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Antibody-Independent Antiviral Function of Memory CD4⁺ T Cells In Vivo Requires Regulatory Signals from CD8⁺ Effector T Cells¹

Trudeau Institute, Saranac Lake, NY 12983

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies have shown that vaccine-primed CD4⁺ T cells can mediate accelerated clearance of respiratory virus infection. However, the relative contributions of Ab and CD8⁺ T cells, and the mechanism of viral clearance, are poorly understood. Here we show that control of a Sendai virus infection by primed CD4⁺ T cells is mediated through the production of IFN- and does not depend on Ab. This effect is critically dependent on CD8⁺ cells for the expansion of CD4⁺ T cells in the lymph nodes and the recruitment of memory CD4⁺ T cells to the lungs. Passive transfer of a CD8⁺ T cell supernatant into CD8⁺ T cell-depleted, hemagglutinin-neuraminidase (HN)_421–436-immune µMT mice substantially restored the virus-specific memory CD4⁺ response and enhanced viral control in the lung. Together, the data demonstrate for the first time that in vivo primed CD4⁺ T cells have the capacity to control a respiratory virus infection in the lung by an Ab-independent mechanism, provided that CD8⁺ T cell "help" in the form of soluble factor(s) is available during the virus infection. These studies highlight the importance of synergistic interactions between CD4⁺ and CD8⁺ T cell subsets in the generation of optimal antiviral immunity.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Respiratory virus infections, such as those mediated by influenza and parainfluenza viruses, can be terminated by cellular immune responses (1, 2). For example, virus-specific CD8⁺ CTL can mediate enhanced clearance of influenza virus or Sendai virus infections via perforin/granzyme- and Fas-mediated destruction of infected lung epithelial cells (3, 4). In addition, cytokines released by virus-specific CD8⁺ CTL effectors, such as IFN- and TNF-, are able to induce cellular proteins that can decrease transcription and/or replication of several viruses, including influenza and parainfluenza virus, thus contributing to the termination of viral infection (5, 6, 7). Recently, several studies have shown that CD8⁺ T cells release both CC () chemokines, such as RANTES, macrophage-inflammatory protein (MIP)³- and MIP-, and CXC () chemokines, such as IL-8 and inflammatory protein-10, following TCR triggering by MHC class I/peptide complexes (8, 9, 10). This suggests that CD8⁺ CTLs are not only the end-product effector cells generated during antiviral immune response, but that they may also be involved in regulation of immune cell interactions as well.

Respiratory virus infection can also be controlled by CD4⁺ T cells and B cells in the absence of CD8⁺ T cells. For example, it has been shown that viral clearance is normal or only slightly delayed after a primary challenge of CD8⁺ T cell-depleted C57BL/6 mice with either influenza virus or Sendai virus (11, 12). Although the underlying mechanisms are not fully understood, it is generally believed that virus-specific CD4⁺ cells operate mainly by providing T cell help for B cells to generate neutralizing Abs and/or secreting antiviral cytokines (IFN-). When both CD8⁺ and B cells are absent, it has been shown that primary virus-specific CD4⁺ T cell responses are unable to terminate an infection caused by either influenza virus or Sendai virus (13, 14). Even influenza virus-specific memory CD4⁺ T cells confer only a very limited control of a secondary viral challenge in CD8⁺ T cell-depleted mice (14). Therefore, it is generally believed that CD4⁺ T cell-mediated mechanisms on their own, or in conjunction with innate immunity, are ineffective at controlling respiratory virus infections. However, this conclusion is based largely on experiments in which the CD8⁺ T cell subset was depleted in vivo by Ab and assumed that the Ab treatment did not impact on the CD4⁺ T cell response.

As an alternative approach to understanding the role of CD4⁺ T cells in antiviral immunity, we recently examined the impact of selective CD4⁺ T cell vaccination on Sendai virus infection (15). Thus mice were vaccinated with an immunodominant class II-restricted hemagglutinin-neuraminidase (HN) peptide to specifically prime virus-specific CD4⁺ T cells. This strategy allowed us to assess the role of memory CD4⁺ T cells in the presence of an intact naive CD8⁺ T cell population. Subsequent infection with Sendai virus resulted in accelerated recruitment of CD4⁺ T cells to the lung and accelerated clearance of Sendai virus in immune competent mice. Interestingly, the primary CD8⁺ T cell response in this system was suppressed, possibly due to the accelerated clearance of Ag from the system. In addition, the virus-specific Ab response was only slightly enhanced under these conditions. These data suggest that primed CD4⁺ T cells may use Ab-independent mechanisms for controlling respiratory virus infections. However, we could not formally exclude a role for CD8⁺ T cells and/or Ab. Therefore, in the present study, we evaluated the capacity of primed CD4⁺ T cells to control a Sendai virus infection in B cell-deficient µMT mice that were additionally depleted of CD8⁺ T cells by treatment with anti-CD8 Abs. Our results demonstrate for the first time that virus-specific memory CD4⁺ T cells make a direct contribution to the clearance of a respiratory virus in the absence of a concurrent CD8⁺ T cell and Ab response, provided that soluble factor(s) produced by CD8⁺ T cells are available during the virus infection. These soluble factors are essential for rapid clonal expansion of the virus-specific memory CD4⁺ T cells in draining mediastinal lymph nodes (MLN) and their subsequent recruitment into the inflamed lung. Furthermore, we show that the antiviral effect of primed CD4⁺ T cells is mediated, at least in part, by IFN-.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Male or female µMT (H-2^b) or C57BL/6 (H-2^b) mice were purchased from either The Jackson Laboratory (Bar Harbor, ME) or the Trudeau Institute (Saranac Lake, NY) and housed under specific pathogen-free conditions before infection with Sendai virus at 6–10 wk of age.

