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Journal of Virology, May 2005, p. 5943-5951, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.5943-5951.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Roles of CD4+ T-Cell-Independent and -Dependent Antibody Responses in the Control of Influenza Virus Infection: Evidence for Noncognate CD4+ T-Cell Activities That Enhance the Therapeutic Activity of Antiviral Antibodies

Krystyna Mozdzanowska, Michelle Furchner, Darya Zharikova, JingQi Feng, and Walter Gerhard*

Immunology Program, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104-4268

Received 18 October 2004/ Accepted 11 January 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have indicated that B cells make a significant contribution to the resolution of influenza virus infection. To determine how B cells participate in the control of the infection, we transferred intact, major histocompatibility complex class II (MHC-II)-negative or B-cell receptor (BCR)-transgenic spleen cells into B-cell-deficient and CD8+ T-cell-depleted µMT mice, termed µMT(–8), and tested them for ability to recover from infection. µMT(–8) mice that received no spleen cells invariably succumbed to the infection within 20 days, indicating that CD4+ T-cell activities had no significant therapeutic activity on their own; in fact, they were harmful and decreased survival time. Interestingly, however, they became beneficial in the presence of antiviral antibody (Ab). Injection of MHC-II(–/–) spleen cells, which can provide CD4+ T-cell-independent (TI) but not T-cell-dependent (TD) activities, delayed mortality but only rarely resulted in clearance of the infection. By contrast, 80% of µMT(–8) mice injected with normal spleen cells survived and resolved the infection. Transfer of BCR-transgenic spleen cells, which contained ~10 times fewer virus-specific precursor B cells than normal spleen cells, had no significant impact on the course of the infection. Taken together, the results suggest that B cells contribute to the control of the infection mainly through production of virus-specific Abs and that the TD Ab response is therapeutically more effective than the TI response. In addition, CD4+ T cells appear to contribute, apart from promoting the TD Ab response, by improving the therapeutic activity of Ab-mediated effector mechanisms.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Many components of the innate and adaptive defense contribute to the control of an influenza virus infection in the immunologically intact mouse. This has been evidenced by increased morbidity and mortality or delayed recovery of mice that have a defective alpha/beta interferon (IFN-{alpha}/ß) response system (17, 22, 36) or complement system (25, 31) or are deficient in major histocompatibility complex class I (MHC-I)-restricted CD8+ T (7) or B (3, 18, 21, 32, 59, 62) cells. Similarly, modifications that decrease the susceptibility of the virus to innate inhibitors in body fluids (52), cellular defense systems induced by IFNs and tumor necrosis factor alpha (TNF-{alpha}) (8, 55, 61), or recognition by CD8+ T cells (62) typically increase viral pathogenicity. However, although an intact multipronged defense is important for recovery from a very severe infection, there is substantial redundancy in the antiviral defense, and no known defense mechanism is absolutely required for effective control of an influenza virus infection of low to moderate severity. Thus, mice lacking IFN-{alpha}/ß receptors (51) or CD8+ T (12, 14, 43, 59), CD4+ T (9, 14, 46), or B (14, 53) cells are all capable of resolving an infection of moderate severity with no or minimal delay.

The relative importance of the various defense activities in the control of the infection in the intact host is not entirely resolved, as no single influenza virus strain has been tested systematically with the same challenge and readout method in the various murine knockout models. Nevertheless, the fact that the 50% lethal doses (LD50s) of three distinct virus strains—the highly pathogenic A/PR/8/34 (H1N1) (18) and A/Japan/305 (H2N2) (21) and the minimally pathogenic plasmid-generated H3N2 reassortant HK-RG (62) strains—were decreased ≥10-fold in B-cell-deficient compared to intact mice provides strong support for the notion that B cells make an important contribution to the control of the primary infection in the intact mouse, although additional defects in B-cell-deficient mice, such as the poor development of splenic T-cell zones and reduced splenic T-cell numbers (47) and possible defects in airway-associated lymphoid tissues (19) and in CD4+ T cells (4, 39), may also contribute to their low resistance to influenza virus infection. A significant role of B cells in the resolution of the infection is consistent also with the kinetics of the primary antibody (Ab) response, whose rise coincides with virus clearance (15, 24, 34, 58).

