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The Journal of Immunology, 2007, 179, 6153 -6159
Copyright © 2007 by The American Association of Immunologists, Inc.
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Rectification of Age-Associated Deficiency in Cytotoxic T Cell Response to Influenza A Virus by Immunization with Immune Complexes

Biao Zheng*, Yongxin Zhang{dagger}, Hongxia He*, Ekaterina Marinova*, Kirsten Switzer*, Daniel Wansley*, Innocent Mbawuike{dagger} and Shuhua Han1,*

* Department of Immunology, {dagger} Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Decline in cellular immunity in aging compromises protection against infectious diseases and leads to the increased susceptibility of the elderly to infection. In particular, Ag-specific cytotoxic T lymphocyte (CTL) response against virus is markedly reduced in an aged immune system. It is of great importance to explore novel strategy in eliciting effective antiviral CTL activity in the elderly. In this study, the efficacy and mechanisms of immunization with immune complexes in overcoming age-associated deficiency in cellular immunity were investigated. In this study, we show that the severely depressed CTL response to influenza A in aged mice can be significantly restored by immunization with immune complexes consisting of influenza A virus and mAb to influenza A nucleoprotein. The main mechanisms underlying this recovery of CTL response induced by immune complex immunization in aged mice are enhanced dendritic cell function and elevated production of IFN-{gamma} in both CD4+ Th1 and CD8+ CTLs. Thus, these results demonstrate that immune complex immunization may represent a novel strategy to elicit effective virus-specific cytotoxic response in an aged immune system, and possibly, to overcome age-related immune deficiency in general.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In the elderly, the state of dysregulated immune functions, or immunosenescence, compromises protection against infectious agents and contributes to the increased susceptibility to infectious diseases. Infectious diseases are major causes of morbidity and mortality in the elderly, accounting for about one third of all deaths in people 65 years of age or older (1, 2, 3, 4, 5). In addition, there is a significant decrease in responsiveness to vaccination in aging. An impaired response to influenza infection and vaccination in the elderly may be the clinically most relevant fact associated with infectious diseases in aging (6, 7, 8). Approximately 90% of the as many as 50,000 to 70,000 annual excess deaths attributed to influenza occur in people aged 65 years or older (4, 5). Even when the antigenic match between influenza vaccine and circulating virus is close, vaccination provides protection for only 30–40% of subjects aged ≥65 years, compared with 70–90% of those <65 years (9). The currently available trivalent inactivated influenza vaccines are particularly ineffective in preventing deaths among elderly persons with associated chronic conditions (8, 9, 10, 11, 12), underscoring the need for influenza vaccines that are more effective in elderly persons that need them most.

Abs specific for influenza surface Ags such as hemagglutinin (HA)2 and neuraminidase (NA) play an important role in protective immunity when the HA and NA of the vaccines closely resemble those of the circulating virus strains. Mutation of HA and NA can result in viral escape from neutralizing Abs (antigenic drift). Occasionally, new viruses emerge with novel HA and NA, against which preexisting Abs are absent in the population (antigenic shift). In these cases, the MHC class I-restricted CD8+ CTL activity directed to more conserved proteins, such as nucleoprotein (NP), matrix protein, and polymerase proteins may contribute to protective immunity against these potentially pandemic viruses (13, 14, 15, 16). In addition, CD8+ CTL activity plays a major role in promoting recovery from severe influenza infection (16, 17, 18, 19). Thus, it will be essential for an effective influenza vaccine to be capable of inducing both high titers of neutralizing Abs and robust CTL activity to influenza.

Our earlier studies demonstrated that influenza virus-specific CTL activity was significantly diminished among elderly persons when compared with the young (20). Results from other groups also indicated an aged-related impairment in CTL activity, showing that elderly persons exhibited significantly lower and shorter-lived CTL response after vaccination with inactivated influenza vaccines (21, 22). Therefore, diminished CTL activity in the elderly may be responsible for poor protection against influenza infection, leading to the occurrence of prolonged and more severe infection in the elderly. This age-related deficiency in CTL activity has also been revealed in animal studies by our earlier studies and others showing delayed development and reduced activity of CTL responses in aged mice compared with young control mice (23, 24, 25). Thus, improvement of the virus-specific CTL responses in the aged may lead to reduced severity of viral infection in this age group.