Peptide vaccination

Sendai virus peptides derived from HN and nucleoprotein (NP) (HN_421–436, HN_559–574, HN_163–178, NP_324–332) have been described elsewhere (16, 17) and were purchased from New England Peptide (Fitchburg, MA). Mice were vaccinated with HN_421–436 peptide as described previously (15) and successful priming was confirmed with a CD4⁺ T cell proliferation assay, as has been described in detail previously (15). Strong HN_421–436-specific CD4⁺ T cell-proliferative responses were routinely obtained after priming, demonstrating that B cell-deficient µMT mice can be successfully primed with the synthetic HN_41–436 peptide (data not shown).

Virus infections and determination of viral load in lung homogenates

The Enders strain of Sendai virus was grown, titrated, and stored as described previously (12). Mice that had been vaccinated with HN_421–436 peptide 20–30 days before were infected intranasally under anesthesia with 500 egg infectious doses (EID₅₀) of Sendai virus. All infected mice were held under Biosafety Level 3 conditions. Virus titers of lung tissues were determined from lung homogenates by endpoint titration in embryonated hen eggs as described previously (12).

In vivo depletion of CD8⁺ T cells with anti-CD8 mAb

Nonthymectomized mice were injected i.p. every third day with 320 µg of purified rat anti-mouse CD8 mAb (clone TIB210) or the same amount of rat IgG2b isotype control (clone LTF-2) in PBS. The depletion started 3 days before viral infection and continued until the tissues were sampled. In some experiments, the depletion started 5 days after viral infection to observe the effect of presence of CD8⁺ T cells at the early stage of infection on CD4⁺ T cell response. The efficiency and specificity of CD8⁺ T cell depletion was monitored by flow cytometry using noncross-reactive mAbs to CD8 as well as mAbs to CD4⁺ T cells and B cells. CD8⁺ T cells were undetectable among lymphocyte-gated populations from lung tissue, MLN, and spleen during the entire 10- to 14-day period of observation, indicating that a sustained deletion of CD8⁺ T cells was achieved by this protocol. CD4⁺ and B220⁺ lymphocyte subsets were not affected by the anti-CD8 mAb depletion.

Analysis of dendritic cell (DC) subpopulations after in vivo treatment of mice with anti-CD8 mAb

Lymph nodes and spleens were pooled from two naive C57BL/6 mice that had been treated with anti-CD8 mAb as described above. Untreated animals from the same batch of mice were used as controls. The organs were cut into small fragments, digested with collagenase D (1 mg/ml; BoehringerMannheim, Mannheim, Germany) for 1 h at 37°C, and then prepared as single cell suspensions. Nonspecific staining was blocked using purified anti-mouse CD16/CD32 (FcRIII/II receptor; PharMingen, San Diego, CA). The cells were then stained with PE-conjugated anti-CD8, FITC-conjugated anti-CD11c, Tricolor-conjugated anti-CD8, and PE-conjugated I-A^b mAbs (PharMingen). Stained samples were acquired on a BD Biosciences FACScan flow cytometer, and the data were analyzed using CellQuest software (BD Immunocytometry Systems, San Jose, CA). To calculate the absolute percentages of CD8⁺ T cells as well as CD8⁺CD11c⁺ and CD8^-CD11c⁺ DC subpopulations in the lymph nodes and spleens, CD8⁺, CD8⁺CD11c⁺, and CD8^-CD11c⁺ events were divided by the total number of events in a live cell gate. The absolute cell numbers of the three individual cell populations per organ were then calculated by multiplying the total cell count per organ with their corresponding percentages.

