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The Journal of Immunology, 2007, 179, 1113-1121
Copyright © 2007 by The American Association of Immunologists, Inc.
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The Antiviral CD8+ T Cell Response Is Differentially Dependent on CD4+ T Cell Help Over the Course of Persistent Infection1

Christopher C. Kemball2,3,*, Christopher D. Pack2,*, Heath M. Guay{ddagger}, Zhu-Nan Li{dagger}, David A. Steinhauer{dagger}, Eva Szomolanyi-Tsuda{ddagger} and Aron E. Lukacher4,*

* Department of Pathology and {dagger} Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322; and {ddagger} Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although many studies have investigated the requirement for CD4+ T cell help for CD8+ T cell responses to acute viral infections that are fully resolved, less is known about the role of CD4+ T cells in maintaining ongoing CD8+ T cell responses to persistently infecting viruses. Using mouse polyoma virus (PyV), we asked whether CD4+ T cell help is required to maintain antiviral CD8+ T cell and humoral responses during acute and persistent phases of infection. Though fully intact during acute infection, the PyV-specific CD8+ T cell response declined numerically during persistent infection in MHC class II-deficient mice, leaving a small antiviral CD8+ T cell population that was maintained long term. These unhelped PyV-specific CD8+ T cells were functionally unimpaired; they retained the potential for robust expansion and cytokine production in response to Ag rechallenge. In addition, although a strong antiviral IgG response was initially elicited by MHC class II-deficient mice, these Ab titers fell, and long-lived PyV-specific Ab-secreting cells were not detected in the bone marrow. Finally, using a minimally myeloablative mixed bone marrow chimerism approach, we demonstrate that recruitment and/or maintenance of new virus-specific CD8+ T cells during persistent infection is impaired in the absence of MHC class II-restricted T cells. In summary, these studies show that CD4+ T cells differentially affect CD8+ T cell responses over the course of a persistent virus infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The generation of a primary CD8+ T cell response to an acute viral or bacterial infection does not require help by CD4+ T cells, in contrast to CD4+ T cell help-dependent CD8+ T cell responses against noninflammatory Ags (1). In sharp contrast, CD4+ T cell help is necessary for driving differentiation of, or maintaining recall-competent, memory CD8+ T cells (2, 3, 4, 5, 6). Although the requirement for CD4+ T cell help to generate and maintain antiviral CD8+ T cell responses has been actively investigated for acutely resolved viral infections (7, 8, 9, 10, 11, 12, 13, 14, 15), far less is understood about the role of CD4+ T cells in maintaining ongoing CD8+ T cell responses to persistent infection. The progressive functional deterioration and deletion of antiviral CD8+ T cells during chronic lymphocytic choriomeningitis virus (LCMV)5 infection is accentuated in CD4+ T cell-deficient hosts (16, 17, 18, 19). CD8+ T cell control of persistent murine gammaherpesvirus 68 ({gamma}-HV-68) infection is gradually lost in the absence of CD4+ T cells (20), despite a long-lived, functional memory CTL response in CD4+ T cell-deficient mice (21, 22). Moreover, given differences in viral load, activation state of APCs, and bystander inflammatory mediators, antiviral CD8+ T cells may exhibit differential requirements for CD4+ T cell help in the context of low-level vs high-level persistent infection. Thus, in a setting where CD8+ T cells repeatedly encounter persistently infected cells, prolonged CD4+ T cell help may be required to sustain CD8+ T cell-mediated immunity.

Using mouse polyoma virus (PyV) as a model for low-level persistent infection, we asked whether CD4+ T cell help is required for the antiviral CD8+ T cell response during acute and persistent phases of infection. PyV is a natural mouse pathogen that establishes silent persistent infection in wild-type mice, but is capable of potent oncogenicity when injected into newborn mice of particular strains (23, 24, 25). PyV-specific CD8+ T cells are needed for control of viral persistence and protection against virus-induced tumors (26, 27, 28). We recently found that the PyV-specific CD8+ T cell response is sustained by the continuous recruitment of naive virus-specific CD8+ T cells during persistent infection (29, 30). CD4+ T cell-deficient mice have been reported to control PyV infection and retain resistance to PyV-induced tumors (31, 32). We sought to reconcile this contradiction between the reported requirement for CD4+ T cell help to foster differentiation of a high-quality memory CD8+ T cell response and the apparent dispensability for CD4+ T cells in anti-PyV CD8+ T cell-mediated surveillance for persistently infected and transformed cells.

In this study we used MHC class II-deficient mice to evaluate the PyV-specific CD8+ T cell response in the absence of CD4+ T cell help during acute and persistent phases of infection. Although PyV-specific CD8+ T cell numbers were markedly lower during persistent infection in MHC class II-deficient than in wild-type mice, the residual antiviral CD8+ T cells were functionally unimpaired. Viral capsid-specific serum IgG, although initially generated, declined, and long-lived PyV-specific Ab-secreting cells (ASCs) were not found in the bone marrow of MHC class II-deficient mice. Moreover, the recruitment and/or maintenance of new naive PyV-specific CD8+ T cells during persistent infection was substantially reduced in MHC class II-deficient hosts. These findings demonstrate that CD4+ T cell help is necessary to sustain long-term PyV-specific humoral immunity and promotes the emergence of newly primed antiviral CD8+ T cells during persistent virus infection, but is not essential for long-term survival of functional antiviral CD8+ T cells.