Fc receptors (FcRs) link the humoral and cellular branches of the immune system and have important functions in the activation and modulation of immune responses. Because both effector cells such as B cell and mast cells, as well as APCs, such as dendritic cells (DCs), follicular dendritic cells (FDCs) and B cells, express various types of FcRs, immune complex (IC) can exert their immunoregulatory functions by direct signaling effector cells and/or by targeting APCs. Thus, the advantages of ICs in inducing immune responses are several fold: ICs can directly activate effector cells, ICs are effectively taken up by professional APCs, and the binding of IC to FcR can act as a natural adjuvant and mediate DC maturation. To explore whether the function of an aged immune system can be improved by Fc receptor signaling and whether IC immunization can overcome age-related immune deficiency including diminished CTL responses, we have investigated the efficacy of IC vaccination in inducing CTL activity against influenza virus. We have found that immunization with ICs can significantly enhance immune responses in aged mice. In particular, our data demonstrate that IC consisting of influenza vaccine and mAb specific for influenza A nucleoprotein can largely overcome the impairment in immune response to influenza and elicit significantly improved CTL responses in aged mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Young (2–4-mo-old) and aged (20–24-mo-old) BALB/c (H-2d) mice were from the Charles River Laboratory (Wilmington, MA) from cohorts maintained by the National Institute on Aging, National Institutes of Health. Animal experimentation was performed in accordance with protocols approved by IACUC of Baylor College of Medicine.

Influenza virus, vaccines, and immunization

Positive controls were young or aged mice immunized with ten 50% minimum infectious doses live mouse-adapted influenza A/Taiwan/1/86 (H1N1) intranasally (i.n.). Purified formalin-inactivated monovalent influenza A/Taiwan/1/86 (Connaught Laboratories) were used to immunize mice alone, or to form ICs with anti-NP mouse mAb (clone AA5H, IgG2a; Serotec) or with isotype control mAb (clone C1, IgG2a). The dosage was the amount of inactivated vaccine containing 5 µg HA/mouse. The amount of anti-NP or control mAb was 5 µg/mouse. The ICs (or vaccine plus control mAb) was prepared by incubating equal amount of Ag and mAbs at 37°C for 2 h, then at 4°C for 18 h. Although NP is an internal protein, we can detect NP in our vaccine preparation by anti-nucleoprotein mAb (ELISA). The HA/NP ratio in exposed surface of the vaccine is ~2,000/1 (unpublished data). Mice were immunized 200 µl:mouse i.p. In experiments studying CTL responses and CD8 T cell cytokine production, a second injection was given 5 wk later.

CTL assay

Influenza-specific CTL activity was measured as we described earlier (23). In brief, 12 days after boost, spleen cells were prepared and stimulated for 6 days with virus-infected syngeneic spleen cells, or medium only. Cells were then washed and titrated in the specific cytotoxicity assay. Target cells were P815 (H-2d) cells infected with live virus, P815 cells exposed to medium only, or EL-4 (H-2b) cells infected with virus. Cytotoxicity was determined after 4 h by measuring released 51Cr. Specific CTL activity will be calculated as: (experimental release – spontaneous release)/(maximum release – spontaneous release) x 100%.

Flow cytometry

For measurement of intracellular production of individual cytokines (IL-4, IL-10, and IFN-{gamma}), cells were first cultured for 3 days in the presence of virus-infected syngeneic spleen cells. Then, cultured cells were stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin for 1 h and with 10 µg/ml Brefeldin A (all from Sigma-Aldrich) for additional 4 h. Cells were recovered, washed, and stained with FITC- or biotin-labeled Abs to CD3, CD4, and CD8, followed by streptavidin-TC. After surface staining, cells were washed and fixed with 4% paraformaldehyde at room temperature for 10 min. Cells were treated with 0.5% saponin at room temperature for 10 min. Finally, cells were washed and incubated with PE-labeled anti-IFN-{gamma} Ab. Dendritic cells were stained with anti-CD11c-APC, anti-CD86-PE, and anti-I-A/I-E-biotin, followed by streptavidin-PerCP. All staining reagents were from BD Pharmingen. Samples were collected on a FACSCalibur machine (BD Biosciences) and analyzed using Flow Jo software (Tree Star).

Detection of influenza-specific Abs by ELISA

Influenza HA- or NP-specific Abs in mouse sera were determined by ELISA as described (26). In brief, microplates were coated with HA- or NP overnight and then blocked with 10% FCS. Samples were added and incubated for 1 h at 37°C and washed. HRP-conjugated goat anti-mouse IgG1, IgG2a, and IgM (Southern Biotechnology Associates) were used as secondary detection reagents. Levels of HA- or NP-specific Abs were calculated using standard sera or mAb to NP.

