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The Journal of Immunology, 1999, 163: 3369-3378.
Copyright © 1999 by The American Association of Immunologists

Visualization of Polyoma Virus-Specific CD8+ T Cells In Vivo During Infection and Tumor Rejection1

Aron E. Lukacher2,3,*, Janice M. Moser3,*, Annette Hadley* and John D. Altman3,4,{dagger}

Departments of * Pathology, and {dagger} Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cells are critical for clearing infection and preventing tumors induced by polyoma virus, a natural murine papovavirus. We previously identified the immunodominant epitope for polyoma virus-specific CTL in tumor-resistant H-2k mice as the Dk-restricted peptide, MT389–397, derived from the polyoma middle T oncoprotein. In this study, we developed tetrameric Dk complexes containing the MT389–397 peptide to directly visualize and enumerate MT389–397-specific CTL during polyoma virus infection. We found that Dk/MT389 tetramer+CD8+ T cells undergo a massive expansion during primary infection such that by day 7 postinfection these Ag-specific CD8+ T cells constitute ~20% of the total and ~40% of the activated CD8+ T cells in the spleen. This expansion of Dk/MT389 tetramer+CD8+ T cells parallels the emergence of MT389–397-specific ex vivo cytolytic activity and clearance of polyoma virus. Notably, Dk/MT389 tetramer+CD8+ T cells are maintained in memory at very high levels. The frequencies of Dk/MT389 tetramer+CD8+ effector and memory T cells in vivo match those of CD8+ T cells producing intracellular IFN-{gamma} after 6-h in vitro stimulation by MT389–397 peptide. Consistent with preferential Vß6 expression by MT389–397-specific CD8+CTL lines and clones, Dk/MT389 tetramer+CD8+ T cells exhibit biased expression of this Vß gene segment. Finally, we show that Dk/MT389 tetramer+CD8+ T cells efficiently infiltrate a polyoma tumor challenge to virus-immune mice. Taken together, these findings strongly implicate virus-induced MT389–397-specific CD8+ T cells as essential effectors in eliminating polyoma-infected and polyoma-transformed cells in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Strong epidemiologic and virologic evidence indicate that a number of human malignancies are linked to infections by DNA viruses, including EBV, hepatitis B virus, and human papillomavirus (HPV)5 (1). Efficient control of acute infection by these oncogenic viruses and immunosurveillance for persistently infected/neoplastic cells are believed to be primarily mediated by CD8+CTL recognizing cell surface class I MHC molecules loaded with virus-derived peptides (2, 3). The emergence of class I MHC epitope-loss and TAP-down-regulated HPV-induced cervical carcinomas in immunocompetent women (4), as well as the inhibition of Ag processing by EBNA-1 (5), a viral protein expressed in all EBV-associated malignancies, point to the critical contribution of CD8+CTL to tumor rejection. The absence of tractable animal models that support productive infection and neoplastic disease by these oncogenic human viruses hampers detailed investigation of the magnitude, regulation, and effector activities of these important antitumor effector T cells.

Polyoma virus is a natural murine DNA virus of the papovavirus family, whose members include SV40 and HPV. When inoculated into newborn mice of particular H-2k strains or into T cell-immunocompromised adult mice, polyoma virus induces a wide spectrum of epithelial and mesenchymal cell-derived tumors that are grossly apparent by 2–4 mo postinfection (6, 7, 8, 9). The high polyoma tumor susceptibility in H-2k mice is a consequence of a hole in the T cell repertoire for polyoma virus-specific CD8+CTL incurred by deletion of Vß6-expressing thymocytes by the endogenous superantigen encoded by the Mtv-7 mouse mammary tumor provirus (10). We recently identified the immunodominant epitope for antipolyoma CTL in H-2k mice as the Dk-restricted peptide derived from amino acids 389–397 of the viral oncoprotein, middle T (MT); this epitope is designated MT389–397 (11). A type II integral membrane protein, MT activates multiple growth-promoting signal transduction pathways (12), and its constitutive expression is required for cellular transformation, tumor induction, and virion assembly (13, 14, 15). By limiting dilution analysis (LDA), we determined that resistant mice possess an ~20-fold higher frequency of precursor CTL directed to MT389–397 than susceptible mice (11), supporting the concept that CTL recognizing this viral epitope mediate immunosurveillance for polyoma virus tumorigenesis.

The recent advent of fluorochrome-conjugated soluble tetramers of class I MHC/peptide complexes has enabled direct ex vivo quantitation of Ag-specific CD8+ T cell responses during acute and persistent microbial infections in nontransgenic hosts (16, 17, 18). In mice acutely infected by lymphocytic choriomeningitis virus (LCMV), a noncytopathic systemically replicating mouse arenavirus, Murali-Krishna et al. (19) showed that up to 70% of the CD8+ T cells in the spleen bound class I MHC/LCMV peptide tetramers. Confirming suspicions of the insensitivity of LDA to determine antiviral CTL frequencies in vivo, these investigators and others have amply demonstrated that tetramer staining detects at least 10-fold higher levels of Ag-specific CD8+ T cells than estimated by LDAs (19, 20, 21).

In this study, we used tetramers of Dk molecules complexed to the immunodominant polyoma MT389–397 epitope to directly visualize polyoma-specific CD8+ T cells during effector and memory phases of primary polyoma virus infection. Systemic infection by this lytic, oncogenic virus induces massive expansion of MT389–397-specific CD8+ T cells, which persist at high levels into memory. We also show, for the first time, ex vivo polyoma-specific cytotoxic activity in acutely infected mice. We further demonstrate efficient infiltration of MT389–397-specific CD8+ T cells into a regressing polyoma tumor challenge to virus-immune mice, providing strong support for the concept that immunosurveillance by antiviral CD8+ T cells protects against virus-induced neoplasia.