Passive transfer of CD8⁺ T cell culture supernatant (CD8⁺ sup) into recipient mice

CD8⁺ sup was generated from activated Sendai virus NP_324–332-specific CD8⁺ T cells. Briefly, CD8⁺ T cells were enriched by negative selection with Dynabeads (Dynal, Oslo, Norway) from pooled spleens of C57BL/6 mice that had recovered from a Sendai virus infection (3 months postinfection). The enriched cell populations routinely comprised 80–87% of CD8⁺ T cells, and <0.2% of CD4⁺ T cells, as determined by flow cytometry. APCs were prepared from the spleens of syngeneic naive mice by depleting the T cell population with anti-Thy1.2 mAb plus a mixture of rabbit and guinea pig complement (Cedarlane Laboratories, Hornby, Ontario, Canada). Flow cytometry analysis confirmed that the resulting cell population contained <0.1% of CD3⁺ cells. Enriched CD8⁺ T cells (5 x 10⁵) were then incubated with T cell-depleted, irradiated (3000 rad) APCs (5 x 10⁵) in the presence of Sendai virus NP_324–332 peptide (1 µg/ml) and 10 U/ml of IL-2 in 200 µl of complete tumor medium at 37°C with 10% CO₂. The culture supernatants were collected after a 5-day incubation, filtered through a 0.45-µM filter, and then frozen at -80°C before use. Strong NP_324–336/K^b-specific cytolytic activity against NP_324–336 peptide-pulsed L-K^b fibroblasts (45–75% of specific ⁵¹Cr release) was detected in the cultures before harvesting the supernatant. Parallel cultures in which APCs and NP_324–332 peptide (1 µg/ml) were cultured in the absence of enriched CD8⁺ T cells were used to generate a control supernatant. Naive or HN_421–436-vaccinated and CD8⁺ T cell-depleted µMT mice were injected i.p. every other day with 300 µl of CD8⁺ sup, starting 1 day before infection with Sendai virus. Control mice received the same amount of control supernatant.

Intracellular IFN- staining following peptide stimulation

Recruitment of Sendai virus-specific CD4⁺ T cells into the bronchoalveolar lavage (BAL) of infected mice was monitored by intracellular IFN- staining as described previously (15). The percentage of IFN--secreting CD4⁺ T cells among total live cells in the BAL was calculated by dividing the number of CD4⁺/IFN-⁺ events by the total number of events in a live cell gate. The results are expressed as the absolute cell number of Sendai virus HN peptide-specific IFN--secreting CD4⁺ T cells per lung. This was calculated by multiplying the total cell count of each lung lavage with the corresponding percentage of IFN--secreting CD4⁺ T cells among the total live cells in the BAL after subtraction of the background obtained with the no peptide control culture (this background was routinely <0.1%).

ELISPOT analysis

The Sendai virus-specific CD4⁺ T cell response was measured by IFN--ELISPOT assay. Ninety-six-well Multiscreen HA plates (Millipore, Bedford, MA) were coated with 10 µg/ml of anti-IFN- capture Ab (clone R46A2; PharMingen). CD4⁺ effector T cells were enriched from the pooled MLN of infected µMT mice (3–5 mice/group) with Dynabeads as described previously (15). CD4⁺ T cells usually accounted for 96–98% of enriched cell populations, as determined by flow cytometry. Serially diluted numbers of enriched CD4⁺ T cells were incubated with irradiated splenocytes from naive C57BL/6 mice (1 x 10⁶ per well) in the presence of Sendai virus HN_421–436 or HN_163–178 peptide (10 µg/ml). Each dilution of effector cells was assayed in duplicate. After a 24-h incubation, the cells were washed with PBS containing 0.05% Tween 20. Biotinylated rat anti-mouse IFN- mAb (10 µg/ml, clone XMG1.2; PharMingen) was added and the plates were incubated overnight at 4°C. The plates were then washed with PBS/Tween and developed with streptavidin-HRP using 3-amino-9-ethyl-carbazole (Sigma, St. Louis, MO) as substrate. The spots in each well were counted using SZH Zoom Stereo Microscope System (Olympus, New Hyde Park, NY). The results were expressed as the number of spot-forming cells (SFC) in each 10⁶ CD4⁺ T cells.