The aim of this study was to determine how B cells contribute to the control of the infection. B cells may participate through several activities, including (i) production of virus-specific Abs, in both T-independent (TI) and T-dependent (TD) fashion; (ii) secretion of cytokines/chemokines with antiviral and/or immunostimulatory activities; and (iii) cognate or noncognate cellular interactions that enhance the response of other cell types, particularly CD4+ T cells. Here, we were interested primarily in identifying the role of the virus-specific Ab response. This was done by adoptive transfer of spleen cells into B-cell-deficient and CD8+ T-cell-depleted hosts, termed µMT(–8), using B cells that differed in ability to generate virus-specific Abs or participate in the TD Ab response. The recipient mice were then tested for ability to resolve a primary infection with influenza virus. The results provide evidence for a major therapeutic role of the TD and lesser role of the TI Ab response. Furthermore, they reveal a significant therapeutic synergism between Ab- and CD4+ T-cell-mediated activities.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. B-cell-deficient mice (C57BL/6-Igh-6, referred to as µMT) (30), anti-HEL B-cell receptor (BCR)-transgenic mice [C57BL/6-TgN(IghelMD4), referred to as MD4] (20), and normal C57BL/6 mice, referred to as B6 mice, were obtained from Jackson Laboratories (Maine). The MD4 mice were bred to B6 mice in the Animal Facility of the Wistar Institute. Progeny mice were screened for expression of transgenic immunoglobulin (Ig) by two-color flow cytometric analysis of blood B cells, using fluorescein isothiocyanate-labeled anti-µb and phycoerythrin-labeled anti-µa (both from PharMingen). MHC-II-deficient mice, backcrossed for five generations to B6 (B6.129-H2-AB1tm1glmN5), and Rag-2(–/–) mice were obtained from Taconic, and athymic nude mice on B6 background from Taconic and NxGen Biosciences. All mice were maintained in the Institute's Animal Facility under specific-pathogen-free conditions.

Virus. The same infectious stock of mouse-adapted influenza type A virus A/PR/8/34 (H1N1), Mt. Sinai strain (PR8), was used throughout this study. It contained 109 50% Madin-Darby canine kidney (MDCK) tissue culture infectious doses and 108.3 50% mouse infectious doses (MID50) per ml. Purified PR8 was used for enzyme-linked immunosorbent assay (ELISA) and for measurement of hemagglutination inhibition titer in serum.

Media and solutions. ISC-CM consists of Iscove's modified Dulbecco's medium (Life Technologies, Gaithersburg, MD), supplemented with 0.05 mM 2-mercaptoethanol, 0.005 mg/ml transferrin (Sigma, St. Louis, MO), 2 mM glutamine (JHR Biosciences, Lenexa, Kans.), and 0.05 mg/ml of gentamicin (Mediatech, Herndon, VA). ISC-CM was further supplemented, as indicated, with bovine calf serum (HyClone Laboratories, Logan, UT) or bovine serum albumin (Sigma). PBSN is phosphate-buffered saline (PBS) containing 3 mM NaN3. PBSN was further supplemented, as indicated, with bovine serum albumin.

Antibodies. The rat monoclonal Abs (MAbs) GK1.5 (anti-mouse CD4, ATCC TIB 207), 53-6-7 (anti-mouse CD8{alpha}, ATCC TIB 105), 187 (anti-mouse C{kappa}, ATCC HB 58), and M4.1 (anti-mouse µ, kindly provided by Fritz Melchers, Biocenter, Basel, Switzerland) were purified from hybridoma culture fluid by adsorption/elution from protein G agarose (Pierce). 14C2-S1-4 and L2-10C1-2 are mouse hybridoma Abs specific for influenza viral M2 and hemagglutinin (HA) proteins, respectively. Both are of {gamma}2a/{kappa} isotype and have been described previously (44, 45).

T-cell depletion, spleen cell transfer, and infection of µMT mice. Purified anti-CD4 and/or anti-CD8{alpha} MAbs were injected intraperitoneally (i.p.) at 0.2 mg per dose into 7- to 12-week-old male µMT mice. The first injection was given 3 days prior to infection and repeated on day 1 and then in 6- to 7-day intervals until termination of experiments. This procedure has been shown previously (43, 46) to consistently deplete the respective T-cell population in spleen and lymph nodes by more than 90% when tested by flow cytometry. Mice treated with anti-CD8{alpha} are designated µMT(–8), and those treated with both anti-CD8{alpha} and anti-CD4 are termed µMT(–4/8). One day prior to infection, mice were injected intravenously (i.v.) with 20 x 106 spleen cells from male donor mice. The spleen cell suspension was depleted of erythrocytes and dead cells by centrifugation (10 min at 600 x g at room temperature) in a discontinuous Percoll gradient (70%/33%). Leukocytes at the interface were collected and washed twice with PBS. In the first experiment, spleen cells were further depleted of CD4+ and CD8+ T cells with magnetic beads prior to injection, but this was omitted in subsequent experiments, as the in vivo anti-CD8{alpha} MAb treatment completely suppressed activation of coinjected CD8+ T cells (data not shown) and coinjection of CD4+ T cells (~4 x 106) appeared to be advantageous by increasing CD4+ T-cell-mediated activities in the recipient mice. One day after spleen cell transfer, mice were infected under ketamine-xylazine anesthesia by application of 50 MID50 of PR8 in a volume of 50 µl to the nares. This procedure initiates an infection throughout the lower respiratory tract.