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

C3H/HeNCr female mice were purchased from the Frederick Cancer Research and Development Center of the National Cancer Institute (Frederick, MD), and C57BR/cdJ female mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were used at 6–9 wk of age.

Viruses and virus inoculation

The wild-type polyoma virus strain A2 was molecularly cloned and plaque purified, and virus stocks were prepared on primary baby mouse kidney cells, as previously described (11). Each mouse was inoculated s.c. in hind footpads with 2 x 106 PFU of virus.

Cell lines

AG104A cells (22) and 6215 cells (11) were maintained in DMEM containing 10% FBS (HyClone, Logan, UT). BALB/3T3 clone A31 cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in DMEM containing 5% bovine calf serum (Summit Biotechnology, Ft. Collins, CO).

Synthetic peptides

Peptides were synthesized by the solid-phase method on a Symphony/Multiplex Peptide Synthesizer (Rainin, Woburn, MA) with F-moc chemistries. HPLC analysis showed that peptides were 90–95% pure. Peptide stock solutions were prepared in water at a concentration of 3 or 6 mM and stored at -20°C. Peptides were diluted in 10% serum-containing medium immediately before use. The following peptides were used in this study: MT389–397 (RRLGRTLLL) and gag88–96 (RRKGKYTGL).

Isolation of polyoma virus-specific CTL

Protocols for establishing polyoma-specific T cell cloned lines are described in detail elsewhere (11). Briefly, draining popliteal and inguinal lymph node cells at ~2 wk postinfection were cocultured with virus-infected, {gamma}-irradiated syngeneic splenocytes. T cells were cloned by limiting dilution from day 7 in vitro secondary or tertiary cultures. T cell lines are maintained by weekly restimulation with virus-infected, irradiated syngeneic splenocytes.

51Cr release assay

Polyo mavirus-infected and peptide-pulsed 51Cr-labeled AG104A target cells were prepared as previously described (11). Target cells were aliquoted at 5000 cells/well into U-bottom 96-well microtiter plates (Costar, Cambridge, MA). Splenocyte effectors were prepared by first lysing erythrocytes using RBC lysing buffer (Sigma, St. Louis, MO), then depleted of adherent cells by incubation for 1 h at 37°C in plastic petri dishes (VWR, Atlanta, GA). After a 4-h incubation at 37°C, one-half the volume of each well was removed and counted in a 1470 Wallac Wizard gamma counter (Turku, Finland).

Percent specific lysis was calculated as follows: ((51Cr release with effector cells - spontaneous 51Cr release)/(total 51Cr release with 1% Triton X-100 - spontaneous 51Cr release)) x 100. Spontaneous 51Cr release from target cells in all assays was 15–20% of the total detergent lysis. The percent specific lysis values represent the mean values of quadruplicate wells. SEMs were always <5% of the mean values and are omitted.

Preparation of H-2Dk tetramers

H-2Dk/peptide tetramers were prepared as previously described (23). Briefly, Escherichia coli strain BL21 (DE3) was transformed with a pET23-Dk-BSP plasmid, and expression of Dk was induced with isopropyl ß-D-thiogalactoside. Human ß2-microglobulin was expressed in the same cell line using the pHN1-ß2m plasmid (57). The folding reaction was performed with either the MT389–397 or gag88–96 peptides. Folding, purification, and biotinylation were performed as previously described (16). Tetramers were made by mixing biotinylated Dk/MT389–397 monomers with allophycocyanin-conjugated streptavidin (Molecular Probes, Eugene, OR) in a 4:1 molar ratio.

Flow cytometry

Single cell suspensions of spleen were prepared, erythrocytes lysed, and 1 x 106 cells stained in phenol red-free RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 2% FBS and 0.01% sodium azide (FACS buffer) for 1 h at 4°C, followed by three washes in FACS buffer, and fixation in PBS containing 1% paraformaldehyde. Cells were surface stained with PE or TRI-COLOR-conjugated rat anti-mouse CD8{alpha} mAb (CT-CD8a; Caltag, South San Francisco, CA) and FITC-conjugated rat anti-mouse CD11a, CD62L, or CD44 mAb (Beckman Coulter, Fullerton, CA). Samples were acquired on a FACSCalibur (Becton Dickinson, San Jose, CA), and data were analyzed using FlowJo software (Tree Star, San Carlos, CA).

For Vß repertoire analysis (see Fig. 7Go), anti-Vß mAbs were either FITC or PE conjugated, and were all purchased from PharMingen (San Diego, CA).



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  		FIGURE 7. Vß repertoire analysis of Dk/MT389 tetramer+CD8+ T cells from polyoma-infected mice. Spleen cells from C3H/HeN mice 7 days postinfection were stained with allophycocyanin-conjugated Dk/MT389 tetramers, TRI-COLOR-conjugated anti-CD8{alpha}, FITC- or PE-conjugated anti-CD11a, and FITC- or PE-conjugated anti-Vß Abs. Values represent the mean percentage ± SEM of Dk/MT389 tetramer+ or Dk/MT389 tetramer-CD11alowCD8+ T cells expressing the indicated Vß of three infected mice. Spleen cells from three naive C3H/HeN mice were also stained with anti-CD8{alpha} and anti-Vß Abs, and the mean percentage of gated CD8+ T cells ± SEM expressing the indicated anti-Vß Abs is shown.

 
For flow cytometry sorting, erythrocyte-lysed spleen cells were stained in phenol red-free RPMI 1640 + 2% FBS (FACS-sorting buffer) containing PE-conjugated anti-CD8{alpha}, FITC-conjugated anti-CD11a, and allophycocyanin-conjugated Dk/MT389 tetramer for 1 h at 4°C. Cells were then washed twice with FACS-sorting buffer, resuspended in FACS-sorting buffer at a concentration of 5 x 107/ml, and immediately acquired and sorted on a FACSVantage (Becton Dickinson).

