Fig 1.
PEP-619WW mice have improved LCMV-cl13 control over PEP-WT mice.
C57BL/6 mice with wild type (PEP-WT, black) or pro autoimmune allele (PEP-619WW, blue) Ptpn22 gene were infected with 1x10^6 PFU chronic viral strain LCMV-clone 13 (LCMV-cl13) (i.v). On indicated days mice were weighed (A), and bled for serum titer (B). Viral load was determined at early (3dpi, C) and late (41dpi, D) within target organs. Using flow cytometry presence of LCMV nucleoprotein (LCMV-NP) (clone VL-4) was determined in multiple myeloid cell subsets at day 3 (E) and day 8 (F). Representative flow plots (which is the median mouse data point in corresponding bar graph) shows LCMV-NP+ cells for different splenic myeloid subset at each time timepoint for mock infected (white bar, circle), infected PEP-WT (black bar, circle), and infected PEP-619WW mice (blue bar, triangles) Quantification for Frequency of LCMV-NP+ cells in corresponding bar graph to the right. Gating strategy for all myeloid subsets is: Lymphocytes> Single cell x2> autofluorescent- >Live> CD3- CD19-> Ly6G- CD11b+/-> (non-neutrophils) > NK1.1- (non NK). Monocytes: myeloid cells> F4/80+ CD11c-> Ly6C+ CD11b+. Marginal Zone Macrophage: myeloid cells> F4/80+ CD11c-> Ly6C- CD11b+> CD209b+. CD8α+ cDCs: myeloid cells> F4/80- >CD11c+ PDCA-1- CD8α+. CD8α- cDCs: myeloid cells> F4/80- >CD11c+ PDCA-1- CD8α-. pDCs: myeloid cells> F4/80- > CD11c+/- PDCA-1+. Weight loss studies pooled from 3 separate experiments: Starting group sizes were PEP-WT n = 15, PEP-619WW n = 19. Serum titer, tissue titer from representative experiment. Each dot indicates an individual mouse. Each dot represents an individual mouse and is from pooled experiments. SEM shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Two Way ANOVA with Sidak Post Hoc Analysis.
Fig 2.
PEP-619WW mice have transcriptionally distinct immune cell subsets from PEP-WT mice during LCMV-cl13 infection.
At 8dpi, pooled splenocytes (by genotype) from age matched, sex matched mice were sorted for live CD45+ cells and submitted for single cell RNA sequencing (scRNAseq) on the 10x genomic platform (diagram created with biorender.com)(A). Relative gene expression of Ptprc indicating immune cell in aggregated data set (both genotypes) (B). Relative gene expression to located immune cell clusters of myeloid Csfr1, neutrophil Ly6g, B cell Cd19, and T cell Cd3e (C). Using Loupe software, each population was reclustered to group like cell subsets among the larger cell type and the proportions compared between genotypes. The frequency of each cluster among total cell population is listed on the graph. Myeloid cell cluster, based on Csf1r expression (D), Neutrophil clusters, based on Ly6g expression (E), and B cell cluster, based on Cd19 expression (F). T cells were further broken down into CD4 T cells (expression of Cd3e>0, Mt2<10, Cd4>0) (G) and CD8 T cells (expression of Cd3e>0, Mt2<10, Cd8b1>0) (H). tSNE plots highlighting Cd4 (G) and Cd8b1 (H) as well as breakdown of cluster for each T cell type in -WT and -619WW cells. Corresponding quantification of proportion of either CD4 T cells or CD8 T cell subsets is next to tSNE plot.
Fig 3.
Top unique genes defining CD4 and CD8 T cell clusters from other clusters.
Using Loupe, we identified the distinguishing gene between the CD4 T cell cluster (A) or CD8 T cell cluster (D). We then calculated the z-score of these genes across the different cluster populations within each T cell subset (-1 black, to +1 brighter blue). Genes associated with each cluster are listed. Cluster ID on bottom. Average gene expression (Log Norm) between CD4 T cell clusters of select genes Cd44, Pdcd1, Bcl6, and Cxcr5 quantified and highlighted on tSNE visualization (B). Log fold change of selected interferon inducible genes, Isg20, Oasl2, Ifi44, Isg15, and Ifi27l2a (C). Average gene expression of selected genes to further identify CD8 T cell clusters Cd69 and Gzmb quantified for each CD8 T cell cluster and highlighted on tSNE visualization (E).
Table 1.
Top Pathways upregulated within each CD8 T cell cluster of LCMV-13 infected mice at 8dpi.
Gene expression data exported from Loupe for each CD8 T cell cluster, as identified in Fig 2, was uploaded to iPathway analysis to determine pathways which were significantly associated with those gene sets. Table shows the top 3 pathways for each indicated cluster, the count of differentially expressed (DE) genes that were expressed in each cluster associated with the pathways, count all genes in that pathway, and the p-value associating that pathway with the gene expression data set.
Fig 4.
PEP-619WW mice have enhanced CD4 T cell activation during LCMV-cl13 infection.