Neutralization of IFN- with anti-IFN- mAb

µMT mice were injected i.p. with either 300 µg of anti-IFN- mAb (clone XMG1.2; Monoclonal Antibody Core Facility, Trudeau Institute) or the same amount of isotype control mAb (clone HRPN) at days -1, 1, 3, 6, and 9 relative to the virus infection. Analysis of cell-free BAL fluid from Sendai virus-infected mice by an IFN- ELISA showed that injection with the anti-IFN- mAb during the course of infection resulted in a complete neutralization of IFN- (data not shown). In contrast, injection with the isotype control Ab did not affect the level of this cytokine in the BAL fluid during infection with Sendai virus. In addition, flow cytometry analysis of the BAL cells confirmed that neutralization of IFN- with the anti-IFN- mAb did not have obvious influence on the recruitment of either CD4⁺ or CD8⁺ T cell subsets into the lung during the virus infection (data not shown).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effective control of Sendai virus infection by HN_421–436 peptide-vaccinated CD4⁺ T cells does not require B cells

We have previously shown that mice vaccinated with an immunodominant Sendai virus HN_421–436 peptide had a significantly enhanced capacity to clear Sendai virus from the lung following a primary challenge (15). The immune response in the lungs of these mice was characterized by an accelerated recruitment of CD4⁺ T cells to the lung and a significantly reduced virus-specific CD8⁺ T cell response. In addition, it was found that CD4⁺ T cell priming resulted in only a slightly enhanced Ab response in the draining lymph nodes. Although these data suggest that primed CD4⁺ T cells alone have the potential to control a respiratory virus infection, we could not completely rule out a role for CD8⁺ T cells and Ab in these experiments. As a first step to determine the capacity of CD4⁺ T cells to control a virus infection in the absence of Abs, we analyzed immune responses in HN_421–436 peptide-vaccinated Ab-deficient µMT mice. As shown in Fig. 1, A and B, HN_421–436 peptide-vaccinated µMT mice were able to control a sublethal Sendai virus infection in the lung much more effectively than control (CFA-vaccinated) µMT mice. Thus, viral loads were significantly reduced in the lungs of vaccinated mice compared with control mice on day 7 of the infection. In addition, virus was completely cleared by day 10 postinfection from the vaccinated mice, whereas there were still high viral loads in the control mice at this time (Fig. 1, A and B). Unprimed control µMT mice ultimately cleared virus from the lung by day 14 (Fig. 1A). These data formally demonstrate that the CD4⁺ T cell-mediated clearance of a Sendai virus infection does not depend on B cells.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Effective control of Sendai virus infection by HN421–436 peptide-vaccinated CD4+ T cells does not require B cells. Control CFA-vaccinated (A and C) or HN421–436-vaccinated (B and D) µMT mice were injected with either anti-CD8 mAb (C and D) or an isotype control mAb (A and B) 3 days before intranasal infection with 500 EID50 of Sendai virus. Lungs were taken at different times postinfection, and virus titers were determined by titrating lung homogenates in embryonated chicken eggs, followed by a hemagglutination assay. The results are expressed as log10EID50. The day 14 data in A and B are from a separate time course and are included to show that control CFA-vaccinated µMT mice are able to clear a Sendai virus infection from the lung.

The virus-specific memory CD4⁺ T cell response is severely impaired in the lung of µMT mice depleted of CD8⁺ T cell subset

The observation that primed CD4⁺ T cells were ineffective in the CD8-depleted mice could be explained by a failure of CD4⁺ T cells in the lung to clear virus, or a failure to recruit activated CD4⁺ T cells to the lung. To distinguish these possibilities, we monitored the virus-specific CD4⁺ T cell accumulation in the airways of HN_421–436 peptide-vaccinated µMT mice during infection with Sendai virus using an intracellular IFN- assay. As shown in Fig. 2, the accumulation of CD4⁺ T cells in the lungs of HN_421–436 peptide-vaccinated µMT mice was much greater than that of control unvaccinated µMT mice on day 7 postinfection (6.9 x 10⁴ vs 0.2 x 10⁴ HN_421–436-specific CD4⁺ T cells per lung). This difference is consistent with previous studies in immunocompetent mice (15). In contrast, the enhanced CD4⁺ T cell accumulation in the lungs of HN_421–436 peptide-primed µMT mice on day 7 postinfection was significantly impaired if the mice were also treated with an anti-CD8 Ab (6.9 x 10⁴ HN_421–436 vs 0.6 x 10⁴ specific CD4⁺ T cells per lung). These data demonstrate that CD8⁺ T cell depletion prevents the accumulation of CD4⁺ T cells to the lung rather than blocking effector functions in the lung. However, this effect was not permanent inasmuch as both naive and HN_421–436-immune µMT mice mounted significant and comparable CD4⁺ T cell responses in their lung on day 10 postinfection, irrespective of CD8⁺ T cell depletion (Fig. 2, middle panels). This result indicates that CD8⁺ T cell depletion appears to preferentially affect the rapid recall of memory CD4⁺ T cell cells rather than the primary CD4⁺ T cell response. It is interesting that more CD4⁺ T cells accumulate in the lungs of CD8-depleted vs nondepleted control mice. This difference probably reflects differences in viral load at this time point (Fig. 1). There was no obvious difference in terms of CD4⁺ T cell response to the subdominant HN_559–574 and to the nonreactive HN_163–178 peptide between naive and HN_421–436-immune µMT mice in these experiments.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Impaired virus-specific memory CD4+ T cell response in the lung of HN421–436 peptide-vaccinated, but CD8+ T cell-depleted mice after Sendai virus infection. µMT mice (upper and middle panels) or C57BL/6 mice (lower panels) were vaccinated with either the HN421–436 peptide or CFA adjuvant alone as a control. The animals were then depleted of CD8+ T cells with anti-CD8 mAb 3 days before they were infected intranasally with Sendai virus. Cells from the BAL were pooled from four to five mice of each group at the indicated time points postinfection, and the frequencies of HN421–436/Ab-specific CD4+ T cells were determined by intracellular IFN- staining. The results are expressed as absolute numbers of the HN421–436/Ab-specific CD4+ T cells per lung and are representative of three independent experiments.