Intracellular IFN-{gamma} staining

Erythrocyte-lysed spleen cells were cultured for 6 h in 96-well flat-bottom microtiter plates (Costar) at 1 x 106 cells/well in 0.2 ml/well Iscove’s modified Dulbecco’s medium (Life Technologies) containing 10% FBS, 50 µM 2-ME, and penicillin/streptomycin and supplemented with 1 µl/ml brefeldin A (Golgiplug; PharMingen), 50 U/ml human rIL-2 (PharMingen), and synthetic peptides at 10 µM. Cells were then surface stained with PE-conjugated rat anti-mouse CD8{alpha} mAb (Caltag) and allophycocyanin-conjugated Dk/MT389 tetramers. After washing, cells were permeabilized and stained for intracellular IFN-{gamma} with FITC-conjugated rat anti-mouse IFN-{gamma} mAb (cloneXMG1.2; PharMingen) or its isotype control Ab (rat IgG1; Beckman Coulter) using the Cytofix/Cytoperm kit, according to manufacturer’s instructions (PharMingen).

Single cell enzyme-linked immunospot (ELISPOT) assay for IFN-{gamma}-secreting cells

The single cell ELISPOT assay was performed as previously described (19). Briefly, 96-well filtration plates (Millipore, Bedford, MA) were coated with rat anti-mouse IFN-{gamma} (clone R4-6A2; PharMingen). Spleen cells from each of four C3H/HeN mice inoculated 7 days earlier with polyoma virus were stained with PE-conjugated anti-CD8{alpha}, FITC-conjugated anti-CD11a, and allophycocyanin-conjugated Dk/MT389 tetramers, and sorted by flow cytometry into three distinct populations, as shown in Fig. 6GoA. Each sorted population was serially diluted 3-fold in the wells to each of which were added 5 x 105 {gamma}-irradiated (2000 rad) syngeneic spleen cells and MT389–397 peptide (0.1 µg/ml) or no peptide. After 36-h incubation at 37°C, plates were washed, then incubated with biotinylated anti-mouse IFN-{gamma} (clone XMG 1.2; PharMingen). Wells were then incubated with HRP avidin D (Vector Laboratories, Burlingame, CA), washed, and developed with freshly prepared substrate buffer (0.03% (w/v) 3-amino-9-ethyl-carbazole, 0.015% (v/v) H202 in 0.1 M sodium acetate, pH 5).



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  		FIGURE 6. Analysis of Ag-specific IFN-{gamma} secretion by Dk/MT389 tetramer-sorted CD8+ T cells. Spleen cells from each of four C3H/HeN mice 7 days after s.c. polyoma inoculation were stained with allophycocyanin-conjugated Dk/MT389 tetramers, and PE-conjugated anti-CD8{alpha} and FITC-conjugated anti-CD11a Abs. A, Representative plot of CD8+ splenic T cells from one of the infected mice, with the three regions of FACS-sorted populations indicated. B, IFN-{gamma} ELISPOT of each sorted population plated at densities titrated in 3-fold steps, starting at 300 cells/well, and cultured in the presence of 0.1 µg/ml MT389–397 peptide for 36 h. No IFN-{gamma} secretion was detected in the absence of MT389–397 peptide stimulation (data not shown). Values represent means of quadruplicate wells at each dilution point for sorted populations from each mouse, with SEMs shown.

 
Polyoma virus plaque assay

Spleen samples were snap frozen in sterile Kontes tubes (Kontes Glass, Vineland, NJ), adjusted to 50 mg/ml in DMEM at 4°C, and homogenized using an overhead stirrer (Wheaton, Millville, NJ) and disposable Teflon pestles (Kontes). Homogenized tissues were then freeze thawed three times, incubated for 45 min at 42°C, and centrifuged to remove cell debris. Supernatants were titered for infectious virus by plaque assay on BALB/3T3 clone A31 cells, as previously described (24). The detection limit for this plaque assay is 1 PFU/mg spleen.

Isolation of tumor-infiltrating lymphocytes (TILs)

A polyoma virus-immune and naive C57BR/cdJ mouse were each injected s.c. with 25 x 106 6215 cells. Nine days later, s.c. tumors were resected, minced, and digested with 500 U/ml collagenase (ICN Biomedicals, Costa Mesa, CA) for 1.5 h at 37°C. Nonadherent cells were collected following a 1.5-h incubation in plastic petri dishes (VWR) at 37°C. Viable mononuclear cells were isolated on LSM (Organon Teknika, Durham, NC); stained with TRI-COLOR-conjugated anti-CD8{alpha}, FITC-conjugated CD11a, and allophycocyanin-conjugated Dk/MT389 tetramers; and analyzed by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ex vivo polyoma-specific cytotoxic activity

We previously identified the immunodominant epitope for polyoma virus-specific CD8+CTL in H-2k mice as a Dk-restricted peptide derived from the sequence spanning amino acids 389–397 of the polyoma MT protein, designated MT389–397 (11). Because syngeneic target cells infected by polyoma virus are recognized less efficiently by MT389–397-specific CTL clones and lines than peptide-pulsed target cells (11), MT389–397-pulsed syngeneic cells were used as targets to optimize sensitivity for detecting polyoma-specific CTL killing directly ex vivo. Spleen cells from adult C3H/HeN (H-2k) mice at days 5 to 12 postinfection were cocultured with MT389–397-coated syngeneic class I MHC+/class II MHC- AG104A (25) target cells in a standard 4-h 51Cr release assay. As shown in Fig. 1Go, MT389–397-specific cytotoxic activity was initially detected at 6 days after infection, reached maximum levels at days 7 to 9 postinfection, and decreased to near baseline levels by day 12 postinfection. No MT389–397-specific cytotoxicity was exhibited by spleen cells from C3H/HeN mice at day 67 postinfection (data not shown). Spleen cells from naive mice did not lyse MT389–397-pulsed targets, nor was killing seen against unpulsed target cells by spleen cells from mice at any of the indicated days postinfection (data not shown).