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

C57BL/6NCr (B6) female mice were purchased from the Frederick Cancer Research and Development Center (National Cancer Institute, Frederick, MD). B6.129-H2-Ab1tm1Gru N12 mice (MHC class II-deficient), B6.SJL-Ptprca/BoAiTac (CD45.1), and B6.SJL-Ptprca/BoAiTac-H2-Ab1tm1GLM N13 mice (MHC class II-deficient, CD45.1) were purchased from Taconic Farms. Mice were housed and bred in accordance with the guidelines of the Institutional Animal Care and Use Committee, and the Department of Animal Resources at Emory University (Atlanta, GA).

Virus preparation and inoculation

PyV strain A2 was molecularly cloned and plaque purified, and virus stocks were prepared on primary baby mouse kidney cells as previously described (27). Each mouse was s.c. inoculated in each hind footpad with 2 x 106 PFU of virus. Mice were infected at 6–12 wk of age. For the purpose of generating a recombinant vaccinia virus (VV) expressing a large T (LT)/hemagglutinin (HA) chimeric protein, a polypeptide containing a CD8+ T cell epitope from PyV LT359–368 was inserted into the influenza A virus HA glycoprotein. PCR was performed using the mouse PyV LT gene as a template with a pair of primers 5'-CACACACAGCTAGCGAATTCAAATGCCTGGTCCATT-3' and 5'-CTCTCTCTTTAATTAAATAGCATTCCATAGGCTTGGTG-3' (the NheI and PacI sites are underlined). PCR products containing the coding region of LT residues from 335 to 386 were digested by NheI and PacI followed by subcloning into the NheI and PacI digested plasmid pRB21-RBD/HA (33). The sequence of the entire coding region was verified. Recombinant VV were generated by a previously described method (34). The LT/HA chimeric proteins expressed by a recombinant VV (VV-LT) were verified using Western blot and ELISA (data not shown), and then a positive plaque was plaque-purified one more time and used to generate the stock viruses in CV1 cells. For rechallenge experiments, each mouse was i.p. inoculated with 1 x 106 PFU of VV-LT or with VV-HA as a control.

Ab treatment

In some experiments, mice received 100 µg of rat anti-mouse CD40 Ab (clone FGK45), provided by Dr. S. Schoenberger (La Jolla Institute for Allergy and Immunology, La Jolla, CA), or 100 µg of ChromPure rat IgG (Jackson ImmunoResearch Laboratories) on days 1, 10, and 20 postinfection (p.i.).

Bone marrow microchimerism induction

The generation of persistently infected CD45 congenic bone marrow chimeras has been previously described (29). Persistently infected, busulfan-treated B6 and I-Ab–/– mice (CD45.2+) received bone marrow i.v. prepared from naive B6.SJL (CD45.1+) and congenic I-Ab–/– (CD45.1+) mice, respectively. The blood, spleen, and lungs of chimeric mice were analyzed by flow cytometry ~7 wk posttransplant.

Peptides and intracellular IFN-{gamma} staining

LT359–368C7Abu, middle T (MT)245–253, and LT638–646 peptides were synthesized and stored as described (29, 35). Cells were stimulated directly ex vivo with 1 or 10 µM synthetic peptide and stained for CD8{alpha} and IFN-{gamma} as described elsewhere (29). The total number of Ag-specific IFN-{gamma}+CD8+ T cells was determined by subtracting unstimulated IFN-{gamma}+CD8+ cells (no peptide) from peptide-stimulated IFN-{gamma}+CD8+ cells. In some experiments, cells were also stained with TNF-{alpha} (Caltag Laboratories) or IL-2 (BD Biosciences) mAbs following peptide stimulation.

Flow cytometry

Cells were stained in PBS containing 2% FBS and 0.1% sodium azide (FACS buffer) for 45 min at 4°C or room temperature, followed by two washes in FACS buffer. Samples were immediately acquired on a FACSCalibur (BD Biosciences) or were fixed in PBS containing 1% paraformaldehyde overnight. Cells were stained with allophycocyanin-conjugated tetramers. CD8{alpha}, NKG2A/C/E (clone 20d5), IFN-{gamma}, and isotype control Abs were purchased from BD Pharmingen. CD45.1, CD27, CD122, CD127, NKG2A (clone 16a11), programmed death receptor-1 (PD-1), and isotype control Abs were purchased from eBioscience. CD11a and CD62L-selectin (CD62L) Abs were purchased from Caltag Laboratories. Data were analyzed using CellQuest software (BD Biosciences).

Quantification of PyV genomes

DNA isolation and TaqMan PCR were performed as described (29). The PyV DNA quantity is expressed in genome copies per milligram of tissue and is calculated based on a standard curve of known PyV genome copy number vs threshold cycle of detection. The detection limit with this assay is 10 copies of genomic viral DNA.

