Cytotoxic T-lymphocyte epitope immunodominance in the control of choroid plexus tumors in simian virus 40 large T antigen transgenic mice

Abstract

The simian virus 40 (SV40) large tumor antigen (Tag) is a virus-encoded oncoprotein which is the target of a strong cytotoxic T-lymphocyte (CTL) response. Three immunodominant H-2(b)-restricted epitopes, designated epitopes I, II/III, and IV, have been defined. We investigated whether induction of CTLs directed against these Tag epitopes might control Tag-induced tumors in SV11(+) (H-2(b)) mice. SV11(+) mice develop spontaneous tumors of the choroid plexus due to expression of SV40 Tag as a transgene. We demonstrate that SV11(+) mice are functionally tolerant to the immunodominant Tag CTL epitopes. CTLs specific for the H-2Kb-restricted Tag epitope IV were induced in SV11(+) mice following adoptive transfer with unprimed C57BL/6 spleen cells and immunization with recombinant vaccinia viruses expressing either full-length Tag or the H-2Kb-restricted epitope IV as a minigene. In addition, irradiation of SV11(+) mice prior to adoptive transfer with unprimed C57BL/6 spleen cells led to the priming of epitope IV-specific CTLs by the endogenous Tag. Induction of epitope IV-specific CTLs in SV11(+) mice by either approach correlated with increased life span and control of the choroid plexus tumor progression, indicating that CTLs specific for the immunodominant Tag epitope IV control the progressive growth of spontaneous tumors induced by this DNA virus oncogene in transgenic mice.

FIG. 1

Progression of choroid plexus tumors in unmanipulated SV11⁺ mice. Serial sections from paraffin-embedded brains of 100-day-old SV11⁻ mice (A and B) and SV11⁺ mice which were 40 (C and D), 60 (E and F), 80 (G and H), and 100 (I and J) days of age were stained with H&E (A, C, E, G, and I) or anti-Tag monoclonal antibody plus peroxidase antiperoxidase (B, D, F, H, and J) to detect Tag⁺ cells (brown). Arrows indicate the location of Tag⁺ cells. Magnifications are indicated.

FIG. 2

SV11⁺ mice were tolerant to Tag CTL epitopes. SV11⁺ (A to D), SV11⁻ (E to H), and SV11⁺ mice reconstituted with 5 × 10⁷ naive B6 spleen cells (I to L) were immunized i.v. with 10⁷ PFU of rVV-941T (A, E, and I), rVV-ES I (B, F, and J), rVV-ES II/III (C, G, and K), or rVV-ES IV (D, H, and L). After 3 weeks, spleen cells were restimulated in vitro with gamma-irradiated B6/WT-19 cells as described in Materials and Methods. Responder cells were tested on day 6 for their ability to lyse ⁵¹Cr-labeled RMA cells pulsed with 1 μM concentrations of the indicated peptides or the Tag-transformed cell lines B6/WT-19 (full-length Tag) and B6/K-1,4,5 (CTL epitope loss Tag) in a 5-h assay. The control peptide for all panels was D^bN5, except that HSV gB498-505 was used in panels D and H and HSV RR1 822-829 was used in panels I and L. Insets show response of spleen cells from the same mice against B6/K-1,4,5 target cells infected with VV-WR or mock infected following 6 days of in vitro restimulation with VV-WR-infected B6 spleen cells as described in Materials and Methods.

FIG. 3

The life span of reconstituted SV11⁺ mice is prolonged by immunization with rVV-ES IV or rVV-941T. SV11⁺ mice remained unmanipulated (long-dashed line) or were reconstituted at 45 days of age with 5 × 10⁷ naive B6 spleen cells and immunized 2 days later with rVV-ES IV (solid line), rVV-941T (dotted line), or rVV-ES-gB498-505 (short-dashed line). Survival was monitored and plotted as percentage of surviving animals versus age. The horizontal dotted line indicates 50% survival. The inset shows survival times and P values as determined for mean survival ages compared to unimmunized animals, with a P value of <0.05 considered significant. The numbers of animals per group are indicated in the figure.

