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

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 × 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, γ-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)) × 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-2D^k tetramers

H-2D^k/peptide tetramers were prepared as previously described (23). Briefly, Escherichia coli strain BL21 (DE3) was transformed with a pET23-D^k-BSP plasmid, and expression of D^k was induced with isopropyl β-d-thiogalactoside. Human β₂-microglobulin was expressed in the same cell line using the pHN1-β₂m 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 D^k/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 × 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 CD8α 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 Vβ repertoire analysis (see Fig. 7⇓), anti-Vβ mAbs were either FITC or PE conjugated, and were all purchased from PharMingen (San Diego, CA).

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-CD8α, FITC-conjugated anti-CD11a, and allophycocyanin-conjugated D^k/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 × 10⁷/ml, and immediately acquired and sorted on a FACSVantage (Becton Dickinson).

Intracellular IFN-γ staining

Erythrocyte-lysed spleen cells were cultured for 6 h in 96-well flat-bottom microtiter plates (Costar) at 1 × 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 CD8α mAb (Caltag) and allophycocyanin-conjugated D^k/MT389 tetramers. After washing, cells were permeabilized and stained for intracellular IFN-γ with FITC-conjugated rat anti-mouse IFN-γ 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-γ-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-γ (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-CD8α, FITC-conjugated anti-CD11a, and allophycocyanin-conjugated D^k/MT389 tetramers, and sorted by flow cytometry into three distinct populations, as shown in Fig. 6⇓A. Each sorted population was serially diluted 3-fold in the wells to each of which were added 5 × 10⁵ γ-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-γ (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).

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 × 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-CD8α, FITC-conjugated CD11a, and allophycocyanin-conjugated D^k/MT389 tetramers; and analyzed by flow cytometry.

Ex vivo polyoma-specific cytotoxic activity

We previously identified the immunodominant epitope for polyoma virus-specific CD8⁺CTL in H-2^k mice as a D^k-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-2^k) 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. 1⇓, 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).

• Download figure
• Open in new tab
• Download powerpoint

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. 2⇓, high viral titers were present in the spleens of C3H/HeN mice as early as 3 days after s.c. inoculation with 2 × 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.

• Download figure
• Open in new tab
• Download powerpoint

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 D^k tetrameric complexes containing the MT389–397 peptide. The specificity of these D^k/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, D^k-restricted viral epitope (Fig. 3⇓A) (11). In addition, D^k/MT389 tetramer binding did not interfere with binding of anti-Vβ mAbs to 8-1 (Vβ6⁺) or 16-5 (Vβ8.1⁺); neither Ab stained the Vβ2⁺ 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 D^k/MT389 tetramer⁺ cells in 1:10 mixtures of clones 8-1 or 16-5 with 15-5 (Fig. 3⇓A). The D^k/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. 2⇑). As shown in Fig. 3⇓B, ∼20% of the CD8⁺ splenic T cells bound the D^k/MT389 tetramer. The specificity for D^k/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 D^k tetramer containing an endogenous retroviral CTL epitope, gag 88–96 (26), to stain spleen cells from a C3H/HeN mouse at day 7 postinfection.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

Specificity of staining with D^k/MT389 tetramers. A, MT389–397-specific CD8⁺CTL clones 8-1 (Vβ6⁺) and 16-5 (Vβ8.1⁺), alone or mixed in a 1:10 ratio with the non-MT389–397-specific CD8⁺CTL clone15-5 (Vβ2⁺), were stained with allophycocyanin-conjugated D^k/MT389 tetramers and PE-conjugated anti-Vβ6 or anti-Vβ8.1/8.2 mAbs. B, Spleen cells from a naive and day 7 postinfection C3H/HeN mouse were stained with allophycocyanin-conjugated D^k/MT389 or D^k/gag88–96 tetramers and PE-conjugated anti-CD8α. Plots are gated on CD8⁺ cells, and values represent the frequency of cells in the indicated regions.

We then used the D^k/MT389 tetramers to directly visualize MT389–397 epitope-specific CD8⁺ T cells through the course of acute polyoma virus infection. As shown in Fig. 4⇓ and summarized in Table I⇓, no splenic D^k/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. 2⇑). 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.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Quantitation of D^k/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 D^k/MT389 tetramers, PE-conjugated anti-CD8α, and FITC-conjugated anti-CD62L, anti-CD11a, or anti-CD44, and assayed by flow cytometry.

At each time point during acute infection, virtually all D^k/MT389⁺CD8⁺ T cells express the CD11a^highCD44^highCD62L^low phenotype indicative of prior TCR activation (Fig. 4⇑). Taken as a percentage of CD11a^highCD8⁺ 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 I⇑ and Fig. 4⇑). 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-2^k mice and similar LDA frequencies of virus- and MT389–397 epitope-specific CTL (11), is here directly confirmed by ex vivo D^k/MT389 tetramer staining of acutely infected mice.