Measurement of serum antibody titers by ELISA. Sera were tested for presence of virus-specific Abs as described previously (45), using purified PR8 (~170 ng) as solid-phase immunosorbent. Bound murine Abs were detected with biotinylated MAbs specific for Cµ or C{kappa} or with goat antisera specific for C{gamma} (ICN Pharmaceuticals, Costa Mesa, CA) or C{alpha} (Southern Biotechnology Associates, Inc., Birmingham, AL). The assays were developed with streptavidin-alkaline phosphatase and p-nitrophenylphosphate (both from Sigma). Purified murine HA-specific MAbs of µ, {gamma}2a, and {alpha} isotypes, respectively, were used for standardization of the assay and quantification of the Ab titers in the test samples. All detection reagents exhibited the designated isotypic specificities (data not shown). Data were collected with the Emax microplate reader and analyzed with the SOFTmax PRO software (both from Molecular Devices, Sunnyvale, CA). Dilutions of serum samples were usually tested in triplicates and in at least two independent assays. Means from independent assays are shown.

Measurement of virus titer in the respiratory tract. The concentration of infectious virus in trachea and lung was determined as described previously (35) by titration of homogenized tissue extracts in microcultures of MDCK cells. Samples that were negative in the MDCK assay were tested also by inoculation of 2 x 50 µl of undiluted extract into the allantoic cavity of two 10-day-old embryonated hen's eggs. The threshold of virus detection is 101.3 and 101 50% egg infectious doses in the case of the lung and trachea, respectively.

Statistical analyses. Data were analyzed for statistical significance using Prism software. Ab and virus titers were compared by parametric and nonparametric t tests, respectively. Survival curves, created by the method of Kaplan and Meier, were compared for statistical difference by the log-rank test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Both TI and TD B-cell activities contribute to recovery from influenza virus infection. The contributions of TI and TD B-cell activities in the control of influenza virus infection were studied in B-cell-deficient µMT mice that were depleted of CD8+ T cells by chronic treatment with the CD8{alpha}-specific MAb 53-6-7. These CD8+ T-cell-depleted mice, termed µMT(–8), were injected i.v. with PBS or various types of spleen cells and 1 day later infected with 50 MID50 of PR8 virus. The mice were then monitored for morbidity (weight loss) and mortality, and all mice surviving until day 20 were killed and tested for virus titer in trachea and lung and Ab titer in serum.

All µMT(–8) mice that had been injected with PBS instead of spleen cells showed a progressive weight loss (Fig. 1B) and died before day 20 (Fig. 1A). This confirmed previous studies (43, 59) showing that CD4+ T cells and innate defense activities of µMT mice could not control this infection. The role of TI B-cell activities was tested by transfer of MHC-II–/– spleen cells into µMT(–8) recipient mice. Because of the lack of MHC-II, these donor B cells cannot engage in a cognate interaction with the recipient CD4+ T cells and can thus only provide TI activities. Compared to nonreconstituted µMT(–8) mice, MHC-II–/–->µMT(–8) mice showed a significant increase in survival (Fig. 1A) and a decrease in morbidity. Six of 14 (43%) mice in this group survived until day 20. The infection was still active in three of these mice but apparently had been resolved in the other three mice (Fig. 1C). A further and significant improvement in the control of the infection was seen in µMT(–8) mice that had received spleen cells from normal B6 mice and thus were expected to generate also a TD B-cell response in addition to the TI B-cell response. Thus, 94% of these mice survived the infection (Fig. 1A), and all surviving mice had cleared it by day 20 (Fig. 1C).



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  		FIG. 1. The role of TI and TD B-cell-mediated activities in the control of influenza virus infection in the absence of CD8+ T cells. B-cell-deficient µMT(B6) mice were depleted of CD8+ T cells by repetitive injection (indicated by arrows marked CD) of CD8{alpha}-specific MAb 53-6-7 (200 µg i.p./dose), starting on day –3 and repeated on days 1, 7, and 14. These mice are referred to as µMT(–8). Some mice were concomitantly depleted of CD4+ T cells by repetitive injection of MAb GK1.5 (200 µg/dose, i.p.) and are referred to as µMT(–4/8). On day –1, mice received an i.v. injection of 0.2 ml PBS (open circles) or 20 x 106 spleen cells (arrow marked Sp) from B6 (closed and open squares) or MHC-II–/– B6 mice (closed triangles). One day later (day 0), mice were infected intranasally with a 50 µl inoculation containing 50 MID50 of PR8. They were subsequently observed for mortality (A) and weight loss (B). The average body weight is shown as percentage of starting weight. On day 20, surviving mice were killed and tested for residual infectious virus in lung and trachea (C). The data are the compilation of six independent experiments, comprising a total of 17 mice in four experiments in the case of B6->µMT(–8), 14 mice in three experiments in the case of MHC-II(–/–)->µMT(–8), 19 mice in four experiments in the case of B6->µMT(–4/8), and nine mice in three experiments in the case of PBS->µMT(–8). (A) Percent survival of all mice within the group. Statistical significance between groups was determined by log-rank test of survival curves created by the Kaplan-Meier method. (B) Body weight as percentage of starting weight at day 0. Means and standard errors of the means of all mice within the group are shown. (C) Virus titer (log10) in lungs of mice that survived until day 20. Dashed horizontal line shows the threshold of detection.

