Fig 1.
Expansion of Late-rising M09 CD4 T cells coincides with the resolution of MCMV persistence in the salivary gland.
(A) Replication of MCMV Smith in the SG determined at various days post infection (dpi) by plaque assay. (B) Overlap of MCMV replication levels (A) (right Y axis) and frequency of MCMV epitope-specific CD4 T cells over time. (C) M09 (red), M25 (blue) and M142 (black) MHC-II tetramer-binding CD4 T cells in the SG from day 0 to day 200 post infection, in percentage (left) and absolute number (right). (D) Representative flow cytometry plot of M09, M25 and M142 CD4 T cells stained with tetramers labeled with 2 different fluorophores (PE and APC) in SG at 0 and 40 D.P.I. All time points represent 4–8 mice and experiments were repeated a minimum of two times. Statistical significance determined by Two-way Anova test is shown between M09 and M25 or M142. **p < 0.01, ****p < 0.0001.
Fig 2.
The M09 133–147 epitope plays a critical role in resolving MCMV persistence in the SG.
(A) MCMVSmith and MCMVK181 replication levels at 55 dpi in the SG. (B) Amino acid sequence of the M09133-147 epitope in the Smith and K181 wild-type strains, and mutations generated in the Smith M09K and K181 M09S ‘swap mutants’. (C) Wild-type (Fig 2A) and epitope-swap mutant replication levels at 55 dpi in the SG. (D) Percentage of tetramer reactive M09 cells in the SG at 48 days of infection. Dashed line represents background of tetramer binding in uninfected mice. Statistical significance was assessed by Mann Whitney test, **p < 0.01, ****p < 0.0001.
Fig 3.
Late-rising CD4 T cells are better cytokine producers during viral persistence.
(A) Representative flow cytometry plot for IFNγ and IL-2 expression by SG resident M09 and M25 reactive cells following ex vivo peptide restimulation. (B) Absolute number of IFNγ (B), IL-2 (C) and TNFα (D) producing splenic CD4 T cells from day 8 to 200 of infection. (E) Percentage of CD4 memory (CD44+) splenocyte expressing IFNγ after peptide stimulation by M09, M25 or M142 peptide at day 8 (left) and day 40 (right) post infection. (F) Euler representation of the poly-functionality of M09, M25 and M142 reactive CD4 T cells from the spleen at day 8 (upper row) and day 40 (lower row) after MCMV infection. Kinetic differences were determined by Two-way Anova test. ****p < 0.0001. Group comparisons were assessed by Mann Whitney test, *p< 0.1**p< 0.01, ***p<0.001, ****p<0.0001.
Fig 4.
Cell surface and transcriptomic phenotypes of M09 and M25 CD4 T cells.
(A) Cell surface phenotypes of conventional (M25, blue) and late-rising (M09, red) tetramer binding CD4 T cells from the SG at day 8, 15 and 40. Total MCMV-specific (CD11ahiCD49d+) (dashed) and naïve (CD11aloCD49d-) (gray filled histogram) CD4 T cells are also shown. (B) M09-CD43lo and M25-CD43hi cells sorted from spleens of day 40 infected mice were subjected to RNA-seq. As IL-10 mRNA levels were found to be low in M09 cells, IPA analyses of core IL-10 inducers was performed followed by gene set enrichment analyses (GSEA). The heatmap shows the IL-10 regulating genes with differences of P<0.2. (C) Bulk RNA-Seq enrichment of expression along genomic coordinates for genes listed in Fig 4B using UCSC genome tracks. Arrows indicate gene orientation and lines within arrows are exons. RPKM, reads per kilobase per million mapped. (D) Absolute number of IL-10GFP+ M09 and M25 SG-resident cells at 40 days of infection. (E) IL-10 expression by CD43hi/lo M09 tet+ CD4 T cells at 40 D.P.I. (F) Splenic total MCMV-specific (CD11ahiCD49d+) CD4 T cells were sorted for CD43Hi and CD43Lo expression at day 45 of infection, 5e4 cells were adoptively transferred into naïve mice, subsequently challenged with MCMV and SG replication was determined at day 15. Statistical significance was assessed by Mann Whitney test, *p<0.1.
Fig 5.
Vaccine induced M09 cells provide dramatic protection.
(A) Mice vaccinated by a single immunization (CFA) with M09, M25 or M142 peptides alone were analyzed for their numbers of tetramer positive cells in the spleen prior to MCMV challenge. (B) Mice vaccinated with M25+M142+M139 peptides (3conv) or 3conv+M09 were challenged with MCMVSmith, and SG replication levels were determined at day 15. (C) Mice were vaccinated with M25+M142, M25+M142+M09 or two CD8 T cell epitopes (M38+IE3), and liver replication levels were determined 8 days post MCMV challenge. (D) Blocking anti-IL-10R antibody was injected at day 0 and 4 (for d8) or at day 0 and 7 (for d15) in vaccinated mice, and SG replication levels were determined at day 8 (upper panel) or 15 (lower panel) after MCMV challenge. Statistical significance was assessed by Mann Whitney test, ***p< 0.001, **p< 0.01, *p<0.1.