Expansion of virus-specific memory CD4⁺ T cells is severely impaired in the draining MLN of µMT mice depleted of CD8⁺ T cell subset

Because influenza or Sendai virus-specific T cell responses are initiated in the draining lymph nodes, we asked whether the dramatically reduced numbers of virus-specific memory CD4⁺ cells in the lungs of CD8-depleted mice reflected an impaired expansion of memory CD4⁺ T cells in the draining lymph nodes. Thus, we determined the clone burst size of virus-specific CD4⁺ T cells in the draining MLN using a single cell ELISPOT assay. As shown in Fig. 3, a high frequency of HN_421–436-specific CD4⁺ T cells could be detected in the MLN of HN_421–436 peptide-vaccinated µMT mice on day 7 postinfection with Sendai virus (5200 SFC per 10⁶ CD4⁺ T cells). However, this expansion of HN_421–436-specific CD4⁺ T cells was dramatically reduced in the MLN of vaccinated mice that had been depleted of CD8⁺ T cells (500 SFC per 10⁶ CD4⁺ T cells). When examined on day 10 after infection, the frequency of HN_421–436-specific CD4⁺ T cells was higher in the MLN of CD8⁺ T cell-depleted HN_421–436 peptide-vaccinated µMT mice than in vaccinated control mice. This suggests that despite the loss of the recall CD4⁺ T cell response due to CD8⁺ T cell depletion, a primary CD4⁺ T cell response occurs normally in these mice (peaking at day 10), as has been shown for CD4⁺ T cell accumulation in the lung (Fig. 2 and Ref. 15). Together, the data show that the decreased numbers of virus-specific memory CD4⁺ T cells in the airways at the early stage of viral infection correlated with an impaired expansion of these CD4⁺ T cells in the draining MLN of CD8⁺ T cell-depleted, vaccinated µMT mice.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Impaired expansion of the virus-specific memory CD4+ T cells in the draining MLN of HN421–436 peptide-vaccinated, but CD8+ T cell-depleted µMT mice after Sendai virus infection. Groups of 4–6 mice were vaccinated with either CFA or HN421–436 peptide. Mice were then treated with either an anti-CD8 or control Ab and infected with Sendai virus. At the indicated times, MLN were isolated, and the frequencies of the HN421–436/Ab-specific CD4+ T cells were determined by a single-cell IFN- ELISPOT assay. The results are expressed as the absolute numbers of HN421–436/Ab-specific CD4+ T cells per MLN and are representative of three independent experiments.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Presence of activated CD8+ effector T cells are required for an effective memory CD4+ T cell response after infection with Sendai virus. HN421–436-vaccinated µMT mice (4–5 mice per group) were treated with either an anti-CD8 or control mAb 5 days after infection with Sendai virus. The frequencies of the HN421–436/Ab-specific CD4+ T cells in the draining MLN and the BAL were then determined on day 7 postinfection by a single-cell IFN- ELISPOT assay. The data represent the absolute numbers of the HN-specific CD4+ T cells per mouse and are representative of three independent experiments.