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  		FIGURE 1. Kinetic analysis of ex vivo MT389–397 epitope-specific lysis by spleen cells during primary polyoma virus infection. Nonadherent spleen cells from uninfected or s.c. virus-inoculated C3H/HeN mice were assayed at the indicated day postinfection for lysis of 51Cr-labeled AG104A target cells pulsed with MT389–397 peptide (10 µM). Each value represents the mean ± SEM percent specific lysis by spleen cells of two mice. No lysis of unpulsed target cells was seen by spleen cells at any postinfection time point (data not shown).

 
We next asked whether the emergence of MT389–397-specific cytotoxic activity parallels the clearance of infectious polyoma virus from the spleen. As shown in Fig. 2Go, high viral titers were present in the spleens of C3H/HeN mice as early as 3 days after s.c. inoculation with 2 x 106 PFU of polyoma virus. Beginning at day 6 postinfection, infectious virus titers progressively decreased such that by day 12 postinfection, titers had dropped by nearly 4 logs. By day 50 postinfection, no infectious virus was detected in the spleen. Thus, the appearance of MT389–397-specific cytotoxicity in the spleen is associated with elimination of infectious virus.



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  		FIGURE 2. Kinetic analysis of polyoma virus clearance from spleens during primary infection. Spleens from C3H/HeN mice inoculated s.c. with polyoma virus were harvested at the indicated day postinfection and homogenized, and infectious virus was titered by plaque assay. Each value represents the mean PFU/mg spleen of two mice. The detection limit is 1 PFU/mg.

 
Enumeration of MT389–397-specific CD8+ T cells in acute polyoma virus infection

The ability to detect MT389–397-specific cytotoxic activity by freshly explanted spleen cells, without prior expansion in vitro, raised the possibility that antipolyoma CD8+CTL may be present in high numbers in the spleen during acute infection. To directly visualize and quantify polyoma-specific CD8+ T cells in vivo, we created soluble Dk tetrameric complexes containing the MT389–397 peptide. The specificity of these Dk/MT389 tetramers is illustrated by their capacity to stain MT389–397-specific CTL clones 8-1 and 16-5, but not clone 15-5, which recognizes another, as yet undefined, Dk-restricted viral epitope (Fig. 3GoA) (11). In addition, Dk/MT389 tetramer binding did not interfere with binding of anti-Vß mAbs to 8-1 (Vß6+) or 16-5 (Vß8.1+); neither Ab stained the Vß2+ clone 15-5. The high sensitivity of this tetramer in identifying MT389–397-specific CD8+ T cells is revealed by the 7–9% frequency of Dk/MT389 tetramer+ cells in 1:10 mixtures of clones 8-1 or 16-5 with 15-5 (Fig. 3GoA). The Dk/MT389 tetramer was then used to probe Ag-specific CD8+ T cells from the spleen of a C3H/HeN mouse at day 7 after s.c. inoculation, when maximal MT389–397-specific lysis was seen (Fig. 2Go). As shown in Fig. 3GoB, ~20% of the CD8+ splenic T cells bound the Dk/MT389 tetramer. The specificity for Dk/MT389 tetramer staining in acute polyoma infection is illustrated by its lack of binding to spleen cells from an uninfected C3H/HeN mouse, and by the inability of another Dk tetramer containing an endogenous retroviral CTL epitope, gag 88–96 (26), to stain spleen cells from a C3H/HeN mouse at day 7 postinfection.



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  		FIGURE 3. Specificity of staining with Dk/MT389 tetramers. A, MT389–397-specific CD8+CTL clones 8-1 (Vß6+) and 16-5 (Vß8.1+), alone or mixed in a 1:10 ratio with the non-MT389–397-specific CD8+CTL clone15-5 (Vß2+), were stained with allophycocyanin-conjugated Dk/MT389 tetramers and PE-conjugated anti-Vß6 or anti-Vß8.1/8.2 mAbs. B, Spleen cells from a naive and day 7 postinfection C3H/HeN mouse were stained with allophycocyanin-conjugated Dk/MT389 or Dk/gag88–96 tetramers and PE-conjugated anti-CD8{alpha}. Plots are gated on CD8+ cells, and values represent the frequency of cells in the indicated regions.

 
We then used the Dk/MT389 tetramers to directly visualize MT389–397 epitope-specific CD8+ T cells through the course of acute polyoma virus infection. As shown in Fig. 4Go and summarized in Table IGo, no splenic Dk/MT389 tetramer+CD8+ T cells were detected at day 3 postinfection above background staining in naive mice, but their numbers dramatically increased over the next 4 days from ~1 in 20 CD8+ T cells at day 5 to nearly 1 in 5 CD8+ T cells in the spleen by day 7 postinfection. Interestingly, the kinetics of expansion of MT389–397-specific CD8+ T cells closely parallels that of Ag-specific CD8+ T cells during acute LCMV infection (19). This day 5 to day 7 expansion phase for antipolyoma CD8+ T cells coincides with the onset of elimination of infectious polyoma virus (Fig. 2Go). From days 9 to 12 postinfection, when infectious virus titers drop precipitously, the number of MT389–397-specific CD8+ T cells contracts to constitute ~1 in 10 splenic CD8+ T cells.