 
The Ab response generated by day 20 in these two groups of mice showed the expected differences: sera from MHC-II–/–->µMT(–8) mice contained relatively small amounts of virus-specific Ab, predominantly of IgM isotype (Fig. 2). These TI Abs originated entirely from the transferred B cells because nonreconstituted µMT mice contained <0.1 µg/ml IgM and IgG (data not shown). By contrast, B6->µMT(–8) mice generated significantly stronger IgM and IgG responses, exceeding those seen in MHC-II–/–->µMT(–8) mice by 13- and 18-fold, respectively. This may explain their much-improved ability to control the infection. Note, however, that the Ab response in B6->µMT(–8) mice differed substantially from the response seen in intact B6 mice 20 days after infection, in that it contained more IgM than IgG while the inverse was the case in intact B6 mice. The sera also contained virus-reactive IgA at concentrations of 0.6 to 1.7 µg/ml. The IgA titers are not shown because they differed only minimally from the IgA titers measured in unmanipulated uninfected µMT mice (0.3 to 0.8 µg/ml) and thus did not appear to reflect significant virus-specific responses of the transferred B cells but rather the range of naturally produced IgA antibodies in µMT mice of B6 background (40).



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  		FIG. 2. Virus-specific Ab titers in serum. Ab titers in serum obtained from the mice shown in Fig. 1 on day 20 were determined by ELISA, using purified PR8 virus as immunosorbent and CHµ-, CH{gamma}-, or CH{alpha}-specific reagents for detection of bound Ab. Sera from intact B6 mice obtained 20 days after PR8 infection were used as positive controls. Each dot is the titer of an individual mouse. Horizontal bars indicate geometric mean titers within each group. The statistical significance of differences between groups was determined by t test. IgA titers are not shown (see text).

 
Taken together, these findings indicated that (i) CD4+ T cells of µMT mice, in conjunction with innate defenses, were incapable of controlling the infection; (ii) the TI B-cell response, in conjunction with the above responses, improved survival; and (iii) the additional TD response further improved the control of the infection and greatly facilitated its resolution.

The significant improvement in the control of the infection seen after transfer of 20 x 106 MHC-II–/– spleen cells into µMT(–8) mice was surprising in view of previous studies showing that nude mice of BALB/c background typically did not survive the infection (54, 56), yet contained 5 to 10 times more B cells than the MHC-II–/–->µMT(–8) mice. To confirm this finding on the B6 background, we compared the course of infection in Rag-2–/– and athymic B6 mice. As expected, all Rag-2–/– mice died within 20 days after infection (Fig. 3A). In the case of athymic mice, 50% survived until day 20. However, in contrast to the MHC-II–/–->µMT(–8) mice (Fig. 1B), the recuperation of body weight stalled in most athymic mice around day 20 (Fig. 3B). By day 28, four of these mice were killed and all were found to have a still-active infection (Fig. 3C, D28 samples). At this time point, virus-specific IgM titers in serum (3.3 ± 1.5 µg/ml) were similar to the titers seen in MHC-II–/–->µMT(–8) mice on day 20 (Fig. 2), but the IgG titers (3.0 ± 3.1 µg/ml) were ~6 times higher. One athymic mouse, which had regained its starting body weight by day 28 postinfection (p.i.), was further observed (Fig. 3A, {blacktriangledown}) and appeared to have recovered from infection; however, upon sacrifice at day 37 p.i., this mouse still contained infectious virus (105.2 50% tissue culture infectious doses) in its lung (Fig. 3C, D37). The results are in agreement with previous studies (56) and support the conclusion that the TI B-cell response can prolong survival but is ineffective in resolving the infection.



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  		FIG. 3. Course of PR8 virus infection in athymic and Rag-2–/– mice. Rag-2 (open squares, n = 5) and athymic B6 (filled triangles pointing up, n = 5) or BALB/c (filled triangles pointing down, n = 5) mice were infected intranasally with a 50 µl inoculation containing 50 MID50 of PR8 and observed for up to 37 days for change in body weight and survival. (A) Body weight (percent, mean ± standard error of the mean). A single athymic BALB/c mouse that fully regained its starting body weight was killed on day 37 for determination of virus titer in the lung. Four athymic B6 mice that survived past day 20 but did not regain their starting weights were killed on day 28 for determination of virus titer. (B) Survival. (C) Lung virus titer (log10) of mice killed on day 28 and 37. Each symbol shows the titer in an individual mouse. Open triangle shows titer from one mouse that died on day 27.