Measurement of Ab-forming cells (AFCs) by ELISPOT assay

The frequencies of specific AFCs from both splenocytes and bone marrow (BM) cells were estimated by ELISPOT assay as described (27, 28). In brief, nitrocellulose filters were coated with 5 µg/ml HA or NP in PBS at 4°C overnight, and then blocked with 10% FCS in PBS. Splenocytes (5 x 105 cells/well) or BM cells (106 cells/well) were incubated on the filters in 96-well plates at 37°C, 5% CO2. After 2-h incubation, filters were washed with PBS containing 50 mM EDTA once, followed by PBS containing 0.1% Tween 20 twice and PBS once. Filters were double-stained with AP-conjugated anti-mouse IgM and HRP-conjugated anti-mouse IgG1 Abs. AP and HRP activities were visualized using 3-amino-9-ethylcarbazole and napthol AS-MX phosphate/Fast Blue BB, respectively.

Dendritic cell isolation, stimulation, and immunization

Splenic DCs were labeled by incubating with anti-CD11c-biotin followed by streptavidin microbeads. DCs were positively isolated passing through a magnetic column twice. Procedures of MACS separation were according to manufacturer’s instructions (Miltenyi Biotec). Purified DCs were incubated for 48 h with immune complex vaccine or vaccine mixed with isotype control Ab. To investigate the efficacy of DCs stimulated with immune complex, DCs cultured above were washed in PBS and injected into the footpads of recipient mice (5 x 106 cells/mouse). Seven days after immunization, draining lymph nodes were taken and lymph node cells (4 x 105 cell/well) were cultured in 96-well flat-bottom plates with medium only or various concentrations of the vaccine for 4 days. Cells were pulsed with 3[H]-thymidine (1 µCi/well) for the last 18 h and incorporation of 3[H]-thymidine was measured with a LKB 1205 Betaplate liquid scintillation counter (Wallac).

Statistical analysis

Student’s t test of unpaired data was used to determine the significance of differences in means. A value of p < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Immunization with immune complex vaccines enhances influenza-specific CTL activity in aged mice

We investigated whether IC vaccination can repair the diminished specific CTL response to influenza A in aged mice by i.p. immunizing aged and young BALB/c (H-2d) mice with ICs consisting of inactivated monovalent influenza A/Taiwan/1/86 (H1N1) and anti-influenza A NP mAb. Control groups include: 1) mice immunized with ten 50% minimum infectious doses live mouse-adapted influenza A/Taiwan/1/86 i.n., 2) mice i.p. immunized with inactivated monovalent influenza A/Taiwan/1/86 only, and 3) mice i.p. immunized with inactivated monovalent influenza A/Taiwan/1/86 plus isotype-matched control mAb.

Our results show that after in vitro stimulation with influenza A virus, effector cells generated from all groups of young mice, either immunized with different forms of inactivated vaccines or infected by live virus, exhibited significant virus-specific CTL activity (Fig. 1A). Interestingly, effector cells generated from young mice immunized with ICs showed higher CTL activity than those from mice immunized with other forms of vaccines including live-virus infection. In marked contrast, effector cells generated from aged mice, immunized either with inactivated vaccines (alone or with control mAb), or even infected with live influenza virus, exhibited little CTL activity (Fig. 1B). These results support earlier findings (20, 21, 22, 23, 24, 25, 29, 30) demonstrating age-related impairment in generating influenza virus-specific MHC class I-restricted CTL activity. Remarkably, effectors generated from aged mice immunized with ICs exhibited significantly enhanced virus-specific CTL activity (Fig. 1B). Therefore, our results demonstrated that immunization with ICs can significantly alleviate age-related deficiency in generating CTL response against influenza A virus.


Figure 1
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  		FIGURE 1. IC vaccination enhances CTL responses against influenza virus-infected target cells in aged mice. Young (A) or aged (B) BALB/c mice were immunized with live influenza virus (•), inactivated virus, i.p. ({square}), inactivated virus with isotype control Ab ({blacksquare}), or IC vaccine ({diamondsuit}, as described in the text. Five wk after primary immunization, the mice received boost injections. Twelve days after boost, spleen cells were prepared and stimulated for 6 days with virus-infected syngeneic spleen cells (upper panels in both figures), or medium only (lower panels). Cells were then washed and titrated in the specific cytotoxicity assay. Target cells were P815 (H-2d) cells infected with live virus (left columns in both figures), P815 cells exposed to medium only (middle columns), or EL-4 (H-2b) cells infected with virus (right columns). Cytotoxicity was determined after 4 h by measuring released 51Cr. Data (mean ± SE) are from triplicate assays from an experiment with six mice in each group. Similar independent experiments have been repeated twice.