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  		FIGURE 4. Quantitation of Dk/MT389 tetramer+CD8+ T cells during primary polyoma virus infection. Spleen cells from C3H/HeN mice at the indicated day after s.c. inoculation with polyoma virus and a naive C3H/HeN mouse were stained with allophycocyanin-conjugated Dk/MT389 tetramers, PE-conjugated anti-CD8{alpha}, and FITC-conjugated anti-CD62L, anti-CD11a, or anti-CD44, and assayed by flow cytometry.

 

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  		Table I. Quantitation of MT389-397 epitope-specific CD8+ T cells during polyoma virus infection1

 
At each time point during acute infection, virtually all Dk/MT389+CD8+ T cells express the CD11ahighCD44highCD62Llow phenotype indicative of prior TCR activation (Fig. 4Go). Taken as a percentage of CD11ahighCD8+ T cells, the MT389–397-specific CD8+ T cells occupy roughly 40% of the Ag-experienced CD8+ T cell population in the spleen by days 7 to 9 postinfection (Table IGo and Fig. 4Go). Thus, the immunodominance of CD8+ T cells recognizing the MT389–397 epitope, predicted from the high frequency of MT389–397-specific CTL cloned lines established from polyoma-immune H-2k mice and similar LDA frequencies of virus- and MT389–397 epitope-specific CTL (11), is here directly confirmed by ex vivo Dk/MT389 tetramer staining of acutely infected mice.

To determine whether the frequency of MT389–397-specific CD8+ T cells identified physically by Dk/MT389 tetramers correlates with that of functionally competent Ag-specific CD8+ T cells, spleen cells taken from C3H/HeN mice on days 3 to 12 of acute polyoma infection were stimulated with MT389–397 peptide for 6 h in vitro in the presence of brefeldin A, and analyzed for intracellular IFN-{gamma} by flow cytometry. Fig. 5Go illustrates the close correspondence between the frequency of MT389–397 peptide-stimulated CD8+ T cells producing IFN-{gamma} and that of CD8+ T cells stained ex vivo by the Dk/MT389 tetramer through the course of acute polyoma virus infection (Fig. 4Go). Furthermore, Fig. 5Go shows that MT389–397 peptide-triggered IFN-{gamma} production coincides with loss of Dk/MT389 tetramer staining of CD8+ T cells, indicative of TCR down-modulation upon engagement by cognate MHC/peptide complexes (27, 28). In the absence of MT389–397, or the presence of gag88–96, no intracellular IFN-{gamma} production or Dk/MT389-TCR down-modulation was observed in CD8+ T cells at any time point postinfection ( (23) and data not shown); naive splenic CD8+ T cells cocultured with MT389–397 peptide were also negative for intracellular IFN-{gamma} (Fig. 5Go). Interestingly, while the frequency of splenic MT389–397-specific CD8+ T cells by day 12 postinfection is only 60% that of mice at day 7 postinfection (Table IGo), day 7 and day 12 postinfection spleen cells exhibit markedly different levels of MT389–397-specific cytotoxic activity (Fig. 1Go), despite the finding that MT389–397-specific CD8+ T cells at both time points are equally capable of producing IFN-{gamma} upon Ag stimulation (Fig. 5Go).



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  		FIGURE 5. Visualization of functional MT389–397 epitope-specific CD8+ T cell responses during primary virus infection by intracellular IFN-{gamma} staining. Spleen cells from uninfected or virus-infected C3H/HeN mice at the indicated days after virus inoculation were cultured for 6 h in the absence or presence of MT389–397 peptide (10-5 M), then stained for surface CD8 and Dk/MT389–397 tetramer, followed by staining for intracellular IFN-{gamma}. Plots are gated on CD8+ cells, and values represent the frequency of cells in the indicated quadrant. Plots shown are representative of three individual naive and polyoma virus-inoculated mice at each of the indicated postinfection time points.

 
To directly validate the correlation between the physical identification of Ag-specific CD8+ T cells by tetramer staining and their functional identification by Ag-stimulated IFN-{gamma} production in this viral system, we used an IFN-{gamma} ELISPOT assay to enumerate functionally competent CD8+ T cells within Dk/MT389 tetramer-positive and tetramer-negative splenic populations from acutely infected mice. Fig. 6GoA shows the conservative placement of gates used to cleanly sort three distinct populations of CD8+ splenic T cells from C3H/HeN mice at day 7 after polyoma infection, based on Dk/MT389 tetramer staining and expression of the CD11a activation marker: Dk/MT389 tetramer+CD11ahigh (gate R4); Dk/MT389 tetramer-CD11ahigh (gate R6); and Dk/MT389 tetramer-CD11alow (gate R5). As shown in Fig. 6GoB, ~70% of the Dk/MT389 tetramer+CD11ahigh population secreted IFN-{gamma} upon MT389–397 peptide stimulation, and no IFN-{gamma} release was detected from the Dk/MT389 tetramer-CD11alow population. Interestingly, ~15% of the Dk/MT389 tetramer-CD11ahigh population was specifically triggered by MT389–397 peptide to secrete IFN-{gamma}. Thus, although most of the tetramer+CD8+ T cells at the peak Ag-specific CD8+ T cell response to acute polyoma virus infection are competent to produce IFN-{gamma}, a significant fraction of the MT389–397-specific T cells among the activated CD8+ T cell population fails to bind the Dk/MT389 tetramer.