ELISA and ELISPOT analysis

PyV major capsid protein VP1-specific ELISA were performed as described (35). Two-fold serial dilutions of a positive control serum sample harvested from PyV-infected B6 mice on day 21 p.i. were used to obtain a standard reference curve on each 96-well plate, and the VP1-specific IgG concentrations of the test samples were expressed in arbitrary units based on comparison with this standard curve. The level of VP1-specific serum IgG is <1 U/ml in uninfected mice. VP1-specific ELISPOT assays were done by a modification of a previously described method for detecting influenza virus-specific ASCs (36). Multiscreen HA plates (Millipore) were coated with 100 µl of purified VP1 protein (0.1 µg/ml) overnight at 4°C and then blocked for 30 min at 37°C with RPMI 1640 plus 10% FBS. Cells were plated in 0.2 ml at 1 x 106, 2.5 x 105, 6.25 x 104, and 1.25 x 104 cells/well, in duplicates, and incubated for 4 h at 37°C. After removing the cells, bound Ab was detected using 100 µl/well of biotin-conjugated goat Abs specific for mouse IgM or IgG (1 µg/ml in PBS containing 1% FBS and 0.1% Tween 20; Vector Laboratories) and streptavidin-conjugated HRP (1 µg/ml in PBS containing 1% FBS and 0.1% Tween 20; Vector Laboratories). Plates were developed with 3-amino-9-ethylcarbazole (Sigma-Aldrich), according to the manufacturer’s instructions. Spots were counted using a dissection microscope. The limit of detection for these assays is ~1 ASC/106 cells.

Statistics

Statistical significance was determined by an unpaired Student’s t test, assuming unequal variances. A value for p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Unhelped antiviral CD8+ T cells are numerically reduced, but functionally competent, during persistent infection

To assess the contribution of CD4+ T cell help to the generation and maintenance of PyV-specific CD8+ T cells, we longitudinally monitored the frequency of Db LT359 tetramer-positive T cells in the blood of individually infected C57BL/6 (B6) and I-Ab–/– mice (Fig. 1A). We elected to use I-Ab–/– instead of CD4–/– mice for these studies because CD4–/– mice can mount an MHC class II-restricted T cell response (37). B6 mice mount a PyV-specific CD8+ T cell response that peaks at day 8 p.i., with a viral epitope-specific dominance hierarchy of LT359 > MT245 > LT638 (29). During the acute phase of infection, the kinetics of LT359-specific CD8+ T cell expansion and contraction were similar between B6 and I-Ab–/– mice, although the depth of contraction was more pronounced in I-Ab–/– mice (Fig. 1A). Interestingly, PyV-infected I-Ab–/– mice mounted a higher magnitude splenic Db LT359-specific CD8+ T cell response at day 8 p.i. than B6 mice, but fewer virus-specific CD8+ T cells remained by day 13 p.i. (Fig. 1B). Although consistently detected at a lower frequency and total number, it is notable that unhelped LT359-specific memory CD8+ T cells were readily detected during the persistent phase of infection as late as 6 mo p.i., indicating that the PyV-specific memory T cell response was long-lived in the absence of CD4+ T cell help (Fig. 1, A and B). Similar results were obtained by quantitating the total number of splenic LT359-specific IFN-{gamma}+CD8+ T cells during the course of infection (data not shown). In addition, PyV-infected I-Ab–/– mice generated greater subdominant PyV-specific CD8+ T cell responses at day 8 p.i. compared to B6 mice, as detected by intracellular IFN-{gamma} production (Fig. 1C). Because the CD8+ T cell compartment is larger in I-Ab–/– than B6 mice (38) and both mice have similar acute infection viral loads (Fig. 1E), the elevated peak magnitude of anti-PyV CD8+ T cells in acutely infected I-Ab–/– mice may reflect availability of more PyV-specific CD8+ T cell precursors. The greater degree of contraction of LT359-specific CD8+ T cells in I-Ab–/– mice between days 8 and 13 p.i. could be associated with a lack of survival factors (e.g., IL-2) in the absence of CD4+ T cell help (39). Another possibility, based on the data presented below (see Fig. 5), is that the more pronounced decrease in the total number of PyV-specific CD8+ T cells by day 13 p.i. may result from a reduction in new antiviral CD8+ T cell priming and/or maintenance following the programmed contraction of effector T cells.


Figure 1
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  		FIGURE 1. MHC class II-deficient mice mount a reduced PyV-specific T cell response. A, The frequency of Db LT359 tetramer-positive cells of total CD8+ PBMCs was monitored longitudinally in B6 and I-Ab–/– mice. Results are the mean (±SD) of n = 3 mice/group and are representative of two independent experiments. *, p < 0.05; **, p < 0.01. B, Total number of splenic Db LT359 tetramer-positive CD8+ T cells on the indicated days p.i. in B6 and I-Ab–/– mice. Results are the average (±SD) of n = 3 mice/group and are representative of two independent experiments. *, p < 0.05; **, p < 0.01. C, The total number of splenic MT245- and LT638-specific IFN-{gamma}+CD8+ T cells at days 8 and 45 p.i. in B6 and I-Ab–/– mice are represented. Values plotted are the average (±SD) of n = 3 mice/group and are representative of two to three independent experiments. *, p < 0.05. D, Cytokine production by LT359-specific CD8+ T cells during acute (day 8 p.i.) and persistent (day 45 p.i.) infection. Values shown in representative dot plots (gated on CD8+ T cells) show the mean percentage (±SD) of IFN-{gamma}-producing cells that coproduce TNF-{alpha} for n = 3 mice. E, The number of PyV genomes in the spleen of B6 or I-Ab–/– mice was quantitated by TaqMan real-time PCR on the indicated days p.i. There is no statistically significant difference in viral load between B6 and I-Ab–/– mice at any time point. Individual mice (n = 3 mice/group) are shown (•) and a bar represents the mean copy number of the group. Data are representative of two to three independent experiments.