 
Immunization with IC enhances IFN-{gamma} production by CD8+ cells from aged mice

IFN-{gamma} is a pivotal cytokine for the induction of anti-viral CTL responses. It has been shown that there is a strong correlation between CD8+ CTL activity and IFN-{gamma} synthesis (31, 32, 33). In earlier work, we and others have observed a significant reduction of IFN-{gamma} production by CD8+ T cells responding to influenza virus in aged mice (33, 34).

To determine whether IC vaccination enhances IFN-{gamma} production by influenza A-specific cytotoxic CD8+ cells from aged mice, we measured the levels of intracellular IFN-{gamma} in CD8+ cells. Splenic cells from aged mice immunized with different vaccines (live influenza A virus, inactivated vaccine alone, ICs containing inactivated vaccine, and specific mAb, or inactivated vaccine plus control mAb) were stimulated with virus-infected stimulator cells for 4 days and examined for IFN-{gamma} production by intracellular cytokine staining. The results show that the levels of IFN-{gamma}-producing CD8+ cells were significantly higher in cultures from IC-immunized aged mice than those from other immunization groups (Fig. 2, A and B). Importantly, CD8+ T cells from IC-immunized aged mice also make more IFN-{gamma} per cell because the mean fluorescence intensity of IFN-{gamma} staining in CD8+ T cells from IC-immunized group was significantly higher than that of other groups (Fig. 2C).


Figure 2
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  		FIGURE 2. IC vaccination enhances IFN-{gamma} production by CD8+ T cells responding to influenza A immunization in aged mice. Immunization of aged mice and stimulation of splenic cells are as described in Fig. 1. Four days after in vitro stimulation with influenza virus-infected splenic cells, cultured cells were harvested and stained for intracellular IFN-{gamma}. A, Profiles of CD8 and IFN-{gamma} staining within the lymphocyte gate. Numbers shown in individual samples indicate percentage of CD8+IFN-{gamma}+ cells in the lymphocyte gate. B, Percentage (mean ± SE) of IFN-{gamma}+ cells in the CD8+ cell gate. C, Mean fluorescence intensity (mean ± SE) of IFN-{gamma} expression in individual samples. Results are representative of two independent experiments with six mice in each experimental group.

 
Immunization with anti-NP IC vaccine enhances Ab response to both NP and HA in aged mice

Another indicator of protection to influenza A infection is the Ab titers of hemagglutination inhibition (35, 36, 37). There is a correlation between low hemagglutination inhibition serum Abs and low efficacy of influenza vaccines in the elderly (40, 41, 42, 43, 44, 45). To test whether IC immunization can enhance Ab responses to both NP and HA in aged mice, we have determined the levels of serum Abs and numbers of AFCs to NP and HA 16 days after immunization. We found that IC vaccine has a significant enhancing effect on Ab levels specific for both NP and HA in aged mice (Fig. 3A). Consistently, the numbers of virus-specific AFCs in the spleen and bone marrow (BM) were significantly increased in aged mice receiving vaccine/anti-NP IC compared with those in mice receiving other forms of vaccines (Fig. 3B). Thus, the results demonstrate that IC vaccination can repair the age-related deficiency in both cellular and humoral immunity to influenza A virus.


Figure 3
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  		FIGURE 3. IC vaccination enhances Ab and AFC responses to influenza A in aged mice. Aged BALB/c mice were immunized with live virus, vaccine, vaccine plus isotype control Ab, or vaccine IC. Sixteen days after primary immunization, serum Ab levels and numbers of splenic or bone marrow AFCs were analyzed. A, Serum IgG Abs specific for HA ({square}) and NP ({blacksquare}) were determined by ELISA. An anti-NP mAb and a pool of HA positive sera were used as standard for NP- or HA-specific Abs, respectively. B, BM ({blacksquare}) or splenic ({square}) IgG1 AFCs secreting specific Abs against influenza A virus were determined by ELISPOT assay. Data (mean ± SE) are from an experiment with six mice in each group. Similar independent experiments have been repeated twice.

 
In vivo priming with IC enhances type 1 cytokine but not type 2 cytokine production by both CD4 and CD8 T cells in aged mice.