Vß repertoire of MT389–397-specific CD8+ T cells

A distinctive feature of the TCRs of MT389–397-specific CTL clones and lines from tumor-resistant (i.e., Mtv-7-) H-2k mice is their strong preferential usage of the Vß6 gene segment (10, 11). Approximately 90% of MT389–397-specific CTL clones isolated from polyoma-immune C3H/He mice express Vß6; of the two non-Vß6 MT389–397-specific CTL clones isolated to date, FACS and Vß cDNA sequencing show that they both express Vß8.1 ((11) and data not shown). We asked whether biased Vß6 expression was also found among MT389–397-specific CD8+ T cells in vivo. Vß repertoire analysis of spleen cells from C3H/HeN mice at day 7 of primary polyoma infection revealed that ~40% and ~20% of the Dk/MT389 tetramer+CD8+ T cells express Vß6 and Vß8.1/8.2, respectively (Fig. 7Go). Other than Vß6 and Vß8.1/8.2, no preferential staining of the MT389–397-specific T cells by the other indicated anti-Vß mAbs was observed. In addition, Fig. 7Go shows that the Vß profiles of the nonactivated (i.e., CD11alow) Dk/MT389 tetramer-CD8+ T cells in the same mice were nearly identical to that of splenic CD8+ T cells in uninfected C3H/HeN mice. This result suggests that polyoma virus infection does not dramatically skew the CD8+ T cell repertoire, but rather triggers selective expansion of Ag-specific CD8+ T cells.

Analysis of polyoma-specific memory CD8+ T cells

We next investigated the frequency of polyoma-specific CD8+ T cells in C3H/HeN mice after clearance of infectious virus. As shown in Fig. 8GoA, ~1 in 10 CD8+ T cells in the spleen by day 48 postinfection is MT389–397 specific, and nearly all of these express the CD11ahighCD44highCD62Llow activated T cell phenotype. It is also interesting to note that MT389–397-specific CD8+ T cells stably constitute ~40% of the activated splenic CD8+ T cell population after viral clearance (Table IGo), which may suggest a coordinate effector-to-memory transition of polyoma virus-specific CD8+ T cells. Consistent with observations in other viral systems that memory antiviral CD8+ T cells rapidly express effector activity upon Ag activation (19, 29, 30), Fig. 8GoB shows that a 6-h in vitro stimulation of day 48 postinfection spleen cells by MT389–397 peptide specifically stimulated intracellular IFN-{gamma} production by most of the Dk/MT389+CD8+ T cells, with concomitant loss of Dk/MT389 tetramer surface staining.



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  		FIGURE 8. Quantitation and analysis of functional activity of memory MT389–397-specific CD8+ T cells. Spleen cells from C3H/HeN mice 48 days postinfection were A, stained with allophycocyanin-conjugated Dk/MT389 tetramers, PE-conjugated anti-CD8{alpha}, and FITC-conjugated anti-CD11a, anti-CD62L, or anti-CD44, or B, cultured for 6 h in the absence or presence of MT389–397 peptide (10-5 M), then stained for surface CD8 and intracellular IFN-{gamma}. Plots are gated on CD8+ cells, and values indicate the percentage of cells in the indicated regions.

 
Ag-specific CD8+ T cells infiltrate a polyoma tumor

We previously showed that CD8+ T cells infiltrating a syngeneic polyoma tumor cell challenge into a virus-immune C57BR/cdJ (H-2k, Mtv-7-) mouse preferentially expressed Vß6 (10), a high-affinity Mtv-7 superantigen-binding Vß domain (31). We revisited this experimental model using Dk/MT389 tetramers to directly ask whether these tumor-infiltrating CD8+ T cells are polyoma specific. This model makes use of the 6215 cell line, which is derived from a polyoma virus-induced salivary gland tumor that developed in a sublethally irradiated C57BR/cdJ mouse (10). The poor immunogenicity of the 6215 cell line is indicated by the finding that as few as 1 x 105 6215 cells injected s.c. into naive C57BR/cdJ mice will give rise to localized tumors (A. Lukacher, unpublished observations); however, 6215 cells are recognized by polyoma virus-specific CD8+CTL (11). Because polyoma virus-immune C57BR/cdJ mice readily reject s.c. challenges of 1–2 x 106 6215 cells (A. Lukacher, unpublished observations), we injected a large inoculum (25 x 106 cells) of 6215 cells to permit outgrowth of palpable tumors in immune mice. TILs were recovered at day 9 postchallenge, at which time a well-encapsulated, 1-cm-diameter tumor had developed in the naive mouse and an irregular, ~0.3-cm-diameter tumor was present in the immune mouse. TILs from tumors in the naive and immune recipients were stained with Abs against CD8 and CD11a, and with Dk/MT389 tetramers. As shown in Fig. 9Go, although all of the CD8+ T cells infiltrating the tumor in the naive mouse were Ag experienced (i.e., CD11ahigh), less than 2% bound Dk/MT389 tetramers. In marked contrast, nearly a quarter of the tumor-infiltrating CD8+ T cells were MT389–397 epitope specific. Vß phenotyping revealed Vß6 expression by 36% of all CD8+ T cells and 50% of Dk/MT389 tetramer+CD8+ T cells infiltrating the tumor in the immune mouse. Similar to the frequencies of Dk/MT389 tetramer+CD8+ T cells in the spleens of day 48 postinfection C3H/HeN mice (Fig. 8Go), ~9% of the CD8+ T cells bound Dk/MT389 tetramers in the spleen of the day 57 postinfection C57BR/cdJ 6215 tumor recipient; comparable Dk/MT389 tetramer+CD8+ T cell numbers were present in a nonchallenged day 57 postinfection C57BR/cdJ mouse (data not shown). This efficient trafficking of MT389–397 epitope-specific CD8+ T cells to the polyoma tumor strongly suggests that antipolyoma CD8+ T cells generated in response to viral infection mediate surveillance against virus-transformed cells.