 
To confirm the protective role of the TI B-cell response, an additional set of experiments was done, in which B6 spleen cells were transferred into CD8+ and CD4+ T-cell-depleted µMT mice. Assuming complete depletion of both T-cell subsets, these B6->µMT(–4/8) mice would be expected to generate only a TI B-cell response. As shown in Fig. 1A, these mice exhibited similar survival as MHC-II–/–->µMT(–8) mice but experienced stronger morbidity (Fig. 1B) and were slightly less effective in clearing the infection by day 20 (Fig. 1C). Unexpectedly, however, Ab titers were significantly higher than in MHC-II–/–->µMT(–8) recipients, presumably because of a functionally incomplete CD4+ T-cell depletion, at least in the six surviving mice. Importantly, the observation that significantly higher Ab titers in the surviving B6->µMT(–4/8) compared to MHC-II–/–->µMT(–8) mice failed to result in a concomitant improvement raised the possibility that Abs and (noncognate) CD4+ T-cell activities may act synergistically in the control of the infection, a point confirmed below.

In conclusion, the results indicated that (i) innate defenses together with CD4+ T-cell-mediated activities of µMT mice were incapable of controlling the infection; (ii) B-cell-mediated TI activities improved recovery, particularly in the presence of concomitant (though not cognate) CD4+ T-cell-mediated activities; and (iii) the additional presence of TD B-cell activities resulted in a further and substantial improvement of the control of the infection and its clearance in most (92%) mice.

Virus-specific B cells are required for the control of the infection. To determine whether virus-specific B cells were important for an effective control of the infection, we tested spleen cells from MD4 mice in the same transfer system. MD4 mice express a transgenic BCR that is specific for hen egg lysozyme, and only ~10% of the B cells in these mice express endogenous V genes (20). Accordingly, spleen cells from MD4+ mice would be expected to contain ~10 times fewer virus-specific precursor B cells than spleen cells from normal mice and produce correspondingly less virus-specific Ab, at least in the initial phase of the response. In agreement with this, virus-specific Ab titers 20 days after infection were ~8 times lower in MD4+ mice than in MD4– littermates (Fig. 4A). Furthermore, the response in µMT(–8) mice injected with MD4+ spleen cells was substantially delayed compared to mice injected with MD4– spleen cells (Fig. 4B). As shown in Fig. 5, transfer of 20 x 106 spleen cells (43% to 59% B220+) from MD4+ mice into µMT(–8) recipients did not improve survival compared to nonreconstituted µMT(–8) mice. By contrast, injection of the same number of cells from MD4– littermates improved survival to the same degree as seen with transfer of spleen cells from normal B6 mice, which were included in some of these experiments as positive control. In addition, recipients of spleen cells from MD4+ and MD4– donors displayed similar morbidity as nonreconstituted and B6-reconstituted µMT(–8) mice, respectively (Fig. 5B). By day 20, all MD4–->µMT(–8) mice had cleared the infection and contained virus-specific Ab titers comparable to the B6 spleen cell recipient mice included in these experiments as positive controls (Fig. 5C).



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  		FIG. 4. MD4+ mice exhibit an impaired virus-specific Ab response. (A) BCR-transgenic (MD4+, open symbols) and nontransgenic (MD4–, closed symbols) littermates were infected intranasally with a 50 µl inoculation containing 50 MID50 of PR8. Sera obtained 20 days after infection were tested in ELISA for concentration of total PR8-specific Ab, using a C{kappa}-specific reagent for detection. Each symbol shows the titer of an individual mouse. Bars indicate geometric mean titers. (B) Spleen cells (20 x 106) from MD4+ (open symbols) or MD4– (closed symbols) donor mice were injected i.v. into µMT(–8) mice. One day later (day 0), the mice were infected intranasally as above with 50 MID50 of PR8. Serum samples obtained 4 and 7 days after infection from two mice within each group were tested in ELISA for concentration of total PR8-specific Ab.

 


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  		FIG. 5. Transfer of HEL-BCR transgenic (MD4+) spleen cells into µMT(–8) mice fails to improve control of PR8 infection. Transfer experiments using spleen cells from MD4+ mice (open circles) and their BCR-transgene-negative (MD4–) littermates (closed circles) were done as described in the legend to Fig. 1. The data are the composite of three independent experiments with a total of 10 recipient mice each for MD4+ and MD4– spleen cells, two experiments and six recipient mice for B6 spleen cells (closed squares), and two experiments and three recipient mice for PBS (open squares). (A) Percent survival. (B) Percent change in body weight, mean, and standard error of the mean of all mice within a group. (C) PR8-specific IgM (open circles) and IgG (closed circles) in serum of surviving mice 20 days after infection were measured by ELISA.

 
These observations indicated that virus-specific B cells were important for control of the infection in this transfer system.