8dpi splenocytes were isolated and examined for CD4 T cell subsets. Th1 cell (Lymphocytes> single cells x2> live> CD3+ Cd19-> CD4+ CD8α-> Foxp3- CD44+> Tbet+) representative flow plot (A), Th1 frequency of total CD4 (B), and gMFI of Tbet in CD44+ CD4 T cells (C) in mock infected (white bar, circles), infected PEP-WT (black bar, circles), and infected PEP-619WW (blue bar, circles). Frequency of Tregs amongst total CD4 T cells (Lymphocytes> single cells x2> Live> CD19- CD3+> CD4+ CD8α-> Foxp3+ CD25+) (D). Frequency TFH cells (Lymphocytes> single cells x2> Live> CD19- CD3+> CD4+ CD8α-> Foxp3- CD25+> CXCR5+ PD-1+) (E). CD4 T cells were also assessed for IFNγ and IL-2 production after peptide (GP61-80) stimulation (F,G,H). IFNγ+ cells representative flow plot (F). IFNγ+ of CD44+ CD4+ T cells (Lymphocytes> single cells x2> live> CD3+ Cd19-> CD4+ CD8α-> CD44+ >IFNγ+) quantification (G). Expression of IFNγ in CD44+ CD4+ T cells, representative histogram (H) and quantification (H). Frequency of IL-2+ IFNγ+ CD44hi CD4+ T cells (Lymphocytes> single cells x2> live> CD3+ Cd19-> CD4+ CD8α-> CD44+ >IFNγ+ IL-2+). Representative experiment or sample shown for A-F, H-I. Pooled data in G. Each dot represents an individual mouse. Experiments were repeated at least 3 times. SEM shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. One Way ANOVA with Tukey Post Hoc Analysis.
Fig 5.
PEP-619WW and PEP-WT mice have comparable function against the LCMV immunodominant epitope GP33-41.
8dpi splenocytes from mock infected (white), PEP-WT (black), and PEP-619WW (blue) were isolated and examined for virus specific CD8 T cell subset, assess CD8 T cell activation and function against GP33-41. Frequency of activated CD8 T cells (Lymphocytes> single cells x2> live> CD3+ Cd19-> CD4- CD8α+> CD44+) (A). Expression of degranulation marker CD017a on activated CD8 T cells (CD44+ CD8 T cells) (representative histogram [left] and gMFI quantification [right] (B). Expression of perforin in activated CD8 T cells (CD44+ CD8 T cells) (representative histogram [left] and gMFI quantification [right] (C). Frequency of Db:GP33 tetramer+ CD8 T cells (Lymphocytes> single cells x2> live> CD3+ Cd19-> CD4- CD8α+> Db:GP33+) (D), and absolute number of Db:GP33 CD8 T cells (E). Whole splenocytes were stimulated with GP33-41 peptide. Representative flow plots showing IFNγ production in response to GP33 peptide (F) and quantification (G). Data in G is pooled from multiple experiments. All other data is from a representative experiment. SEM shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. One Way ANOVA with Tukey Post Hoc Analysis. Experiments repeated 3 independent times. Individual data points represent a single animal.
Fig 6.
Ptpn22 pro-autoimmune allele promotes more immunostimulatory-like CD8α- cDCs during LCMV-cl13 infection.
8dpi spleens were harvested from mock infected (white, circle), infected PEP-WT (black, circles), and infected PEP-619WW (blue, triangles) mice. Representative flow plot (A) and quantification (B) of PD-L1+ among CD11c+ cells (Lymphocytes> Single cell x2> Live> CD3- CD19-> Ly6G- CD11b+/->CD11c+ F4/80-). PD-L1 expression on CD11c+ cells representative histogram (C) and quantification (D). Absolute number of CD8α- cDCs (Lymphocytes> Single cell x2> Live> CD3- CD19-> Ly6G- CD11b+/->CD11c+ F4/80->CD8α- PDCA-1->MHC-II (I-Ab)+) (E), CD8α+ cDCs (Lymphocytes> Single cell x2> Live> CD3- CD19-> Ly6G- CD11b+/->CD11c+ F4/80->CD8α+ PDCA-1->MHC-II (I-Ab)+) (F), and pDCs ((Lymphocytes> Single cell x2> Live> CD3- CD19-> Ly6G- CD11b+/->CD11c+ F4/80->CD8α+/- PDCA-1+> Ly6C+ B220+) (G). Expression (gMFI) of PD-L1 on CD8α- cDCs (H), CD8α+ cDCs (I) and pDC (J). Expression (gMFI) of CD86 on CD8α- cDCs (K), CD8α+ cDCs (L), and pDCs (M). Representative experiment or sample shown. Each dot represents an individual mouse. Experiments were repeated at least 3 times. SEM shown. ns = not significant *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. One Way ANOVA with Tukey Post Hoc Analysis.
Fig 7.
Ptpn22 alternative allele enhances anti-viral T cell activation through T cell intrinsic and T cell extrinsic mechanisms.
CD45.2 C57BL/6 mice (PEP-WT or PEP-619WW) received 5x10^4 CD45.1 SMARTA CD4 T cells prior to being infected with LCMV-cl13 (diagram created with biorender.com) (A). Frequency of CD45.1 SMARTA T cells (Lymphocytes> single cells x2> Live> CD3+ CD19-> CD4+ CD8α-> CD44+> CD45.1+ CD45.2-) for each transfer condition, representative flow plot (B) and quantified (C). Following peptide stimulation with GP61-80 peptide, IFNγ and IL-2 production of SMARTA cells were measured (D-G). Representative flow plot showing IFNγ+ CD45.1+ CD4 T cells (D), quantification IFNγ+ CD45.1 Cd4 T cells (E), expression (gMFI) of IFNγ in CD45.1 SMARTA cells with representative histogram (F), frequency of IFNγ+ IL-2+ CD45.1 CD4 T cells (G). PEP-WT or PEP-619WW FLT3L-differentiated DCs were pulsed with GP61-80 peptide then incubated with PEP-WT or PEP-619WW SMARTA CD4 T cells for 3 days. Following incubation, SMARTA T cell function was determined through IFNγ production by intracellular cytokine staining and measured on a flow cytometer (H). C, E, G, and H from pooled data. F is a representative experiment. Experiments were repeated at least 3 times. SEM shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Two Way ANOVA with Sidak post hoc analysis.