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  		FIGURE 9. Visualization of Ag-specific CD8+ T cells infiltrating a polyoma tumor. TILs were isolated from s.c. tumors developing in naive or polyoma virus-immune C57BR/cdJ mice 9 days after injection of 25 x 106 6215 polyoma tumor cells, and stained for expression of CD8 and CD11a, and with Dk/MT389 tetramers. Plots are gated on CD8+ T cells, and the values indicate the percentage of Dk/MT389 tetramer+ cells. For CD11a staining, the gate was set based on distinct high and low CD11a-expressing CD8+ T cells in the spleen of a C57BR/cdJ mouse at day 7 postinfection.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate that systemic infection by polyoma virus, an oncogenic murine DNA virus, elicits massive expansion of polyoma-specific CD8+ T cells. Nearly one-half of the Ag-experienced (i.e., CD11ahighCD44highCD62Llow) CD8+ T cell population in the spleen at the peak of the CD8+ T cell response during acute infection (days 7 to 9 postinfection) binds tetrameric complexes of Dk and the immunodominant MT389–397 viral epitope. By day 50 postinfection, when no infectious virus is detectable by plaque assay, the number of Ag-experienced Dk/MT389 tetramer+CD8+ T cells contracts to only about 16% of those present at days 7 to 9 postinfection. Single CD8+ T cell effector function analysis, based on peptide-stimulated intracellular IFN-{gamma} production, revealed that most of the MT389–397 epitope-specific CD8+ effector and memory T cells were functionally competent. In addition, MT389–397-specific cytotoxic activity was detected directly ex vivo in acutely infected mice during the maximum expansion phase of Dk/MT389 tetramer+CD8+ T cells. In support of their probable dual role in tumor immunosurveillance, MT389–397-specific CD8+ T cells, induced by viral infection, were found to efficiently infiltrate a polyoma tumor.

The kinetics and magnitude of the Ag-specific CD8+ T cell response to primary polyoma virus infection described in this study virtually parallel those seen in acute infection by moderately replicating strains of LCMV (19). Both viruses elicit large-scale expansions of antiviral CD8+ T cells within the first 8 days of systemic infection, with CD8+ T cells directed to immunodominant epitopes accounting for 30–50% of their respective activated CD8+ T cell population in the spleen. The contraction phase of the Ag-specific CD8+ T cell response to each virus takes place over a similar time frame, and coincides with clearance of infectious virus (days 8 to 15 postinfection). This tight correspondence in antiviral CD8+ T cell responses is striking considering that polyoma and LCMV, while both natural murine pathogens, are unrelated viruses having different cellular tropisms and lytic and noncytopathic fates, respectively, for productively infected cells. A remarkably similar pattern of dramatic expansion and contraction of Ag-specific CD8+ T cells is observed in the peripheral blood of individuals undergoing infectious mononucleosis (20), the clinical manifestation of primary EBV infection. In experimental murine influenza pneumonia, in which productive viral infection is primarily restricted to the respiratory epithelium, tetramer staining of bronchoalveolar lavage inflammatory cells has also shown large numbers of anti-influenza CD8+ T cells in the respiratory tract during primary infection (32). Taken together, these studies point toward a general pattern of transient large-scale expansion of Ag-specific CD8+ T cell responses to control the acute phase of productive viral infection.

Despite clearance of nearly 4 logs of infectious virus by day 50 postinfection, there was only a 6-fold decrease in the number of splenic Dk/MT389 tetramer+CD8+ T cells from the peak MT389–397 CD8+ T cell response. This contrasts with the anti-LCMV CD8+ T cell response, in which greater than 90% of the activated T cells apoptose after viral clearance (33). This rather high homeostatic anti-polyoma CD8+ T cell level may reflect chronic, low-level antigenic stimulation by cells nonproductively or productively infected by polyoma virus. Persistence of virus-derived CD8+ T cell epitopes is suggested by the finding that approximately one-third of Dk/MT389 tetramer+CD8+ memory T cells express high levels of CD69 (J. Moser, unpublished observations), a very early activation marker whose expression is transient and depends on continuous TCR activation (34, 35). polyoma virus infection is endemic in wild mice, in which it establishes a persistent silent infection; upon irradiation, however, polyoma-induced tumors may develop (36). Using a modified PCR approach and a novel highly sensitive bioluminescence RT-PCR methodology, we recently discovered long-lived persistence of polyoma viral DNA as well as mRNA for viral nonstructural and capsid proteins in multiple organs in immunocompetent adult-inoculated mice (24). In this connection, it is worth noting that human polyoma viruses persist as life-long asymptomatic infections in most individuals, yet can induce debilitating lytic lesions upon immunosuppression (37). Continuous CD8+CTL effector immunosurveillance and eradication of cells expressing polyoma proteins are most likely required to prevent emergence of polyoma-induced tumors, as highlighted by the importance of Ag-specific CD8+ T cells in controlling development of EBV-induced lymphomas from latently infected B cells (3, 38). Reduction in HIV burdens in asymptomatic individuals following administration of highly active antiretroviral therapy has recently been shown to cause a significant reduction in the frequency of HIV epitope-tetramer+ memory CD8+ T cells (39, 40). Thus, it is possible that chronic exposure to polyoma CD8+ T cell epitopes may contribute to the maintenance of the large polyoma-specific memory CD8+ T cell pool, which, in turn, is required to supply sufficient CTL effectors against polyoma-transformed cells and their nonproductively infected progenitors.

The efficient recruitment of Dk/MT389 tetramer+CD8+ T cells to the polyoma tumor challenge in a virus-primed recipient (Fig. 9Go) strongly supports the importance of anti-polyoma CD8+ T cells in immunosurveillance against polyoma-induced neoplasia. Because MT is essential not only for virion assembly (15), but also for induction and maintenance of cellular transformation (13, 14), MT epitope-specific CD8+ T cells would be expected to have a dual role in targeting both productively and nonproductively infected cells. Conversely, infected mice whose immunodominant anti-polyoma CD8+ T cells are directed against epitopes from viral capsid proteins, expressed by productively infected cells and transiently by some polyoma tumors (41), may be predisposed to polyoma virus tumorigenesis.