The therapeutic activity of passive MAbs is enhanced in mice that contain CD4+ T cells. We wanted to pursue the above noted discrepancy in Ab titers and infection control between MHC-II–/–->µMT(–8) mice and B6->µMT(–4/8) mice. An obvious difference between these mice was the treatment of the latter group with anti-CD4 MAb, which suppressed at least partially the functional activity of CD4+ T and perhaps additional CD4+ cell types. Although these CD4+ cells could not resolve the infection on their own [Fig. 1A, see PBS->µMT(–8) mice], they may have provided activities that operated synergistically with Abs and thereby improved Ab-mediated control of the infection. To test this hypothesis, we compared the therapeutic activity of passive virus-specific MAbs in recipient µMT(–8) and µMT(–4/8) mice. The experiments were essentially performed like those shown in Fig. 1 and 5 except that the recipient mice did not receive spleen cells prior to infection but instead were injected chronically with virus-specific Ab after infection. The main purpose of these experiments was to assess various Ab treatment protocols for possible enhancement of therapeutic activity in µMT(–8) compared to µMT(–4/8) mice. This included treatment with a suboptimal dose of an HA- or M2e-specific Ab, both known from previous studies to be ineffective in resolving on their own the infection in SCID mice (44, 45), starting on day 1 p.i., and treatment with a mixture of both Abs starting on day 6 p.i. All recipient mice that received PBS instead of antiviral Abs died within 10 to 20 days after infection (Fig. 6A2). Note that µMT(–8) control mice died earlier (P < 0.01) than µMT(–4/8) mice and also showed faster loss of body weight (Fig. 6A). This appears to be a reflection of an augmented inflammatory response promoted by the CD4+ T cells which failed to control the infection and instead resulted in increased morbidity and mortality (11, 29, 33, 56, 63). By contrast, when treated with virus-specific MAbs, µMT(–8) mice consistently fared better than µMT(–4/8) mice: they showed relatively less weight loss (Fig. 6B1 and C1) or better recuperation of body weight (Fig. 6D1), lower mortality (Fig. 6B2 and C2) and, at termination of experiments, more effective clearance of virus from the respiratory tract (Fig. 6B3, C3, and D3). The results clearly showed that antiviral Abs were therapeutically significantly more effective in µMT(–8) than in µMT(–4/8) mice, in spite of the fact that control µMT(–8) exhibited greater morbidity than control µMT(–4/8).



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  		FIG. 6. The therapeutic activity of passive Ab is enhanced in the presence of CD4+ T cells. µMT mice were depleted of CD4+ and/or CD8+ T cells by repetitive i.p. injection of anti-CD4- and/or anti-CD8{alpha}-specific MAbs (indicated by arrows marked CD). On day 0, they were infected intranasally with a 50 µl inoculation containing 50 MID50 of PR8 and repetitively injected i.p. with PR8-specific MAbs (arrows marked with Ab). The mice were monitored for 30 days for morbidity (change in body weight, A1, B1, C1, and D1) and mortality (A2, B2, C2, and D2). At termination of experiments, lung and trachea were tested for presence of infectious virus (B3, C3, and D3). Three independent experiments with different Ab treatments are shown. (A1 and A2) Pooled data from all three experiments showing the course of infection in control mice that received PBS instead of antiviral Ab (n = 10/group). (B1, B2, and B3) Treatment with 15 µg HA-specific Ab L2-10C1 (G2a), starting on day 1 after infection. n = 6/group. (C1, C2, and C3) Treatment with 30 µg M2e-specific MAb 14C2-S1-4 (G2a), starting on day 1 after infection. Three mice in the µMT(–8) and two in the µMT(–4/8) group. (D1, D2, and D3) Treatment with a mixture of M2e- and HA-specific MAbs (as above), starting on day 6 after infection with 30 µg and 10 µg, respectively. The M2e- and HA-specific MAbs were used at 45 µg and 15 µg in subsequent injections. n = 6/group.

 
To mimic the conditions of an actual primary Ab response, an additional experiment was performed in which PR8-infected Rag-2(–/–) and µMT(–8) mice were treated 7 days p.i.—the time when the active Ab response becomes clearly detectable—by i.p. injection of 0.35 ml serum obtained from B6 mice 9 days after primary infection with PR8. The serum contained 18 µg PR8-specific IgM and 30 µg/ml IgG Abs, thus providing a single dose of ~6 µg IgM and 10 µg IgG per mouse. As observed in the comparison of µMT(–8) and µMT(–4/8) mice, µMT(–8) mice showed slightly greater weight loss than Rag-2(–/–) mice (Fig. 7A). On day 10 p.i., 3 days after Ab treatment, all surviving mice were killed and tested for virus titer in the lung. Virus titers were not different between control Rag-2(–/–) and µMT(–8), again showing that CD4+ T cells, on their own, had no significant effect on virus load. However, the virus load was significantly lower in Ab-treated µMT(–8) than in Ab-treated Rag-2(–/–) mice, again indicating a higher therapeutic activity of an actively produced early antiviral Ab preparation in the presence of CD4+ T cells. Thus, CD4+ T cells appear to contribute to the control of the infection not only by promotion of a TD virus-specific Ab response but also through additional B-cell-independent activities which are beneficial in conjunction with virus-specific Abs but appear to be ineffective and even harmful when they operate on their own.