 

Figure 5
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  		FIGURE 5. Late priming and/or maintenance of PyV-specific CD8+ T cells during persistent infection is diminished in the absence of CD4+ T cell help. A, Persistently infected, busulfan-treated B6 and I-Ab–/– mice (CD45.2+) received bone marrow from naive B6.SJL (CD45.1+) and congenic I-Ab–/– (CD45.1+) mice, respectively. Seven weeks posttransplant (day 113 p.i. for B6 hosts and day 95 p.i. for I-Ab–/– hosts), the frequency of the host-derived (CD45.1–) and donor-derived (CD45.1+) LT359-specific CD8+ T cell response in the spleen and lungs was quantitated by IFN-{gamma} intracellular staining after peptide stimulation. B, The frequency of the host-derived (CD45.1–) and donor-derived (CD45.1+) LT359-specific CD8+ T cell response in the blood, spleen, and lungs of I-Ab–/– hosts was quantitated by Db LT359 tetramer staining. Values shown in representative dot plots (gated on CD8+ T cells) show the mean percentage (±SD) of IFN-{gamma}+ (A) or tetramer-positive (B) cells of total CD45.1–CD8+ (host) or CD45.1+CD8+ (donor) cells (atop the left and right quadrants, respectively) for n = 3 mice. Data are representative of two independent experiments.

 
In persistently infected mice, however, the total number of PyV-specific CD8+ T cells targeting both immunodominant and subdominant epitopes was significantly (p < 0.05) reduced in I-Ab–/– mice, as detected by intracellular IFN-{gamma} production and by tetramer staining (Fig. 1, B and C). During acute and persistent phases of infection (days 8 and 45 p.i., respectively), PyV-specific CD8+ T cells were phenotypically indistinguishable in B6 and I-Ab–/– mice (based on the expression of CD11a, CD62L, CD122, CD127, and CD94/NKG2A; data not shown). In addition, a similar proportion of IFN-{gamma}-producing LT359-specific CD8+ T cells in B6 and I-Ab–/– mice coproduced TNF-{alpha} or IL-2 at both acute and persistent infection time points (Fig. 1D and data not shown). Despite dramatic differences between B6 and I-Ab–/– mice in PyV-specific CD8+ T cell numbers during acute and persistent infection, it is notable that B6 and I-Ab–/– mice carried similar viral loads in the spleen throughout infection (Fig. 1E). There was also no significant (p < 0.05) difference in viral genome copy numbers found in B6 vs I-Ab–/– mice in the kidneys, lungs, or thymus at days 13 and 42 p.i. (data not shown). Thus, it was not surprising that I-Ab–/– mice, like the highly PyV tumor-resistant B6 mice (24), were tumor-free at necropsy at 6 mo p.i., raising the possibility that unhelped CD8+ T cells were able to efficiently mediate antitumor surveillance. Taken together, these data suggest that PyV-specific CD8+ T cells generated in the absence of CD4+ T cells, though few, are present in sufficient numbers and retain the functional competence necessary to control this low-level persistent viral infection.

CD4+ T cell help maintains anti-PyV humoral immunity during low-level persistent infection

Unlike many viruses that require T cell help to generate protective, isotype-switched antiviral Ab responses, PyV infection elicits a virus-neutralizing, isotype-switched, T cell-independent type 2 Ab response directed to VP1, the major capsid protein (40, 41). Because CD40–/– and T cell-deficient mice mount VP1-specific IgG responses that are 10-fold lower than wild-type mice (42), we asked whether PyV-infected I-Ab–/– mice would likewise mount a smaller VP1 Ab response. Unexpectedly, VP1 serum IgG titers were similar in B6 and I-Ab–/– mice at day 8 p.i. (Fig. 2A), but dramatically declined in I-Ab–/– mice by 23 days p.i. (Fig. 2A). In contrast, serum anti-VP1 IgG titers increased ~100-fold in B6 mice from day 8 p.i. (Fig. 2A). These data indicate that the T cell-independent PyV-specific Ab response was not sustained during persistent infection in the absence of CD4+ T cell help. This difference in serum anti-VP1 IgG titer was recapitulated by the number of bone marrow-resident VP1-specific ASCs. VP1-specific IgG ASCs were present in the bone marrow of persistently infected B6 mice, but were below the limit of detection in nearly all infected I-Ab–/– mice (Fig. 2B). In addition, VP1-specific IgG ASCs were not detected in the spleens of persistently infected B6 and I-Ab–/– mice, and VP1-specific IgM ASCs were not detectable in either group of mice (data not shown). These data support two conclusions: first, the T cell-independent PyV Ab response deteriorates in the absence of CD4+ T cells; and second, that CD4+ T cells are required for long-term maintenance of the PyV-specific IgG response.


Figure 2
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  		FIGURE 2. MHC class II-deficient mice mount a reduced PyV-specific humoral response. A, VP1-specific serum IgG titers were measured by ELISA on the indicated days p.i. in B6 and I-Ab–/– mice. Data are the mean ± SD of n = 2–3 mice/group and are representative of two to three independent experiments. *, p < 0.05; **, p < 0.01. B, Number of VP1-specific IgG ASCs at days 83 and 89 p.i. in the bone marrow of B6 and I-Ab–/– mice. Individual mice (•) are represented, and bar indicates the mean number per group (n = 6 mice/group, pooled from two independent experiments). *, p < 0.05.

