8 and CD4/CD8/CD90 depletion groups in B6 mice did not reach significance until late in the infection, which agrees with the proposed role of cells in contributing to recovery via tissue repair. We cannot rule out the possibility that the observed differences between B6 and ^-/- mice were attributable not to cells, but instead to genetic differences remaining after back-crossing the knockout to the B6 background for 12 generations, or to the 10-fold higher challenge dose used for ^-/- mice. The challenge doses were chosen based on our preliminary studies, which showed that ^-/- mice were less susceptible to X-79 challenge than B6 mice, perhaps due to a compensatory mechanism resulting from the lack of the population since birth. Although further study is needed, our results point to the exciting possibility that T cells contribute to heterosubtypic immunity. One potential mechanism is the secretion of factors that promote the repair and regrowth of respiratory epithelium.

The very fact that robust heterosubtypic immunity is multifactorial, and persists despite knockout or depletion of one or another immune effector mechanism, is its strength. The immune system is highly redundant, and different effectors may be important under different circumstances. Experimental systems highlighting the contribution of particular effectors allow us to explore which effectors are necessary and which are not, which are important under what conditions, and which should be monitored in vaccine trials. Future vaccines must be designed to induce as many effector mechanisms as possible. Although the potency and durability of heterosubtypic immunity in humans are unknown, the live influenza vaccines currently under development may provide a more useful level of this type of broad cross-protection than inactivated virus vaccine.

	Acknowledgments

 
We thank Howard Mostowski for performing flow cytometry analyses, and Anthony Ferrine and other staff in the Center for Biologics Evaluation and Research animal facility for expert animal care. We thank Drs. Kanta Subbarao, Karen Elkins, and Lynn Cooper for critical review of the manuscript. R. Brutkiewicz gratefully acknowledges the support and encouragement of Drs. Jonathan Yewdell and Jack Bennink, in whose laboratories the CD1^-/- mouse work was initiated.

	Footnotes

 
¹ This work was supported in part by a grant from the National Vaccine Program Office to S.L.E. K.A.B. was supported in part by an appointment to the Research Participation Program at the Center for Biologics Evaluation and Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.

² Address correspondence and reprint requests to Dr. Kimberly Benton, Food and Drug Administration, Center for Biologics Evaluation and Research, Office of Therapeutics Research and Review, Division of Cellular and Gene Therapies, 1401 Rockville Pike, HFM-521, Rockville, MD 20852. E-mail address: bentonk{at}cber.fda.gov

³ Current address: Division of Allergy, Immunology, and Transplantation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.

⁴ Abbreviations used in this paper: HA, hemagglutinin; DI, doubly inactivated; i.n., intranasal(ly); M, matrix protein; MDCK, Madin-Darby canine kidney; NA, neuraminidase; NP, nucleoprotein; TCID₅₀, tissue culture 50% infectious dose.

Received for publication May 9, 2000. Accepted for publication April 10, 2001.

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