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  		FIG. 7. Transfer of antiviral Abs actively produced during the early response to influenza virus infection. Rag-2(–/–) (squares) and µMT(–8) mice (circles) were infected intranasally with a 50 µl inoculation containing 50 MID50 of PR8 and monitored for weight loss (A) and mortality (B). Seven days after infection, mice (five/group) were treated by i.p. injection of 0.35 ml PBS (controls, open symbols) or pooled sera obtained from immunocompetent B6 mice 9 days after primary infection with 50 MID50 of PR8 (closed symbols). Three days later, surviving mice were killed and virus titers in lungs determined (C). In panel C, each symbol indicates the titer of an individual mouse. Bars show the geometric mean virus titer within each group. The significance between groups was determined by Mann-Whitney test and is indicated.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Both CD8+ T-cell-dependent and -independent activities contribute to the control of influenza virus infection in the immunologically intact host, and both are necessary, in addition to innate defense activities, for survival from a severe virus challenge with 1 LD50. In this study, the nature and relative importance of CD8+ T-cell-independent therapeutic activities were investigated by testing how short-term reconstitution of B- and CD8+ T-cell-deficient mice with various types of splenic B and CD4+ T cells affected their ability to control the virus infection. The virus challenge dose used in all experiments corresponded to 0.2 LD50 for immunologically intact mice. This is a substantial challenge that none of the nonreconstituted but 81% of the µMT(–8) mice reconstituted with 20 x 106 B6 spleen cells survived. The high rate of recovery seen in µMT(–8) mice after transfer of only 20 x 106 spleen cells confirms the remarkably high therapeutic efficacy of CD4+ T- and B-cell-mediated activities in this infection. Three main types of therapeutic activities may operate in this situation, in addition to innate defense: CD4+ T-cell- and TI and TD B-cell-mediated activities.

All µMT(–8) mice that received PBS instead of spleen cells died before day 20. This confirms previous studies showing that naïve (43) and even memory CD4+ (59) T cells are essentially incapable of controlling a pulmonary influenza virus infection in the absence of B cells. This cannot be attributed to an ineffective stimulation or recruitment of CD4+ T cells into the infected lung, as previous studies have not revealed substantial differences between CD4+ T-cell responses in influenza virus-infected B-cell-deficient and intact mice (32, 43, 59), with the possible exception of a diminished proliferative response in vitro of cells isolated from the spleen, though not draining lymph nodes, 9 to 10 days after infection (32). A pulmonary influenza virus infection is apparently less controllable by CD4+ T-cell-mediated defense activities than cutaneous herpes simplex virus infection (41), cytomegalovirus infection of the salivary gland (37), or pulmonary infection with Mycobacterium tuberculosis (10). In fact, CD4+ T-cell-mediated activities on their own were harmful, increased morbidity, and accelerated death (Fig. 6). Analogous observations have been made in other studies (11, 33, 56, 63). Importantly, however, CD4+ T-cell-mediated activities became beneficial in the presence of virus-specific Abs. This beneficial effect was clearly evidenced by enhancement of the therapeutic activity of passive MAb in µMT(–8) compared to µMT(–4/8) recipient mice (Fig. 6) and probably also by the finding that MHC-II–/–->µMT(–8) mice controlled the infection better than B6->µMT(–4/8) mice, in spite of smaller Ab titers in the former mice (Fig. 1 and 2). The basis of this therapeutic synergism between Ab and CD4+ T-cell activities remains unknown but could very well be due to greater availability of activated effector cells for mediating Ab-dependent cell-mediated cytotoxicity or enhanced phagocytosis and destruction of Ab-opsonized virus. The latter is supported by a recent study showing that passive Ab was more effective in intact mice than in FcR{gamma}(–/–) mice, which lack Fc{gamma}RI, Fc{gamma}RIII, and Fc{varepsilon}RI (28). An analogous observation was made in a study of rabies virus infection in which the efficacy of passive Ab appeared to be affected by the size of the concomitant inflammatory response (27) and in a recent study of protection against Friend murine leukemia virus where passive virus-neutralizing Abs were found to work synergistically with vaccine-primed T cells (42). Cooperative interactions between T cells and passive Ab have been observed also in the control of Cryptococcus neoformans infection in mice (5, 64).

