Figure 1.
(A) Schematic representation of the breeding strategy used to produce G3 pedigrees. Details of the breeding procedure are described in the Materials and Methods (ENU mutagenesis and breeding section). (B) G3 pedigrees were screened for their susceptibility to HSV-1 infection. Mice were infected i.p. with 1×104 pfu of HSV-1 strain 17 and survival was monitored for two weeks post-infection; susceptible A/J and BALB/c control mice are represented by a blue line, resistant C57BL/6J and C57BL/10J control mice are represented by a green line, all G3 mice by a red line and P43 G3s derived from two G2 daughters (G2a and Gb) and one G1 male by brown and black lines. “n” indicates the number of infected mice for each group.
Figure 2.
Identification and characterization of an HSV-1 susceptible ENU mutant.
(A and B) Genome-wide linkage analysis was performed in 45 G3 mice from the P43 pedigree (11 susceptible, 34 resistant) using polymorphic markers distinguishing the B6 and B10 genetic backgrounds. (A) Genome-wide linkage quantitative trait loci (qtl) analysis revealed a significant peak on chromosome 1 (LOD score = 6.7, calculated using survival as the phenotype). The horizontal line represents the threshold of significant LOD scores genome-wide, at a p-value = 0.05. (B) Haplotype analysis of the distal portion of chromosome 1 are shown for all G3 mice from the P43 pedigree (A, dark gray shading = B6 homozygote; B, white = B10 homozygote; H, light gray = B6/B10 heterozygote; S = susceptible animal; R = resistant animal). (C) Genomic DNA from two G3 P43 pedigrees (one resistant and one susceptible) was amplified by polymerase chain reaction and the star indicates the position of the PtprcL3X mutation. DNA sequence electropherograms are shown for both resistant and susceptible mice and each of the four nucleotides were labeled with a unique fluorescent dye. Amino acid (aa) positions are displayed in bold; the black arrow indicates the direction of transcription. (D) PtprcL3X, PtprcL3X/+, Ptprc+/+ and Ptprcex9−/− mice were infected i.p. with 1×104 pfu of strain 17 and monitored for survival. “n” indicates the number of infected mice for each group. (E) Blood from Ptprc+/+ and PtprcL3X mice was collected and PBMCs were isolated and stained for B220 (n = 3). The expression of B220 was quantified by FACS (upper panel) and represented as a percentage of total cells (lower panel).
Table 1.
Exome sequencing analysis.
Figure 3.
The HSV-1 susceptibility associated with the PtprcL3X genotype is independent of the infection route and HSV-1 strain.
(A) PtprcL3X and heterozygous littermates were infected i.n. with 5×103 pfu of HSV-1 and monitored for survival. (B and C) PtprcL3X/+ and PtprcL3X mice were infected i.p. with 1×104 pfu of either McIntyre (B) or F strain (C) and monitored for survival. n≥6 for each group and data represent at least two independent experiments. (A, B and C).
Figure 4.
Expression of viral and inflammatory host genes in brain tissue collected from PtprcL3X/+ and PtprcL3X infected mice.
(A) PtprcL3X/+ and PtprcL3X mice were infected i.p. with 1×104 pfu of HSV-1. At the indicated day (D3, D6 and D10), total brains were harvested and mechanically homogenized for a plaque assay (n≥3). Viral titers are presented as pfu/gram of brain. The dotted line indicates the threshold of detection. (B and C) PtprcL3X/+ and PtprcL3X mice were infected i.p. with 1×104 pfu of HSV-1 (n = 9). Following infection, these mice were weighed two times daily. The brain stems of PtprcL3X mice that had lost at least 15% of their pre-infection weight were harvested. PtprcL3X/+ mice were sacrificed and their brain stems were collected at days 7, 9, and 11 p.i. (n = 3 for each time point). The expression of the ICP4 viral gene (B) and the indicated cellular genes (C, upper panels) was normalized to that of hprt. Data are presented as a fold increase relative to infected B6 samples. *, p-value (p)<0.05 and **, p<0.005. Correlations of expression levels were determined by comparing ICP4 and the indicated cellular genes (C, lower panels).
Figure 5.