The finding that the presence of CD8⁺ cells during the first 5 days after infection failed to rescue a virus-specific memory CD4⁺ T cell response suggests that CD8⁺ cells may provide some form of "help" for memory CD4⁺ T cells after they are activated. This help could be mediated by direct contact between CD4⁺ and CD8⁺ T cells, by releasing soluble factors, or both. To test this, we generated a supernatant from memory CD8⁺ T cells that had been stimulated in vitro with Sendai virus NP_324–332 peptide. We then asked whether this supernatant would restore the memory CD4⁺ T cell response in HN_421–436-vaccinated mice that had been CD8⁺ T cell-depleted. As shown in Fig. 5A, repeated injection of the CD8⁺ sup into anti-CD8-treated, HN_421–436-immune µMT mice during infection with Sendai virus resulted in nearly a complete restoration of the virus-specific memory CD4⁺ T cell response in the draining MLN of the recipients. The numbers of IFN--secreting CD4⁺ T cells in the MLN of CD8⁺ T cell-depleted µMT mice that received the CD8+ sup was 90% of that from control µMT mice in which CD8⁺ T cells were present. In addition, the numbers of CD4⁺ T cells accumulating in the lung were substantially restored (Fig. 5, B and C). In contrast, a control supernatant generated from the same APCs and NP_324–332 peptide, but which had been CD8⁺ T cell-depleted, did not restore the CD4⁺ T cell response in the MLN or CD8⁺ T cell accumulation in the lung. These data demonstrate for the first time that in vivo-primed memory CD4⁺ T cells are able to control a Sendai virus infection in the lung, but that they require help from CD8⁺ T cells in the form of soluble factors.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Restored virus-specific memory CD4+ T cell response in both MLN and BAL after passive transfer of CD8+ T cell supernatant into HN421–436 peptide-vaccinated, but CD8+ T cell-depleted µMT mice. Groups of HN421–436-vaccinated µMT mice were infected with Sendai virus. Indicated groups were depleted of CD8+ T cells by Ab depletion beginning day -3 and/or CD8+ T cell supernatant was prepared from Sendai virus NP324–336-specific CD8+ CTL effectors on day -1. The animals were repeatedly injected with both anti-CD8 mAb and CD8+ T cell supernatant during infection. MLN and BAL were sampled from groups of 4–5 mice on day 7 postinfection for analysis of the HN-specific CD4+ T cells, as described in Materials and Methods.

We next asked whether the supernatant-restored secondary CD4⁺ T cell response in CD8⁺ T cell-depleted µMT mice was able to clear a Sendai virus infection. Thus, HN_421–436-vaccinated, CD8⁺ T cell-depleted, µMT mice were treated with the CD8 supernatant and then infected with Sendai virus. Ten days postinfection, the viral load in lung homogenates was determined and compared with control naive µMT mice that were CD8⁺ T cell-depleted, but did not receive the CD8⁺ sup during the viral infection. As shown in Fig. 6, the CD8⁺ sup substantially enhanced viral clearance. In contrast, the virus titers in unvaccinated µMT mice that were depleted of CD8⁺ T cell population but received CD8⁺ sup was comparable to that of unvaccinated mice, indicating that the CD8⁺ sup alone did not have any direct antiviral effects. Again, µMT mice previously vaccinated with HN_421–436 peptide, but not CD8⁺ T cell-depleted, cleared the virus effectively on day 10 after infection in the presence of CD8⁺ T cells. Together, the data demonstrate that primed CD4⁺ T cells can make significant contributions to the clearance of Sendai virus in the absence of both the CD8⁺ and B cell subsets, provided that regulatory signals required for rapid clonal expansion of memory CD4⁺ T cells are provided by virus-specific CD8⁺ CTLs during viral infection.

View larger version (12K): [in this window] [in a new window]   FIGURE 6. Significantly reduced viral load in the lung of CD8+ T cell-depleted, HN421–436-immne µMT mice following passive transfer of CD8+ T cell supernatant. Naive or HN421–436-immune µMT mice were treated as described in Fig. 5. The lung virus titers were determined on day 10 after infection as described in Fig. 1.

Accelerated clearance of Sendai virus in vaccinated mice depends on IFN-

The mechanism by which primed CD4⁺ T cells clear Sendai virus is not known. However, we have shown that primed CD4⁺ T cells are recruited rapidly to the lung and secrete substantial amounts of IFN- (Fig. 2 and data not shown), which is reported to have strong antiviral effects (5, 6, 7). Thus, we asked whether IFN- contributed to viral clearance in this system by neutralizing IFN- during the course of the virus infection in HN_421–436 peptide-vaccinated µMT mice. As shown in Fig. 7, control vaccinated µMT mice completely cleared the virus from the lung tissues by day 10 postinfection. In contrast, vaccinated µMT mice that additionally received neutralizing IFN- Ab during the infection had variable titers of Sendai virus in the lung at day 10 postinfection (an average of 4.2 EID₅₀/lung). As shown previously (Fig. 1), all unvaccinated µMT mice failed to clear the virus by day 10 after infection. These data demonstrate that IFN- plays an important role in the control of Sendai virus infection in HN_421–436 peptide-vaccinated mice.