FIG. 4

Reconstituted SV11⁺ mice develop epitope IV-specific memory CTLs following active immunization. The scheme for determining the effect of epitope IV-specific CTL response on choroid plexus tumor development in SV11⁺ mice is shown in the upper portion of the figure. SV11⁺ mice were reconstituted with 5 × 10⁷ naive B6 spleen cells i.v. at 45 days of age followed 2 days later by no immunization or immunization with rVV-941T, rVV-ES IV, or rVV-ES-gB498-505. At 100 days of age, mice were sacrificed and spleen cells were restimulated in vitro with gamma-irradiated B6/WT-19 (A to C) or B6/350gB (D) cells. After 6 days, responder cells were tested for lysis of RMA target cells which had been pulsed with a 1 μM concentration of the indicated peptide in a 5-h ⁵¹Cr release assay. Each curve within a given panel represents the response of an individual mouse to RMA cells pulsed with the indicated target or control peptide. The number of mice in each group is indicated.

FIG. 5

Reconstituted SV11⁺ mice immunized with rVV-ES IV and rVV-941T have reduced choroid plexus tumor burdens by 100 days of age. Brains were harvested from SV11⁺ and SV11⁻ mice at 100 days of age, sectioned, and stained with H&E as described in Materials and Methods. All sections are shown at ×40 magnification unless indicated otherwise. (A) Grade IV tumor from an SV11⁺ mouse which received naive B6 spleen cells at 45 days of age but remained unimmunized. (B) Grade IV tumor from an SV11⁺ mouse which received naive B6 spleen cells at 45 days of age followed by immunization with rVV-ES-gB498-505. (C and D) Grade I and II choroid plexus tumors, respectively, from two different SV11⁺ mice reconstituted with naive B6 spleen cells at 45 days of age and immunized with rVV-941T. (E and F) Grade II choroid plexus tumors from two representative SV11⁺ mice reconstituted with naive B6 spleen cells at 45 days of age and immunized with rVV-ES IV. Insets in panels D and F show immunohistochemical staining for Tag of parallel paraffin-embedded sections. The dashed boxes indicate the relative location of Tag staining with the corresponding H&E-stained sections. The magnification of the insets is ×100. Arrows indicate the location of small tumors.

FIG. 6

Epitope-specific CTLs are primed by endogenous Tag in irradiated SV11⁺ mice. SV11⁺ mice were irradiated at 450 rads at 40 days of age and then reconstituted with 5 × 10⁷ B6 (A to C) or SV11⁺ (D) spleen cells. Some mice remained unimmunized (C and D) or were immunized 2 days later with rVV-941T (A) or rVV-ES-IV (B) After 3 to 5 weeks, spleen cells were restimulated in vitro with B6/WT-19 cells and tested on day 6 in a ⁵¹Cr release assay against RMA target cells pulsed with 1 μM concentrations of the indicated peptides or the Tag-transformed cell lines B6/WT-19 (full-length Tag) and B6/K-1,4,5 (CTL epitope loss Tag). Data represent the response of individual mice from representative groups of three to five mice.

FIG. 7

Irradiated SV11⁺ mice given adoptive transfers with naive B6 spleen cells have significantly increased life spans. SV11⁺ mice were irradiated at 450 rads at 40 days of age and reconstituted the following day with 5 × 10⁷ naive B6 (dotted line) or SV11⁺ (solid line) spleen cells. Some SV11⁺ mice remained unmanipulated (dashed line). Survival was monitored and plotted as percentage of surviving animals versus age. The inset shows survival times and statistics for each group. P values were determined for mean survival ages compared to unmanipulated SV11⁺ mice, with a value of <0.01 considered significant. The number of animals per group are indicated in the figure. The dashed horizontal line indicates 50% survival.

FIG. 8

Irradiated SV11⁺ mice given adoptive transfers with naive B6 spleen cells have reduced tumor burdens by 100 days of age. SV11⁺ mice were irradiated at 450 rads at 40 days of age and reconstituted the following day with 5 × 10⁷ naive B6 spleen cells. Brains were harvested at 100 days of age, fixed, paraffin embedded, sectioned, and stained with H&E (A) or by immunohistochemistry for Tag (B). The arrow indicates the location of Tag-positive cells (brown), and the box in panel A indicates the relative location of the tissue stained in panel B. Magnifications are indicated.