Control of the infection was significantly improved in MHC-II–/–->µMT(–8) mice compared to PBS->µMT(–8) (Fig. 1). However, as these mice generated not only a TI B-cell but also a (noncognate) CD4+ T-cell response, the improvement seen in these mice overestimated the true therapeutic efficacy of the TI Ab response because of the above-mentioned synergism between CD4+ T-cell activities and Ab. This is supported by the finding that mice depleted of both CD4+ and CD8+ T cells controlled the infection somewhat less well than the MHC-II–/–->µMT(–8) mice in spite of having significantly higher serum Ab titers. Apparently, an incomplete depletion of CD4+ T cells, at least in the mice that survived until day 20, resulted in an Ab response that exceeded the true TI response but lacked the full synergistic contribution by CD4+ T cells. It is consistent also with the finding that athymic nude mice did not resolve the infection more effectively than MHC-II–/–->µMT(–8), in spite of having 5 to 10 times more B cells available for the TI response than the latter after transfer of 20 x 106 spleen cells. Thus, although the TI B-cell response clearly made a significant contribution to the control of the infection by delaying death, it lacked the ability to resolve it, at least in the case of the highly pathogenic mouse-adapted PR8 virus. The TI B-cell response appears to be more effective, however, in resolving a PR8 infection of the nasal epithelium or a pulmonary infection by a less pathogenic influenza virus, such as X31 (our unpublished preliminary observations). In addition, the TI B-cell response appears to be more effective in mice that carry a functional Mx gene and thus have a stronger innate defense (23).

It is interesting that some viral infections, e.g., by polyomavirus (57), rotavirus (16), and Sindbis virus (26), can readily be resolved in athymic nude mice. Furthermore, the TI Ab response has been reported to make a substantial contribution also to the control of Semliki Forest virus, in which case it can convert an infection that is lethal in SCID mice into a persistent subclinical infection of the central nervous system in athymic nude mice (1). Another example is vesicular stomatitis virus, where the TI Ab response, in conjunction with the complement system, has been reported to prevent the infection from spreading into the central nervous system (2, 49). Compared to the above examples, the TI Ab response appears to be of lesser therapeutic importance, at least in the control of a pulmonary infection by the pathogenic PR8 virus.

By far the most effective antiviral defense was seen in µMT(–8) mice that had been reconstituted with normal B6 spleen cells. Compared to the MHC-II–/–->µMT(–8) mice, these mice would be expected to differ mainly by the additional contributions from the TD B-cell response. The cognate T-B interaction augmented virus-specific IgM and IgG titers 13- and 18-fold, respectively, compared to the TI response seen in MHC-II–/–->µMT(–8) mice. However, the response was only two to three times higher than in B6->µMT(–4,8) mice (Fig. 2), which had a high mortality (Fig. 1A) and resolved the infection by day 20 only in one out of six mice (Fig. 1C). The large discrepancy in morbidity and control of the infection between these groups provides yet another example of the therapeutic synergism between Ab and CD4+ T-cell-mediated activities.

Although the Ab response in B6->µMT(–8) mice was largely the result of a cognate T-B interaction, it was clearly not a normal TD response, as its IgM titer was eight times higher and its IgG titer 13 times lower than seen in normal intact B6 mice 20 days after infection (Fig. 2). This could be due to an inherent defect in CD4+ T cells from B-cell-deficient mice as suggested for instance by the finding that these T cells promoted IgM but not IgG production by B cells in vitro (39) and inhibited in vivo reconstitution by bone marrow-derived cells (4). In addition, it is noteworthy that TNF-{alpha} and lymphotoxin {alpha}-deficient mice, which, like µMT mice, lack follicular dendritic cells and B-cell follicles, have been reported to produce normal or increased IgM and generally decreased IgG responses compared to intact mice after primary immunization with sheep red blood cells and trinitrophenylated-keyhole limpet hemocyanin (50) or after influenza virus infection (38). The TD response generated in the adoptive transfer system used here is therapeutically probably less effective than the normal TD response.

In conclusion, this study showed that a moderately severe pulmonary infection initiated with ~0.2 LD50 of the highly pathogenic influenza virus strain PR8 could be resolved in more than 80% of B-cell-deficient and CD8+ T-cell-depleted mice after reconstitution with only 20 x 106 naïve syngeneic spleen cells (~12 x 106 B cells). An effective control required a TD Ab response. However, it did not only depend on the size of the Ab response but also on concomitant noncognate CD4+ T-cell-mediated activities that augmented Ab-mediated therapeutic activities. This therapeutic synergism between antiviral Abs and CD4+ T-cell activities appears to operate also in other host-pathogen interactions (5, 27, 42, 64). In the case of influenza virus infection, it may account also for some of the CD8 T-cell-independent protection of the immune host against drifted or heterosubtypic viruses that circumvent neutralization by HA-specific Abs (6, 13, 35, 48, 60). The underlying mechanisms and their dependence on Ab specificity, isotype, and dosage remain to be identified.


   ACKNOWLEDGMENTS
 
We thank N. Baumgarth and W. Weninger for comments on the manuscript.

This work was supported by NIH grant AI13989 and the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health.


   FOOTNOTES
 
* Corresponding author. Mailing address: Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104-4268. Phone: (215) 898-3840. Fax: (215) 898-3868. E-mail: gerhard{at}wistar.org. Back


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 Introduction
 Materials and Methods
 Results
 Discussion
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Journal of Virology, May 2005, p. 5943-5951, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.5943-5951.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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