To determine the effect of IC immunization on cytokine production and Th1/Th2 responses, we have investigated the cytokine profiles of both CD4+ and CD8+ T cells after in vivo priming with IC or control influenza A vaccine. One week after immunization, draining lymph node cells were stimulated with the immunizing influenza vaccine and Ag-specific cytokine production was measured by intracellular cytokine staining. The results showed that immunization with IC enhanced IFN-{gamma} production in both CD4+ and CD8+ T cells in aged mice (Fig. 4A). The percentages of IFN-{gamma}-producing CD4+ and CD8+ T cells in the mice immunized with IC were increased by 2-fold and two-thirds, respectively, compared with the control mice immunized with vaccine and control Ab. However, there were no differences in the frequencies of cells producing IL-4 or IL-10 between IC-immunized mice and control animals (Fig. 4, B and C). These findings suggest that in vivo priming with IC predominantly promotes Th1 response and has less effect on Th2 response. It is interesting that there were significant subpopulations of CD8 T cells expressing IL-4 or IL-10. Experiments costaining different cytokines showed that the IL-4 or IL-10 producing cells did not produce IFN-{gamma} (data not shown). The exact role or functions of these T cells in vivo are not clear.


Figure 4
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  		FIGURE 4. IC immunization promotes Th1 cytokine production. Seven days after primary immunization, draining lymph node cells from mice primed with immune complex ({blacksquare}) or vaccine plus isotype control Ab ({square}) were stimulated with influenza A vaccine for 3 days. Cytokine profiles were determined by intracellular cytokine staining. Data shown were percentages of cells positive for individual cytokines (IFN-{gamma} in A, IL-4 in B, and IL-10 in C) in total lymphocyte gate. Similar results were obtained from three independent experiments with four mice in each experimental group.

 
IC promotes maturation and function of DC from aged mice

To further determine the mechanisms underlying the immune enhancing effects of IC vaccine in aged mice, we investigated the effects of ICs in promoting DC maturation and function. Splenic DCs were isolated and incubated with IC vaccine or vaccine/control Ab for 2 days. The expression levels of MHC class II and costimulatory molecule CD86 were evaluated by flow cytometry. Our results show that the frequencies of DCs with higher expression levels of MHC class II molecule or CD86 were significantly increased in DCs cultures with ICs compared with that in DC cultures with vaccine plus control Ab (Fig. 5A). In addition, the overall expression of MHC class II and CD86 was significantly up-regulated on DCs stimulated with IC vaccine (Fig. 5B). To test whether these immune complex-pulsed DCs are more efficient in presenting Ag and stimulating T cells, we primed T cells in vivo with DCs pulsed with immune complex or control preparation. We found that DCs pulsed with immune complex elicited significantly more robust T cell proliferative response than DCs cultured with control preparation (Fig. 5C). Thus, these findings demonstrate that enhanced DC maturation and function may contribute to the mechanisms that improve immune responses to influenza A by IC vaccination.


Figure 5
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  		FIGURE 5. IC enhances DC maturation and function. Purified DCs were cultured with immune complex vaccine or vaccine mixed with isotype control Ab for 48 h. A, Percentages of CD11c+ DCs with different levels of MHC class II or CD86 expression are shown. B, Mean fluorescence intensity of MHC class II or CD86 expression on DCs under different culture conditions are shown. {square}, cultured with medium only; Figure 5, stimulated with vaccine and control Ab mix; {blacksquare}, stimulated with IC. Data are representative of three independent experiments. C, T cell proliferation following DC immunization. Seven days after immunization with DCs cultured with vaccine and control Ab mix ({square}) or DCs cultured with IC ({blacksquare}), draining lymph node cells were stimulated in vitro with medium or 300 ng/ml vaccine. 3[H]-thymidine incorporation was measured for cell proliferation. Data are representative of two independent experiments with four mice in each group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our current study demonstrates that age-associated impairment in generating functional influenza-specific CTLs can be significantly alleviated by immunization with immune complex vaccine. This improved CTL function, together with enhanced virus-specific humoral immunity, results in an overall improved influenza-specific immune response in aged animals.