 
Unhelped PyV-specific CD8+ T cells in persistently infected mice retain recall potential

Because the defect of CD8+ T cells primed in the absence of CD4+ T cells is realized by a defective memory recall response (2, 3, 4, 5, 6), we evaluated the recall potential of PyV-specific CD8+ T cells in B6 and I-Ab–/– mice. Persistent PyV-infected mice were challenged by VV-LT, or the control recombinant VV-HA, and T cell responses in the spleen were analyzed 4 days later. The total number of LT359-specific CD8+ T cells in mice receiving the control challenge virus closely approximated that of unchallenged PyV-infected animals at a similar time postinfection (Fig. 3A and 1B). Compared with mice receiving the control VV, the number of Db LT359 tetramer-positive CD8+ T cells expanded ~10-fold in both B6 and I-Ab–/– mice given VV-LT (Fig. 3A). Similar results were obtained by enumerating LT359-specific IFN-{gamma}+CD8+ T cells in the spleen after peptide stimulation, both in IFN-{gamma}+ cell number and mean fluorescence intensity, indicating that the expanded populations of helped and unhelped virus-specific T cells were functional (Fig. 3B and data not shown). Phenotypic analysis of Db LT359 tetramer-positive CD8+ T cells in all groups of mice revealed that CD62L expression was uniformly low (data not shown). However, Ag rechallenge was associated with phenotypic changes in the expression of CD27, CD127 (IL-7R{alpha}-chain), and the inhibitory receptors CD94/NKG2A and PD-1 by Db LT359 tetramer-positive CD8+ T cells in both persistently infected B6 and I-Ab–/– mice (Fig. 3C). In particular, a substantially greater percentage of recalled tetramer+ cells in I-Ab–/– than B6 mice were CD27high and CD127low, a phenotype indicative of effector T cell differentiation (Fig. 3C). Additionally, a larger percentage of unhelped tetramer-positive cells expressed PD-1 (Fig. 3C), a receptor that is up-regulated on recently activated effector CD8+ T cells during acute infection, as well as on functionally impaired CD8+ T cells during chronic viral infection in mice and humans (43, 44, 45, 46, 47, 48). Interestingly, the two inhibitory receptors examined in this study, PD-1 and CD94/NKG2A, showed inverse expression profiles on LT359-specific CD8+ T cells upon Ag rechallenge in mice of both strains; the frequency of tetramer-positive cells expressing PD-1 increased (markedly so in I-Ab–/– mice), whereas fewer cells expressed CD94/NKG2A (Fig. 3C). Taken together, these data suggest that unhelped PyV-specific CD8+ T cells in persistently infected mice are capable of robust expansion and cytokine production following Ag reencounter, but have phenotypic differences from those primed and maintained in a CD4+ T cell-sufficient environment.


Figure 3
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  		FIGURE 3. Unhelped PyV-specific memory CD8+ T cells mount a functional recall response. Persistently PyV-infected B6 and I-Ab–/– mice (day 61 p.i.) were rechallenged with a recombinant VV encoding the LT359–368 epitope (VV-LT) or a control recombinant VV (VV-HA), and T cell responses were analyzed 4 days later. A, Total number of Db LT359 tetramer-positive CD8+ T cells in the spleen. B, Total number of LT359-specific IFN-{gamma}+CD8+ T cells in the spleen. Data are the mean ± SD of n = 3 mice/group. *, p < 0.05. C, Percentage of Db LT359 tetramer-positive CD8+ T cells in the spleen expressing CD27, CD127, NKG2A, or PD-1. Values shown in representative dot plots (gated on CD8+ T cells) show the mean percentage (±SD) of tetramer-positive cells that are CD27high, CD127low, NKG2A+, or PD-1+ (n = 3 mice). Dot plot quadrant gates are set from isotype controls. All data are representative of two independent experiments.

 
CD40 stimulation partially restores the PyV-specific CD8+ T cell response in MHC class II-deficient mice

Because I-Ab–/– mice generated numerically depressed PyV-specific CD8+ T cell memory, we considered whether administration of agonistic CD40 Ab (clone FGK45) could boost the number of PyV-specific CD8+ T cells in persistently infected mice. It had been reported that two injections of FGK45 mAb during acute {gamma}-HV-68 infection prevented the reactivation of latent virus in I-Ab–/– mice in a CD8+ T cell-dependent fashion (49). We hypothesized that administration of multiple doses of FGK45 during the immune response to PyV infection would augment antiviral CD8+ T cell numbers in I-Ab–/– mice. The LT359-specific CD8+ T cell response in I-Ab–/– mice that received control rat IgG was significantly (p < 0.01) reduced compared with B6 mice that received rat IgG, in agreement with previous data (Fig. 4A and 1B). Compared with IgG-treated I-Ab–/– mice, LT359-specific T cell numbers significantly increased (p < 0.05) in I-Ab–/– mice that received three injections of FGK45 (Fig. 4A). Similar results were obtained by quantifying LT359 peptide-stimulated IFN-{gamma}+CD8+ T cells (data not shown). Of note, FGK45 administration was associated with phenotypic changes in the expression of CD27, CD127, and PD-1 by Db LT359 tetramer-positive CD8+ T cells in I-Ab–/– mice, such that the phenotype of these T cells approximated that seen in persistently infected B6 mice (Fig. 4B). In particular, the proportion of PD-1-expressing unhelped tetramer-positive cells declined in FGK45-treated mice (Fig. 4B). Thus, interventions that improve CD40 engagement can partially override the quantitative defect and phenotypic profile imparted to PyV-specific CD8+ T cells by the absence of CD4+ T cell help.


