Expression of mouse mammary tumor virus superantigen mRNA in the thymus correlates with kinetics of self-reactive T-cell loss

Abstract

Mouse mammary tumor virus (MMTV) encodes a superantigen (Sag) that is expressed at the surface of antigen-presenting cells in conjunction with major histocompatibility complex (MHC) type II molecules. The Sag-MHC complex is recognized by entire subsets of T cells, leading to cytokine release and amplification of infected B and T cells that carry milk-borne MMTV to the mammary gland. Expression of Sag proteins from endogenous MMTV proviruses carried in the mouse germ line usually results in the deletion of self-reactive T cells during negative selection in the thymus and the elimination of T cells required for infection by specific milk-borne MMTVs. However, other endogenous MMTVs are unable to eliminate Sag-reactive T cells in newborn mice and cause partial loss of reactive T cells in adults. To investigate the kinetics of Sag-reactive T-cell deletion, backcross mice that contain single or multiple MMTVs were screened by a novel PCR assay designed to distinguish among highly related MMTV strains. Mice that contained Mtv-17 alone showed slow kinetics of reactive T-cell loss that involved the CD4(+), but not the CD8(+), subset. Deletion of CD4(+) or CD8(+) T cells reactive with Mtv-17 Sag was not detected in thymocytes. Slow kinetics of peripheral T-cell deletion by Mtv-17 Sag also was accompanied by failure to detect Mtv-17 sag-specific mRNA in the thymus, despite detectable expression in other tissues, such as spleen. Together, these data suggest that Mtv-17 Sag causes peripheral, rather than intrathymic, deletion of T cells. Interestingly, the Mtv-8 provirus caused partial deletion of CD4(+)Vbeta12(+) cells in the thymus, but other T-cell subsets appeared to be deleted only in the periphery. Our data have important implications for the level of antigen expression required for elimination of self-reactive T cells. Moreover, these experiments suggest that mice expressing endogenous MMTVs that lead to slow kinetics of T-cell deletion will be susceptible to infection by milk-borne MMTVs with the same Sag specificity.

FIG. 1

Scheme for generation of Mtv single-positive mice. Each animal was analyzed in a series of three PCRs containing primers that were specific for the MMTV proviruses in C58/J or BALB/cJ parents. Animals negative for all proviruses were checked for the integrity of DNA samples by PCRs with GAPDH primers.

FIG. 2

PCR assay for specific endogenous Mtv proviruses. BALB/cJ (lanes 3 to 5) or C58/J (lanes 7 to 9) DNA was used in reactions containing primers specific for the indicated MMTV proviruses. The primers used to detect Mtv-3 and Mtv-6 were the same. Reaction mixtures were analyzed on a 2% agarose gel and stained with ethidium bromide. Lanes 2 and 6 show PCR mixtures lacking template DNA. Molecular size markers are shown in lane 1.

FIG. 3

Kinetics of T-cell deletion in N₁ mice from the C58/J × PERA cross. Percentages of CD4⁺Vβ3⁺ (A and E), CD4⁺Vβ7⁺ (B and F), CD4⁺Vβ11⁺ (C and G), and CD4⁺Vβ12⁺ (D and H) T cells were determined. All values were compared to the percentage of T cells in Mtv-negative mice from the same cross. Each point represents the average of values from 3 to 12 mice, except that two animals with Mtv-7 alone were tested at 3.5 months. No N₁ animals with Mtv-3, -7, and -17 were tested at 7 months. Standard deviations are represented by vertical bars spanning each point.

FIG. 4

Kinetics of T-cell deletion in N₁ mice from the BALB/cJ × PERA cross. Percentages of CD4⁺Vβ3⁺ (A and E), CD4⁺Vβ5⁺ (B and F), CD4⁺Vβ7⁺ (C and G), and CD4⁺Vβ12⁺ (D and H) T cells were determined. Each point represents the average of values from 3 to 11 mice, except that two animals with Mtv-8 alone were tested at 3.5 months; two animals with both Mtv-6 and Mtv-8 were tested at 3.5 and 7 months; two animals with Mtv-6, -8, and -9 were tested at 7 months; and one animal with Mtv-8 and -9 was tested at 3.5 months. All values were compared to the percentage of T cells in Mtv-negative mice from the same cross. Standard deviations are given by vertical bars spanning each point.

FIG. 5

Sag-specific T-cell deletion in CD4⁺ and CD8⁺ thymocytes from mice containing Mtv-3, Mtv-7, and Mtv-17 only. Percentages of CD4⁺ (A) and CD8⁺ (B) thymocytes in mice containing single MMTV proviruses were compared to those in Mtv-negative mice derived from the same cross. Most animals tested (three to six mice from each strain) were 4 weeks old.

FIG. 6

Sag-specific T-cell deletion in CD4⁺ and CD8⁺ thymocytes from mice containing Mtv-6, Mtv-8, and Mtv-9 only. Percentages of CD4⁺ (A) and CD8⁺ (B) thymocytes in mice containing single MMTV proviruses were compared to those in Mtv-negative mice derived from the same cross. Animals tested (three mice from each strain) were 4 to 8 weeks old.

FIG. 7

RNase protection assays for the expression of endogenous MMTVs. Total RNA (40 μg) was hybridized to a riboprobe containing the Sau3A fragment of the Mtv-17 LTR (24), including the polymorphic region of the sag gene. An actin probe (Ambion, Austin, Tex.) was used as a control for the quality of the RNA. Hybridizations contained RNA from the following sources: tissues from an MMTV-negative backcross mouse (lanes 1 and 2), tissues from a backcross animal containing Mtv-17 only (lanes 3 to 5), yeast RNA (lane 6), tissues from a male (C58/J × PERA)F₁ animal (lanes 7 to 10), and tissues from a female (C58/J × PERA)F₁ animal (lanes 11 to 14). Abbreviations: SG, salivary gland; MG, mammary gland. The faster-migrating bands in these lanes are due to partial protection of the riboprobe by Mtv-3- and Mtv-7-specific RNA transcripts. The Mtv-3 and Mtv-7 transcripts cannot be distinguished in this assay.

FIG. 8

Detection of sag-specific spliced transcripts from the LTR and envelope promoters. RT-PCRs were performed with RNA extracted from the salivary gland (SG) (lanes 1, 4, and 7), spleen (lanes 2, 5, and 8), and thymus (lanes 3, 6, 9, and 10). RNA samples were derived from Mtv-negative animals (lanes 1 to 3), Mtv-3-only animals (lanes 4 to 6), Mtv-17-only animals (lanes 7 to 9), or Mtv-7-only animals (lane 10). RNAs were derived from pools of organs from 6-week-old mice (three to five animals). Each cDNA was used in three separate PCRs containing primers for the LTR promoters (A), the envelope promoter (B), and GAPDH (C). The PCRs in panel A were performed for 20 cycles, and the PCRs in panel B were performed for 35 cycles. RT-PCR mixtures (one-third of the reaction mixture) were analyzed on 2% NuSieve agarose gels. After electrophoresis, DNA was transferred to nylon membranes, hybridized to an MMTV LTR probe (A and B), and subjected overnight to autoradiography. Longer exposures of the autoradiogram in panel A show Mtv-7 expression in the thymus. Mtv-17 expression in the thymus was not detected after 35 PCR cycles with primers for the LTR promoters or long exposures of the autoradiograms. GAPDH reaction mixtures were stained with ethidium bromide after electrophoresis as a control for the integrity of cDNAs.