To determine whether the frequency of MT389–397-specific CD8⁺ T cells identified physically by D^k/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-γ by flow cytometry. Fig. 5⇓ illustrates the close correspondence between the frequency of MT389–397 peptide-stimulated CD8⁺ T cells producing IFN-γ and that of CD8⁺ T cells stained ex vivo by the D^k/MT389 tetramer through the course of acute polyoma virus infection (Fig. 4⇑). Furthermore, Fig. 5⇓ shows that MT389–397 peptide-triggered IFN-γ production coincides with loss of D^k/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-γ production or D^k/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-γ (Fig. 5⇓). 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 I⇑), day 7 and day 12 postinfection spleen cells exhibit markedly different levels of MT389–397-specific cytotoxic activity (Fig. 1⇑), despite the finding that MT389–397-specific CD8⁺ T cells at both time points are equally capable of producing IFN-γ upon Ag stimulation (Fig. 5⇓).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

Visualization of functional MT389–397 epitope-specific CD8⁺ T cell responses during primary virus infection by intracellular IFN-γ 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 D^k/MT389–397 tetramer, followed by staining for intracellular IFN-γ. 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-γ production in this viral system, we used an IFN-γ ELISPOT assay to enumerate functionally competent CD8⁺ T cells within D^k/MT389 tetramer-positive and tetramer-negative splenic populations from acutely infected mice. Fig. 6⇓A 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 D^k/MT389 tetramer staining and expression of the CD11a activation marker: D^k/MT389 tetramer⁺CD11a^high (gate R4); D^k/MT389 tetramer⁻CD11a^high (gate R6); and D^k/MT389 tetramer⁻CD11a^low (gate R5). As shown in Fig. 6⇓B, ∼70% of the D^k/MT389 tetramer⁺CD11a^high population secreted IFN-γ upon MT389–397 peptide stimulation, and no IFN-γ release was detected from the D^k/MT389 tetramer⁻CD11a^low population. Interestingly, ∼15% of the D^k/MT389 tetramer⁻CD11a^high population was specifically triggered by MT389–397 peptide to secrete IFN-γ. 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-γ, a significant fraction of the MT389–397-specific T cells among the activated CD8⁺ T cell population fails to bind the D^k/MT389 tetramer.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 6.

Analysis of Ag-specific IFN-γ secretion by D^k/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 D^k/MT389 tetramers, and PE-conjugated anti-CD8α 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-γ 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-γ 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.

Vβ 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-2^k mice is their strong preferential usage of the Vβ6 gene segment (10, 11). Approximately 90% of MT389–397-specific CTL clones isolated from polyoma-immune C3H/He mice express Vβ6; of the two non-Vβ6 MT389–397-specific CTL clones isolated to date, FACS and Vβ cDNA sequencing show that they both express Vβ8.1 ((11) and data not shown). We asked whether biased Vβ6 expression was also found among MT389–397-specific CD8⁺ T cells in vivo. Vβ repertoire analysis of spleen cells from C3H/HeN mice at day 7 of primary polyoma infection revealed that ∼40% and ∼20% of the D^k/MT389 tetramer⁺CD8⁺ T cells express Vβ6 and Vβ8.1/8.2, respectively (Fig. 7⇓). Other than Vβ6 and Vβ8.1/8.2, no preferential staining of the MT389–397-specific T cells by the other indicated anti-Vβ mAbs was observed. In addition, Fig. 7⇓ shows that the Vβ profiles of the nonactivated (i.e., CD11a^low) D^k/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.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 7.

Vβ repertoire analysis of D^k/MT389 tetramer⁺CD8⁺ T cells from polyoma-infected mice. Spleen cells from C3H/HeN mice 7 days postinfection were stained with allophycocyanin-conjugated D^k/MT389 tetramers, TRI-COLOR-conjugated anti-CD8α, FITC- or PE-conjugated anti-CD11a, and FITC- or PE-conjugated anti-Vβ Abs. Values represent the mean percentage ± SEM of D^k/MT389 tetramer⁺ or D^k/MT389 tetramer⁻CD11a^lowCD8⁺ T cells expressing the indicated Vβ of three infected mice. Spleen cells from three naive C3H/HeN mice were also stained with anti-CD8α and anti-Vβ Abs, and the mean percentage of gated CD8⁺ T cells ± SEM expressing the indicated anti-Vβ Abs is shown.

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. 8⇓A, ∼1 in 10 CD8⁺ T cells in the spleen by day 48 postinfection is MT389–397 specific, and nearly all of these express the CD11a^highCD44^highCD62L^low 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 I⇑), 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. 8⇓B shows that a 6-h in vitro stimulation of day 48 postinfection spleen cells by MT389–397 peptide specifically stimulated intracellular IFN-γ production by most of the D^k/MT389⁺CD8⁺ T cells, with concomitant loss of D^k/MT389 tetramer surface staining.

• Download figure
• Open in new tab
• Download powerpoint

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 D^k/MT389 tetramers, PE-conjugated anti-CD8α, 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-γ. 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-2^k, Mtv-7⁻) mouse preferentially expressed Vβ6 (10), a high-affinity Mtv-7 superantigen-binding Vβ domain (31). We revisited this experimental model using D^k/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 × 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 × 10⁶ 6215 cells (A. Lukacher, unpublished observations), we injected a large inoculum (25 × 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 D^k/MT389 tetramers. As shown in Fig. 9⇓, although all of the CD8⁺ T cells infiltrating the tumor in the naive mouse were Ag experienced (i.e., CD11a^high), less than 2% bound D^k/MT389 tetramers. In marked contrast, nearly a quarter of the tumor-infiltrating CD8⁺ T cells were MT389–397 epitope specific. Vβ phenotyping revealed Vβ6 expression by 36% of all CD8⁺ T cells and 50% of D^k/MT389 tetramer⁺CD8⁺ T cells infiltrating the tumor in the immune mouse. Similar to the frequencies of D^k/MT389 tetramer⁺CD8⁺ T cells in the spleens of day 48 postinfection C3H/HeN mice (Fig. 8⇑), ∼9% of the CD8⁺ T cells bound D^k/MT389 tetramers in the spleen of the day 57 postinfection C57BR/cdJ 6215 tumor recipient; comparable D^k/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.

• Download figure
• Open in new tab
• Download powerpoint

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 × 10⁶ 6215 polyoma tumor cells, and stained for expression of CD8 and CD11a, and with D^k/MT389 tetramers. Plots are gated on CD8⁺ T cells, and the values indicate the percentage of D^k/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.