Figure 4
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  		FIGURE 4. CD40 stimulation partially restores the PyV-specific CD8+ T cell response in MHC class II-deficient mice. A, PyV-infected B6 and I-Ab–/– mice were treated with an agonist CD40 Ab (clone FGK45) or control rat IgG as indicated, and the total number of Db LT359 tetramer-positive CD8+ T cells in the spleen was quantitated at day 39 p.i. Data are the mean ± SD of n = 3 mice/group. *, p < 0.05; **, p < 0.01. B, Effect of FGK45 administration on expression of CD27, CD127, and PD-1 by Db LT359 tetramer-positive CD8+ T cells. Representative dot plots (gated on CD8+ T cells) show the mean percentage (±SD) of tetramer-positive cells that are CD27high, CD127low, or PD-1+ for n = 3 mice. Dot plot quadrant gates are set from isotype controls. Data are representative of two independent experiments.

 
Persistent infection-phase priming and/or maintenance of PyV-specific CD8+ T cells is impaired in the absence of CD4+ T cell help

Using a persistently infected bone marrow chimera model, we previously demonstrated that naive LT359-specific CD8+ T cells are primed and expand during the persistent phase of PyV infection (29). In contrast to antiviral CD8+ T cells primed early after infection, we asked whether CD8+ T cells primed within the microenvironment of persistent virus infection required CD4+ T cell help for expansion and differentiation. Because virus-induced inflammatory signals that could bypass the requirement for T cell help during acute infection are likely to be much lower (or absent) during persistent infection, it is conceivable that the generation of virus-specific T cells at this stage of infection might be similar to helper-dependent CD8+ T cell responses directed against noninflammatory Ags. To test this hypothesis, we created two sets of persistently infected CD45.1+ bone marrow chimeras in which CD4+ T cell help was present or absent during infection. Persistently infected, busulfan-conditioned B6 and I-Ab–/– mice (CD45.2+) received bone marrow from naive B6.SJL (CD45.1+) and congenic I-Ab–/– (CD45.1+) mice, respectively. The minimally myeloablative dose of busulfan used in these studies permits stem cell engraftment without irradiation and does not perturb viral load or established T cell populations (29, 30). Seven weeks posttransplant, by which time microchimerism is established and donor-derived LT359-specific CD8+ T cells are detectable in a B6 host (29), the magnitude of the host-derived (CD45.1–) and donor-derived (CD45.1+) LT359-specific CD8+ T cell response in the spleen and lungs was enumerated. Newly primed donor-derived LT359-specific IFN-{gamma}-producing CD8+ T cells were clearly present in the B6 chimeras, in addition to endogenous host-derived LT359-specific CD8+ T cells (Fig. 5A). Host-derived LT359-specific CD8+ T cells were also detected in I-Ab–/– chimeras (Fig. 5A). However, the frequency of donor-derived LT359-specific CD8+ T cells in I-Ab–/– chimeras was markedly lower than in B6 chimeras (Fig. 5A). Similar results were obtained by quantifying Db LT359 tetramer-positive CD8+ T cells in the blood, spleen, and lungs directly ex vivo, excluding the possibility that a substantial number of nonfunctional donor-derived LT359-specific CD8+ T cells were present in I-Ab–/– chimeras (Fig. 5B). In addition, there was no significant (p < 0.05) difference in viral genome copy number between B6 and I-Ab–/– mice in the thymus at day 42 p.i. (data not shown), arguing against central tolerance as a explanation for the low frequency of late-primed PyV-specific CD8+ T cells. Importantly, because bone marrow microchimerism induction requires several weeks to detect a donor-derived T cell response, the resulting population represents the sum of all virus-specific cells that were primed and subsequently maintained over this time period. Taken together, these data show that de novo priming and/or maintenance of PyV-specific CD8+ T cells during persistent infection is diminished without cognate CD4+ T cell help.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we used the mouse PyV model to investigate the CD4+ T cell help-dependence of virus-specific CD8+ T cell and humoral responses to a low-level systemic persistent viral infection. CD4+ T cell deficiency affects antiviral CD8+ T cell and Ab responses to PyV infection in a manner distinct from that seen in other well-established murine models of persistent viral infections. For example, in persistent {gamma}-HV-68 infection, I-Ab–/– and B6 mice mount antiviral CD8+ T cell responses of similar magnitude at all stages of infection (21). In chronic LCMV infection in B6 mice, virus-specific memory CD8+ T cells decrease in number over time and suffer progressive functional deterioration (16, 17, 18, 19, 50). The pace of CD8+ T cell exhaustion is accelerated and the deficits more profound in CD4–/– hosts, with deletion of functionally inert T cells and chronic high-level systemic viral replication (17, 19). None of these features applies to PyV infection in CD4+ T cell-deficient mice.