The absence of MT389–397-specific cytotoxicity but intact IFN-{gamma} production by polyoma-specific memory CD8+ T cells, and the presence of both activities at the peak of MT389–397-specific CD8+ T cell expansion, suggest a dissociation in these effector functions by CD8+ T cells after viral clearance. Although CTL activity is assayed at the bulk level in a conventional 51Cr release assay, loss of MT389–397-specific cytotoxicity in polyoma-immune mice is associated with only a 2-fold decrease in the frequency of Dk/MT389 tetramer+CD8+ T cells compared with that at days 7 to 9 postinfection, when ex vivo MT389–397-specific cytotoxic activity is readily detectable. Lack of ex vivo virus-specific cytotoxicity by CD8+ T cells from immune hosts, and their acquisition of antiviral cytotoxic activity upon in vitro restimulation with infected APCs, have been taken to indicate that virus-specific memory CD8+ T cells lack cytotoxic effector function (39). Other studies have shown that memory CD8+ T cells possess cytoplasmic perforin, and are capable of ex vivo Ag-specific killing (42, 43). Perhaps the inflammatory milieu created in response to acute viral infection may provide the appropriate microenvironment to induce cytotoxic effector function by antiviral CD8+ T cells (44, 45, 46, 47, 48).

Confirming our previous Vß phenotypic analyses of MT389–397-specific CD8+CTL clones and lines (11), we found that Dk/MT389 tetramer+CD8+ T cells from acutely infected mice preferentially express Vß6. Although 40% of these directly explanted Ag-specific CD8+ T cells expressed Vß6, roughly 20% also stained with a mAb directed against an epitope common to Vß8.1/8.2 (Fig. 6Go). By RT-PCR analysis using Vß-specific oligonucleotide primers (49), we identified Vß8.1 as the other preferentially expressed Vß gene segment by MT389–397-specific CD8+ T cells (data not shown). Biased usage of Vß8.1 in addition to Vß6 is particularly interesting given that the endogenous Mtv-7 superantigen, which confers susceptibility of H-2k mice to polyoma virus-induced tumors (10), reacts with both of these Vß domains (50). Consistent with this finding, Vß8.1 is the only Vß domain other than Vß6 expressed by MT389–397-specific CTL clones that have been isolated from virus-immune C3H/HeN mice (11).

A potential limitation in the sensitivity of MHC tetramer detection of Ag-specific T cells is the level of cell surface TCR expression. TCR engagement by cognate MHC/peptide ligand triggers TCR down-modulation (27). By combining Dk/MT389 tetramer staining with MT389–397 peptide-stimulated intracellular IFN-{gamma} production, we demonstrated specific peptide dose-dependent reduction in TCR expression of freshly explanted MT389–397-specific CD8+ T cells during a 6-h in vitro MT389–397 peptide stimulation (23). In the early phases of acute infection, repetitive encounters with large numbers of virus-infected cells may induce transient TCR down-modulation of antiviral CD8+ T cells. This possibility is supported by the finding that ~15% of Ag-experienced Dk/MT389 tetramer-CD8+ T cells sorted from day 7 postinfection mice secreted IFN-{gamma} upon exposure to MT389–397 peptide in ELISPOT assays lasting 36 h (Fig. 6Go), over which time Ag-specific TCRs could reexpress on the cell surface (51) and engage Dk/MT389–387 complexes. Alternatively, the failure of Dk/MT389 tetramers to stain Ag-specific CD8+ T cells could be attributed to their inability to bind to or their rapid dissociation from low-affinity TCRs (52, 53). In either case, the actual frequency of antiviral CD8+ T cells elicited during acute infection may be higher by 10–20% than directly visualized by class I MHC tetramer staining.

A dominant theme emerging from class I MHC tetramer analyses of CD8+ T cell responses in a number of viral systems, including polyoma virus, is the enormous expansion of Ag-specific CD8+ T cells induced by infection. Although the enhanced sensitivity afforded by class I MHC tetramers for detecting Ag-specific T cells has forced a reexamination of the contribution of bystander CD8+ T cells to the overall CD8+ T cell expansion in viral infection, a role for bystander CD8+ T cells in host protection against viral infection has not been excluded. Moreover, the fact that this Ag-specific CD8+ T cell expansion occurs in response to infection by unrelated viruses with distinct life cycles and cellular tropisms may implicate common innate inflammatory processes in fostering proliferation and differentiation of antiviral effector CD8+ T cells (54, 55, 56).


   Acknowledgments
 
We thank Joseph Blattman for helpful discussions, Joseph Miller for technical assistance in tetramer construction, and Robert Karaffa for technical assistance for flow cytometry.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants CA71971 (to A.E.L.) and AI42373 (to J.D.A.). Back

2 Address correspondence and reprint requests to Dr. Aron Lukacher, Department of Pathology, Woodruff Memorial Research Building, Room 7301, Emory University School of Medicine, 1639 Pierce Drive, Atlanta, GA 30322. E-mail address: Back

3 A.E.L., J.M.M., and J.D.A. contributed equally to this work. Back

4 Address correspondence and reprint requests to Dr. John Altman, Department of Microbiology and Immunology, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322. E-mail address: Back

5 Abbreviations used in this paper: HPV, human papillomavirus; ELISPOT, enzyme-linked immunospot; LCMV, lymphocytic choriomeningitis virus; LDA, limiting dilution analysis; MT, middle T protein; Mtv, mouse mammary tumor provirus; TIL, tumor-infiltrating lymphocyte. Back

Received for publication April 16, 1999. Accepted for publication June 29, 1999.


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 Results
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