View larger version (11K): [in this window] [in a new window]   FIGURE 7. Clearance of Sendai virus in vaccinated mice is mediated by IFN-. CFA or HN421–436-vaccinated µMT mice were injected i.p. with either anti-IFN- mAb or the isotype control mAb starting at day 3 before infection with Sendai virus. The animals were repeatedly receiving the mAbs during infection with Sendai virus. The lung virus titers were determined on day 10 after infection as described in Fig. 1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our observation that primed CD4⁺ T cells can mediate effective antiviral immunity appear to directly contradict the studies of other groups that have reported that CD4⁺ T cells are ineffective at controlling influenza or Sendai virus infections in mice (13, 14). A key difference between the two approaches lies in the use of anti-CD8 Abs to deplete CD8⁺ T cells in vivo. Thus, our studies used a peptide priming approach that allowed us to analyze a memory CD4⁺ T cell response in immunologically intact mice with a full complement of naive CD8⁺ T cells. In contrast, the other studies have relied on Ab depletion of CD8⁺ T cells in live virus-primed animals. However, the data presented in the current study indicate that CD8⁺ T cell depletion impairs the recall of memory CD4⁺ T cells. Thus, it is likely that studies involving CD8⁺ T cell depletion inadvertently miss the contribution of CD4⁺ T cells to the cellular immune response to respiratory virus infections. The data presented in the current report confirm that CD4⁺ T cells can mediate strong antiviral activity and resolve the discrepancy in the literature.

It currently remains unclear what role CD8⁺ T cells play in promoting the CD4⁺ T cell response. One possibility is that there is an effect of CD8⁺ T cell depletion on a non-T cell population of cells. In this regard, it has been shown that DC can be divided into CD8⁺ and CD8^- subpopulations (21, 22). Recent data suggests that Ag-pulsed CD8⁺ DCs may direct Th1 polarization of CD4⁺ T cells after injection into mice (23, 24). Therefore, it is possible that the impaired CD4⁺ T cell response observed under our experimental condition might be due to the codeletion of the CD8⁺ DC subpopulation. However, this explanation is unlikely. First, although we found that the anti-CD8 mAb can bind to the CD8⁺ DC population, this did not lead to physical depletion of this DC population in vivo. Thus, the absolute numbers of DC remained constant following CD8 depletion, although we could not detect CD8 expression on these cells (data not shown). Second, in vitro proliferation of HN_421–436-specific CD4⁺ T cells from anti-CD8 mAb-treated µMT mice was normal following restimulation with the corresponding peptide Ag, indicating that in vivo depletion of CD8⁺ DC (either physically or functionally) does not appear to affect the proliferative potential of the virus-specific CD4⁺ T cell population (data not shown). Third, we found that only the secondary HN_421–436-specific Th1 CD4⁺ T cell response was impaired after treatment of the µMT mice with the anti-CD8 mAb. Th1 polarization of naive CD4⁺ T cells was normal following challenge with Sendai virus, independent of treatment with the anti-CD8 mAb in vivo (data not shown). Fourth, and most importantly, we found that a supernatant prepared from Sendai virus-specific CD8⁺ T cells was able to restore the recall CD4⁺ T cell response of HN_421–436-immune µMT mice treated with anti-CD8 mAb. This treatment was effective when given as late as 5 days into the infection and argues strongly against the idea that CD8⁺ T cells are involved in the induction phase of the response. Therefore, the impaired memory CD4⁺ T cell response following in vivo treatment of the HN_421–436-immune µMT mice with the anti-CD8 mAb is most probably due to depletion of CD8⁺ T cell subset, rather than the CD8⁺ DC subpopulation.

Although the recall of a memory CD4⁺ T cell response appears to be dependent on CD8⁺ T cells, it is interesting to note that the CD8⁺ T cell response in the lung is actually suppressed in vaccinated mice that have been infected with Sendai virus (15). This apparently contradictory effect is actually consistent with the anatomical compartmentalization of the immune response. Thus, the induction of CD4⁺ T cell response occurs in the local draining lymph node and is apparently dependent on CD8⁺ T cells at that site (Fig. 4). In contrast, the accelerated accumulation of CD4⁺ T cells in the lung results in rapid clearance of the virus and, consequently, a reduced or aborted recruitment of effector CD8⁺ T cells in the lung airways (Fig. 3). In this regard, it should be noted that the primary CD8⁺ T cell response develops with slower kinetics relative to the memory CD4⁺ T cell response, in this system.