Complex changes in the immune response occur as species including mouse and man undergo postmaturational aging. The most profound changes in an aged immune systems are in the T cell compartment (46, 47, 48, 49, 50). The age-related decline in T cell function results in a shift in the phenotype of circulating CD4+ T cells, with a decrease in naive CD4+ T cells and relative accumulation of memory CD4+ T cells. In addition, the memory T cells include a spectrum of normal functioning and hypofunctioning T cells, compared with memory T cells in young controls. The decrease in functioning cells results in impaired proliferating capacity and impaired expression of IL-2/IL-2 receptor (46). Although less numerous, studies on CD8+ T cells have also found some age-related changes (51, 52). It has been demonstrated that influenza virus-specific class I-restricted CD8+ CTL activity was significantly diminished in elderly persons (20, 21, 22) as well as in aged mice (23, 24, 25). Existing evidence suggests that there are age-related changes in DCs. It has been shown that although the elderly are able to generate large numbers of DCs from PBMC and that these cells have a phenotype and Ag presentation capacity similar to those of DCs from young controls, DCs in the elderly may have an impaired capacity to cross tissue barriers and to trigger cytokine production from specific T cells (53). Therefore, the ability of DCs to differentiate after interaction with T cells may be impaired with aging, and this circumstance may be related to the observation that production of GM-CSF, a key DC growth factor, was diminished in the elderly (54). Thus, improvement of DC maturation and function may be paramount to overcoming age-related impairment in immunity (55, 56, 57).

Immune cells express four types of Fc{gamma}R: Fc{gamma}RI, IIB, III, and IV (58, 59, 60). Binding of Fc{gamma}R can lead to either activating or inhibitory signaling depending on which specific Fc{gamma}R is being engaged. Activating FcRs (Fc{gamma}RI, III, and IV) associate with the ITAM motif-containing {gamma}-chain and their engagement results in src and syk kinase-mediated activation. In contrast, the inhibitory FcR (Fc{gamma}RIIB) is a receptor containing a cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM) that inhibits ITAM-mediated signals through the recruitment of the inositol-phosphatase SHIP (61, 62). Balanced signaling through activating and inhibitory FcR intimately regulates the activity of various cells in the immune system (58, 59, 63, 64). Recent work has demonstrated that different subclasses of IgG have differential affinities for specific activating Fc{gamma}Rs compared with their affinities for the inhibitory Fc{gamma}R, leading to substantial differences in their ability to mediate effector functions (60, 65). An activating-to-inhibitory ratio can be used to describe this differential affinity for functional distinct FcRs by a specific IgG subclass (65, 66). It has been shown that the hierarchy of in vivo biological activity for the IgG subclasses is IgG2a ≥ IgG2b > IgG1 >> IgG3, mirroring the hierarchy based on the activating-to-inhibitory ratios (60, 65).

In the current study, we used an IgG2a mAb specific for the NP of influenza A virus to form ICs. Our data demonstrated that when aged mice were immunized with this IgG2a IC, the influenza-specific immunity was significantly improved compared with that in aged mice that received other forms of vaccines, including the same vaccine plus an isotype control Ab. In particular, IC vaccination in aged mice induced a significant virus-specific CTL response that was almost undetectable in aged mice immunized with other forms of vaccination, including live-virus infection. Both viral specific cytotoxicity and IFN-{gamma} production by CD8+ T cells were significantly enhanced in aged animals immunized with IC vaccine. In addition, Ab responses against both surface Ag (HA) and core Ag (NP) were improved by IC immunization. In vivo priming experiments showed that our IgG2a IC predominantly promotes type 1 cytokine production in both CD4+ and CD8+ T cells, suggesting that both Th1 and cytotoxic T cell functions are improved by IC vaccination. We have also investigated the possible mechanisms underlying the immune enhancing effect by IC vaccines. Our results have shown that, when stimulated with IC vaccine, DCs from aged mice expressed significantly higher levels of MHC class II and costimulatory molecules. In addition, when used to prime T cells in vivo, IC-pulsed DCs elicited a significantly enhanced T cell proliferation than DCs cultured with control vaccine preparation. Thus, our data indicate that ICs can enhance the maturation and function of aged DCs.

In summary, our findings indicate that impaired antiviral responses in aging can be significantly improved by immunization with IC vaccines, which may have important implications in designing vaccine compositions and immunization protocols for the elderly population and certain T cell deficient patients, such as AIDS.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Shuhua Han, Department of Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail address: shan{at}bcm.edu Back

2 Abbreviations used in this paper: HA, hemagglutinin; NA, neuraminidase; NP, nucleoprotein; FcR, Fc receptor; DC, dendritic cell; FDC, follicular DC; IC, immune complex; i.n., intranasally; AFC, Ab-forming cell; BM, bone marrow. Back

Received for publication March 22, 2007. Accepted for publication August 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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