A numerically small, but functionally intact, antiviral CD8+ T cell response is maintained during persistent PyV infection in I-Ab–/– mice, which control viral replication as efficiently as wild-type mice. Moreover, these unhelped PyV-specific CD8+ T cells underwent vigorous expansion and expressed effector activities (e.g., IFN-{gamma} production) upon re-encounter with cognate Ag. Our findings are in agreement with a previous report showing that continuous Ab-mediated depletion of CD4+ T cells did not affect the capacity of vesicular stomatitis virus-specific memory CD8+ T cells to undergo secondary expansion and effector differentiation (15). An intriguing possibility is that distinct populations of PyV-specific CD8+ T cells that differ in their dependence on CD4+ T cell help for priming and/or maintenance are elicited at different phases of infection. Because de novo CD8+ T cell priming and/or maintenance during persistent PyV infection was reduced in the absence of MHC class II-restricted T cells (Fig. 5), a substantial portion of antiviral CD8+ T cells maintained in persistently infected I-Ab–/– mice likely represent CD4+ T cell-independent CD8+ T cells that were generated during acute infection. Whether long-term maintenance of these T cells is dependent upon chronic Ag stimulation (51) or particular common {gamma}-chain cytokines (52, 53, 54, 55) remains to be clarified. In this connection, the absence of CD4+ T cells in I-Ab–/– mice may favor an IL-2-independent pathway for CD8+ T cell clonal expansion. Fearon and colleagues (56) recently reported that in vitro CD27 costimulation promotes IL-7-dependent CD8+ T cell proliferation in the absence of IL-2R signaling, without inducing effector differentiation. A similar mechanism may enable unhelped antiviral CD8+ T cells avoid replicative senescence and maintain themselves long-term through self-renewal.

Given that persistently infected I-Ab–/– mice do not have a demonstrable defect in their ability to control viral persistence despite fewer late-primed antiviral CD8+ T cells, these newly recruited T cells do not appear to be required to control persistent infection. As with most viruses, control of PyV infection is multifactorial and redundant. We previously reported that beta2-microglobulin-knockout mice clear PyV with similar efficiency to wild-type animals; others have also shown that CD4–/– and CD8–/– mice (single or combined) control PyV (26, 31, 32, 57). In the setting of depressed T cell expansion and maintenance (i.e., Ab-mediated costimulation blockade of B7-CD28 and CD40-CD40L pathways), we previously described multiorgan differences in PyV load (35). Importantly, PyV is among a small group of viruses that elicits a protective T cell-independent antiviral Ab response (58). Although VP1-specific Ab titers fell over time in I-Ab–/– mice, it is likely that this T cell-independent Ab response provided protection over the course of infection, as transfer of serum IgG from PyV-infected T cell-deficient mice into SCID mice renders them resistant to PyV infection (58). An alternative argument that may be raised is that early events decide the fate of persistent PyV infection and, depending on this level, the predisposition to developing tumors. Because B6 and I-Ab–/– mice mount strong effector CD8+ T cell responses to acute PyV infection, it is possible that a similar set-point for persistent infection is achieved, over which a reduced number of functionally competent, unhelped memory CD8+ T cells are capable of maintaining long-term control.

How do unhelped PyV-specific CD8+ T cells avert functional debilitation despite continuous Ag encounter during persistent infection (29, 30)? A number of factors (e.g., high viral load, APC dysfunction, immune deviation, up-regulation of T cell inhibitory receptors, elevated regulatory T cell activity) may come into play to down-modulate antiviral CD8+ T cell effector activities, either as a viral immune evasion strategy or host defense against immunopathology (59). One explanation is that T cell-independent VP1-specific Abs may check virus levels during acute infection in I-Ab–/– mice sufficiently to rescue a fraction of the unhelped anti-PyV CD8+ T cells from functional destitution. However, VP1-specific serum IgG titers progressively decline in persistently infected I-Ab–/– mice, in association with an absence of anti-VP1 ASCs in the spleen and bone marrow. Because humoral immunity is sustained by long-lived bone marrow-resident plasma cells and memory B cells (60), our data suggest that these cell types are not formed in the absence of CD4+ T cell help. Consistent with this idea, signaling lymphocytic activation molecule-associated protein expressing CD4+ T cells were shown to be necessary for the generation of long-lived plasma cells and memory B cells in response to LCMV and influenza virus infections (61, 62).

Recent studies have documented PD-1 receptor expression by virus-specific CD8+ T cells during acute and high-level chronic viral infection in mice and humans, with PD-1 blockade resulting in restoration of T cell function (63). We have extended this finding by showing that PD-1 is also expressed by PyV-specific CD8+ T cells during the low-level persistent infection phase of the virus. Whether PD-1 operates at the level of the effector T cell or influences programming of T cell differentiation is currently unknown. In support of the latter possibility, a large fraction of the unhelped anti-PyV CD8+ T cells expressed PD-1, but these cells were unimpaired in their capacity to produce IFN-{gamma}. This finding is in line with a recent report showing that PD-1 receptor engagement on HIV-specific CD8+ T cells does not directly inhibit T cell effector function, but may regulate T cell proliferation, survival, and susceptibility to apoptosis (46). In addition, although PD-1 is transiently up-regulated on activated effector CD8+ T cells during acute LCMV Armstrong infection, its expression is not associated with inhibition of effector T cell function and PD-ligand 1 blockade does not increase virus-specific CD8+ T cell numbers in this model (43). Thus, it is not unexpected that PD-1+ Db LT359-specific CD8+ T cells are capable of cytokine production. Augmented CD40 signaling via FGK45 mAb administration resulted in fewer PD-1+ PyV-specific CD8+ T cells, but whether this result can be attributed to altering T cell differentiation or reducing Ag load remains to be determined. It has also been suggested that PD-1 may be up-regulated to restrain ongoing antiviral CD8+ T cell responses and thereby limit immunopathology (64). Expression of another T cell inhibitory receptor, CD94/NKG2A, by PyV-specific CD8+ T cells has also been shown to dampen cytopathic effector activity (65). Interestingly, we found that expression of PD-1 and CD94/NKG2A was inversely modulated upon Ag rechallenge of persistently infected I-Ab–/– mice. PyV-specific memory CD8+ T cells lose expression of CD94/NKG2A after Ag-driven proliferation to alleviate the block to killing infected cells (65, 66). It is interesting to speculate that PD-1 may play a counterbalancing effect to CD94/NKG2A by controlling secondary T cell effector differentiation.