The finding that only the secondary, and not the primary, HN_421–436-specific CD4⁺ T cell response was impaired in the anti-CD8 mAb-treated mice following viral challenge suggests that depletion of CD8⁺ T cells preferentially impairs virus-specific memory Th1 CD4⁺ effector T cells. It has been well established that memory T cells acquire distinct profiles of cell surface markers and possess the potential to migrate to an inflammatory site with accelerated kinetics (25, 26). Recent studies suggest that these properties of T cells, at least for CD4⁺ T cells, are related to the differential expression of chemokine receptors. For example, it has been shown that CCR5, CCR7, and CXC chemokine receptor 3 are expressed preferentially on Th1-polarized CD4⁺ T cells, and not on naive T cells (27, 28, 29). These chemokine receptors recognize type I chemokines, including RANTES, MIP-1 and MIP-1, and inflammatory protein-10 (30), suggesting that distinct chemokine signals are required for the recruitment of Th1 cells to the sites of inflammation. This idea is further supported by a recent study showing that Th1-polarized, but not Th2-polarized vesicular stomatitis virus glycoprotein G-specific transgenic CD4⁺ T cells migrated to peripheral target organs in vivo in response to chemokines produced at local inflammatory sites (31). In addition, recent data suggest that CC chemokines not only play an important role in regulating T cell trafficking to inflammatory sites, but also in participating in T cell activation via costimulating these T lymphocytes (32, 33). A biologically potent source of CC chemokines in vivo has now been shown to be the CD8⁺ T cell population (34, 35). Based on these recent findings, we hypothesize that removal of the CD8⁺ T cell population results in a diminished level of CC chemokines in vivo following infection with Sendai virus. Therefore, the expansion of the HN_421–436-specific memory CD4⁺ T cells in draining lymph nodes and/or the recruitment into the inflammatory lung are significantly impaired. Additional experiments are currently under way in our laboratory to characterize the chemokine receptor profiles of the primed HN_421–436-specific CD4⁺ T cells and to determine chemokine patterns and source in the pneumonic lung of the CD8⁺ T cell-deleted and virus-infected animals. It is hoped that these studies will provide new insight into the mechanisms of how CD8⁺ T cells regulate the CD4⁺ T cell subset during a respiratory virus infection.

The mechanism by which CD4⁺ T cells mediate viral control in this system is poorly understood. Some studies have indicated that cytolytic CD4⁺ T cells can be generated under certain conditions (12). However, we have not been able to detect cytolytic CD4⁺ T cells in this vaccination system following virus infection (data not shown). In some viral infections, such as those induced by HSV, lymphocytic choriomeningitis virus, and mouse hepatitis virus-68, the generation and maintenance of effective antiviral CD8⁺ T cell immunity is dependent on CD4⁺ T cell help (36, 37, 38). However, the accelerated clearance of Sendai virus infection in this report could not be attributed to an enhancement of the primary CD8⁺ T cell response by primed CD4⁺ T cells, as both absolute CD8⁺ T cell numbers and cytotoxic activity were significantly reduced in HN_421–436-vaccinated mice. Another possibility is that CD4⁺ T cells mediate their effects through the production of antiviral cytokines. Our previous studies have shown that primed CD4⁺ T cells in this system are almost exclusively of the Th1 phenotype and produce substantial amounts of IFN- (15). In the present study, we found that in vivo neutralization of IFN- diminished the capacity of peptide-vaccinated mice to clear Sendai virus from the lung. However, the mechanism by which IFN- mediates this effect is unclear because this cytokine has multiple activities in vivo, including effects on the recruitment of other cells to the lung. In addition, it is unclear whether effector CD4⁺ T cells in the lung are the key producers of IFN-, or whether they simply enhance IFN- production by other cell lineages. However, CD4⁺ T cells have been shown to clear experimental vesicular stomatitis virus infection by secreting antiviral cytokines such as IFN- and TNF- in an Ag-dependent manner (39, 40).

In summary, the present study documents for the first time that a secondary Sendai virus-specific CD4⁺ T cell response can make significant contributions to the viral clearance in the absence of functional B cells. However, soluble factors released by the CD8⁺ T cell subset are essential for the rapid expansion and subsequent recruitment into the inflammatory lung tissues of these CD4⁺ T cells. This represents the first demonstration that CD8⁺ T cells can regulate the functional properties of CD4⁺ T cell subset during a virus infection. Furthermore, the data again emphasize that caution must be used when conclusions on the role of individual lymphocyte populations in antiviral immunity are drawn based on the data generated from either knockout mice or mice depleted of T cell subsets with mAbs. Therefore, effective antiviral immunity appears to depend on synergistic interaction between individual arms of the immune system (41).

	Acknowledgments

 
We thank Simon Monard for assistance with the flow cytometry and Dr. Marcy Blackman for critical review of the manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant R01 AI-37597 (to D.L.W.) and funds from the Trudeau Institute.

² Address correspondence and reprint requests to Dr. David L. Woodland, Trudeau Institute, P.O. Box 59, Saranac Lake, NY 12983. E-mail address: dwoodland{at}trudeauinstitute.org

³ Abbreviations used in this paper: MIP, macrophage-inflammatory protein; BAL, bronchoalveolar lavage; EID₅₀, 50% egg infectious dose; HN, hemagglutinin-neuraminidase; MLN, mediastinal lymph node; NP, nucleoprotein; CD8⁺ sup, CD8⁺ T cell culture supernatant; SFC, spot-forming cells; DC, dendritic cell.

Received for publication March 2, 2001. Accepted for publication June 4, 2001.

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