Reducing persistent polyomavirus infection increases functionality of virus-specific memory CD8 T cells

Abstract

Mouse polyomavirus (MuPyV) causes a smoldering persistent infection in immunocompetent mice. To lower MuPyV infection in acutely and persistently infected mice, and study the impact of a temporal reduction in viral loads on the memory CD8 T cell response, we created a recombinant MuPyV in which a loxP sequence was inserted into the A2 strain genome upstream of the early promoter and another loxP sequence was inserted in cis into the intron shared by all three T antigens. Using mice transgenic for tamoxifen-inducible Cre recombinase, we demonstrated that reduction in MuPyV load during persistent infection was associated with differentiation of virus-specific CD8 T cells having a superior recall response. Evidence presented here supports the concept that reduction in viral load during persistent infection can promote differentiation of protective virus-specific memory CD8 T cells in patients at risk for diseases caused by human polyomaviruses.

Keywords: CD8 T cells; Memory; Mouse; Persistent viral infection; Polyomavirus.

Fig. 1. Generation of mutant A2-Flx carrying double loxP sequence insertions

(A) The schematic design of loxP sequence insertions at BglI and STIN sites within the MuPyV genome. (B) Plaque formation at day 6 p.i. by A2, A2 mutants with one loxP sequence insertion at the BglI site (A2_BglI), STIN (A2_STIN), or both sites (A2-Flx) as shown in (A) on A31 cells. (C) One-step growth curve assays for A2 and A2-Flx in NMuMG cells infected at MOI 1. Viral titers were measured by plaque assay at indicated days p.i.

Fig. 2. Viral protein expression byA2-Flx-infected cells

NMuMG cells were infected (MOI 1) with the indicated parental A2 virus or single or double loxP-containing recombinant A2 viruses, as shown in Fig. 1. At 24 h, lysates were prepared and analyzed by Western blotting with a pan-T mAb, polyclonal antibodies for VP2 and VP3, or a mAb for β-actin as a loading control. M, mock infection.

Fig. 3. Insertion of loxP sequence interferes with splicing of transcripts for MT and ST of A2-Flx

(A) Donor (GT) and acceptor (AG) splicing sites of the primary transcript for generating LT, MT, and ST mRNAs in parental A2 virus are indicated in bold. The nucleotide sequence of the intronic region shared by all three T antigen genes for parental A2, and the inserted loxP sequence is highlighted in red at the same region in A2-Flx. (B) Sequencing of transcripts for LT and ST from A2-Flx infected cells. Transcripts for LT and ST isolated from infected NMuMG cells were reverse transcribed, and cDNA amplified with LT_For and LT_Rev primers (Table 1) as shown in (A). Two bands amplified from LT and ST cDNA were separated on agarose gels. P, positive control plasmid containing the complete MuPyV genome; M, mock-infected cells. From the sequencing electropherogram for the LT band, arrow indicates the ligation site between two exons as shown in (A), with the shaded area indicating 5’end of the second exon for LT. In the electropherogram for the ST band, the shaded area indicates the intact intron that contains the loxP sequence; arrows indicate splicing donor and acceptor sites shown in (A).

Fig. 4. Comparison of the MuPyV-specific CD8 T cell response in mice infected with A2 and A2-Flx viruses

(A) Viral DNA was measured by qPCR in spleens at indicated days p.i. after A2 or A2-Flx infection. Values are mean ± SEM of at least 3 mice/group. Dashed line indicates the limit of detection. *p < 0.05. (B) Frequency of splenic D^bLT359 tetramer⁺ CD8⁺ cells at indicated day p.i. Data are the mean ± SEM of at least three mice per group and are representative of two experiments. *p < 0.05. (C) Frequency of LT359-specific CD8 T cells with the MPEC (KLRG1^loCD127^hi), DPEC (KLRG1^hiCD127^hi), SLEC (KLRG1^hiCD127^lo), and EEC (KLRG1^loCD127^lo) phenotype at day 104 p.i. (D) Frequency of T-bet⁺ and Eomes⁺ D^bLT359 tetramer⁺ CD8 T cells. (E) Ratio of splenic LT359 peptide-stimulated CD8 T cells producing intracellular IFN-γ⁺ to D^bLT359 tetramer⁺ CD8 T cells at day 104 p.i. (F) Ratio of intracellular TNF-α⁺ of IFN-γ⁺ CD8 T cells after LT359 peptide stimulation, at day 104 p.i. Values indicate mean ± SEM from 3–4 mice/group of two experiments. p > 0.05 indicates that a given difference is not significant.

Fig. 5. LoxP sites in A2-Flx are recognized by Cre-recombinase in vitro

(A) NMuMG cells were co-infected with A2 or A2-Flx virus and a recombinant adenovirus expressing Cre recombinase. Two days later, viral genomic DNA was isolated and amplified with primers as shown in A. (B) PCR products were separated by agarose gel and bands of the expected length are shown.

Fig. 6. Reduction of viral loads in A2-Flx infected Rosa-Cre mice correlates with lower magnitude of virus-specific CD8 T cell response

(A) Rosa-Cre mice were injected with Tamoxifen (TM) (3mg/day) or corn oil vehicle control (Ctl) daily for 2 days before mice were infected with A2-Flx, and then treated daily for another 5 days. At days 7 and 10 p.i., mice were bled. (B) At day 5 p.i., viral genome copies in brain, spleen, and kidney were determined by qPCR. Values indicate mean + SEM from 5 mice from each group. Dash line indicates the limit of detection. * p <0.05. (C) Gated region of contour plots show splenic D^bLT359 tetramer⁺ CD8⁺ cells at the indicated days p.i., with values indicating frequency of D^bLT359 tetramer⁺ CD8⁺ cells of total CD8⁺ cells. Each panel is representative of 3 mice/group of two experiments.

Figure 7. Reducing viral load during persistent infection increases recall response of CD8 T cells

(A) Rosa-Cre mice infected with A2-Flx virus were injected at day 34 p.i. with Tamoxifen or vehicle control daily for 6 days. Tamoxifen-treated and control mice were euthanized at day 60 p.i. to quantify splenic viral DNA. Numbers of D^bLT359 tetramer⁺ CD8 T cells in blood were determined before rVSV-LT359 infection at day 60 p.i. (pre-challenge) and 5 days after infection with rVSV-LT359 (post-challenge) (B) Numbers of splenic viral genomes by qPCR assay. Values indicate mean + SEM. Dash line indicates the limit of detection. (C) Numbers of LT359⁺ CD8 T cells/100 µl blood were determined by flow cytometry pre- and post-challenge infection with rVSV-LT359. ** p < 0.005. (D) pre- vs post-challenge fold expansion of LT359⁺ CD8 T cells of data in (C); values indicate mean + SEM of 4–5 mice/group, of three experiments. **, significant difference.