Another finding of this study is that de novo priming and/or maintenance of virus-specific CD8+ T cells during persistent infection is diminished in the absence of CD4+ T cell help. Work from several groups has shown that CD4+ T cell help is necessary for driving differentiation of, or maintaining recall-competent, memory CD8+ T cells (2, 3, 4, 5, 6). More recently, Williams et al. (67) have proposed that CD4+ T cells promote the survival of memory CD8+ T cells by sustaining IL-7R and IL-15R expression and thereby retaining their responsiveness to IL-7 and IL-15. Memory CD8+ T cell survival and homeostatic proliferation depend on IL-7 and IL-15 (52, 53, 54, 55). We have previously shown that continuous priming of new thymic emigrants is required for the maintenance of virus-specific CD8+ T cells during persistent infection (30). Therefore, in the absence of CD4+ T cell help, antiviral CD8+ T cell numbers wane over time in I-Ab–/– mice during persistent infection in part because of a lack of recruitment or maintenance of new virus-specific CD8+ T cells. It is important to point out that the donor T cell response that develops following bone marrow microchimerism induction represents a heterogeneous population generated over a 7-wk engraftment period. Thus, this approach cannot distinguish whether CD4+ T cell help is necessary for priming or maintenance of antiviral CD8+ T cells recruited during the persistent phase of infection. Recent evidence indicates that influenza viral CD4+ and CD8+ T cell epitopes persist in secondary lymphoid organs for up to 2 mo after resolution of acute infection and are available to stimulate naive donor TCR transgenic cells throughout this period (68, 69). Thus, the accentuated decrease in PyV-specific CD8+ T cells by day 13 of infection in I-Ab–/– mice (Fig. 1, A and B) may reflect the consequence of reduced CD4+ T cell-dependent de novo priming of PyV-specific CD8+ T cells even at early stages of persistent PyV infection.

Although CD4+ T cells promote the recruitment and/or maintenance of persistent infection-primed virus-specific CD8+ T cells, CD40-CD40L costimulation is dispensable for generating and/or maintaining PyV-specific CD8+ T cells during persistent infection (35). The mechanism of CD4+ T cell help of virus-specific T cells is potentially multifaceted, and signaling pathways other than CD40-CD40L could be involved. T cell help and CD40-CD40L signals play distinct roles in regulating the differentiation of proliferation-competent memory CD8+ T cells (70). In addition, CD4+ T cells maintain LCMV-specific memory CD8+ T cells independently of CD40-CD40L signaling (67). Nonetheless, we have demonstrated that agonist CD40 Ab stimulation partially overrides the quantitative defect and phenotypic profile imparted to PyV-specific CD8+ T cells by the absence of CD4+ T cell help (Fig. 4). Our findings warrant future investigation into the efficacy of interventions to boost CD40 signaling to improve antiviral T cell responses in persistent infection. Caution should be taken with such therapy, however, because excessive treatment could lead to deletion of virus-specific CD8+ T cells (71).

In summary, our studies reveal a novel contribution by CD4+ T cells in fostering long-lived memory CD8+ T cell and humoral responses to low-level persistent virus infection. Although newly primed CD8+ T cells may not be crucial for maintaining viral control in the setting of PyV infection, where unhelped memory CD8+ T cells together with T cell-independent antiviral Ab appear to be sufficient to mediate protective immunosurveillance, our findings may be particularly relevant for high-level chronic viral infections. Thus, CD4+ T cells may be critical for maintaining a steady influx of antiviral CD8+ T cells to replace those exhausted and/or deleted by HIV, hepatitis B virus, or hepatitis C virus infection.


   Acknowledgments
 
We thank Dr. Christian Larsen and Dr. Thomas Pearson for providing busulfan, Dr. Stephen Schoenberger for providing CD40 Ab, and Dr. Eun Lee and Annette Hadley for expert technical assistance.


   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 This work was supported by Grants R01CA71971 and R01CA100644 (to A.E.L.), R01CA66644 (to E.S.-T.), AI66870 (to D.A.S.), T32AI007610 (to C.D.P.), and T32AI07272 (to H.M.G.) from the National Institutes of Health. Back

2 C.C.K. and C.D.P. contributed equally to this work. Back

3 Current address: Molecular and Integrative Neurosciences Department, The Scripps Research Institute, 10550 North Torrey Pines Road, Mail Code SP30-2110, La Jolla, CA 92037. Back

4 Address correspondence and reprint requests to Dr. Aron E. Lukacher, Department of Pathology, Emory University School of Medicine, Woodruff Memorial Research Building Room 7307, 101 Woodruff Circle, Atlanta, GA 30322. E-mail address: alukach{at}emory.edu Back

5 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; ASC, Ab-secreting cell; {gamma}-HV-68, murine gammaherpesvirus 68; HA, hemagglutinin; LT, large T; MT, middle T; p.i., postinfection; PD-1, programmed death receptor-1; PyV, polyoma virus; CD62L, CD62L-selectin; VV, vaccinia virus. Back

Received for publication April 25, 2007. Accepted for publication May 10, 2007.


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 Introduction
 Materials and Methods
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