(A, B, C and D) The spleen of PtprcL3X/+ and PtprcL3X mice was collected and specific immune cell populations were analyzed by FACS (n = 3). Isolated splenocytes were stained for CD3, DX5 and CD19 as well as IgD and IgM; their expressions were quantified by FACS and represented as a percentage of total cells. (E) Thymocytes isolated from PtprcL3X/+ and PtprcL3X mice were stained for CD4 and CD8 and their expression were represented as a percentage of total cells. (F) Thymocytes were gated on CD4−CD8− (double negative, DN); they were then analyzed based on their CD25 and CD44 expression profiles (DN1: CD25−CD44+; DN2: CD25+CD44+; DN3: CD25+CD44−; DN4: CD25−CD44−). CD25 and CD44 expression are presented as a percentage of total CD4−CD8− double negative cells and are averaged over three animals. *, p<0.05; **, p<0.005 and ***, p<0.0005. PtprcL3X/+ and PtprcL3X are shown in white and grey, respectively (A, B, C, D, E and F).
Figure 6.
CD4+, without CD8+ T cells, are incapable to protect mice against lethal HSV-1 infection.
(A) Schematic representation of the adoptive transfer strategy. Details of this procedure are described in the Materials and Methods (Cell transfer experiments and NK depletion section). (B) PtprcL3X/+ and PtprcL3X mice received 2×107 total splenocytes from either PtprcL3X/+ or PtprcL3X mice. After two hours, these mice were infected i.p. with 1×104 pfu of HSV-1 and their survival was monitored for two weeks (n = 6). (C) PtprcL3X/+ mice were treated with either anti-asialo GM1 antibody or PBS. After 24 hours, these mice were infected i.p. with 1×104 pfu of HSV-1 and their survival was monitored for two weeks (n = 6). The injection of either anti-asialo GM1 antibody or PBS was performed every three days until the experimental endpoint. (D) PtprcL3X mice received either 5×106 T cells or 1.2×107 B cells from PtprcL3X/+ mice. PtprcL3X/+ and PtprcL3X mice that received only PBS respectively correspond to the positive and negative controls. After two hours, all mice were infected i.p. with 1×104 pfu of HSV-1 and survival was monitored for two weeks (n≥6). (E) PtprcL3X mice received either 2.5×106 CD8+ or 2.5×106 CD4+ T cells from either PtprcL3X/+ or B6.H2-DbKb knock-out (only for the CD4+ T cells transfer) mice, whose H2-DbKb make them depleted in CD8+ T cells (CD8−/−). PtprcL3X/+ mice that received only PBS correspond to the positive control. After two hours, all mice were infected i.p. with 1×104 pfu of HSV-1 and survival was monitored for two weeks (n≥6). Data represent two independent experiments. (B–E).
Figure 7.
Important effect of IFN-γ production by the CD4+ T cells.
(A, B) PtprcL3X mice received 2.5×106 CD4+ T cells from PtprcL3X/+ mice. PtprcL3X/+ and PtprcL3X mice that received only PBS correspond to the positive and negative controls, respectively. After two hours and when indicated, mice were infected i.p. with 1×104 pfu of HSV-1 and sacrificed at days 2 and 7 p.i (n = 3 for each group). At the indicated day, peritoneal cells were collected, stained for CD45.2, CD3, CD4 and CD8, quantified by FACS and gated on CD45.2+CD3+ (A, left panels, only shown for PtprcL3X mice at day 7 p.i.); they were then analyzed based on their CD4 and CD8 expression profiles (A, right panel, only shown for PtprcL3X infected mice that received CD4+ T cells from PtprcL3X/+ mice, day 7 p.i.). Proportion of CD45.2+CD3+ T cells was also represented as a percentage of total peritoneal cells (B, left panel), while expression of CD4 and CD8 were represented as a percentage of total CD45.2+CD3+ T cells (B, right panel, left axis). Triangles and squares correspond to PtprcL3X/+ and PtprcL3X mice, respectively. The expression of the ICP4 viral gene (B, right panel, right axis) was determined in total peritoneal cells collected from PtprcL3X/+ mice at days 2 and 7 p.i. ICP4 expression was normalized to that of hprt. Data are presented as a fold increase relative to infected PtprcL3X/+ samples at day 7 p.i. (C) PtprcL3X/+ and IFN-γ−/− mice were infected i.p. with 1×104 pfu of HSV-1 and sacrificed at day 7 p.i. Then, peritoneal cells were collected and were gated on CD45.2+CD3+. CD4 and CD8 expression are presented as a percentage of total CD45.2+CD3+ cells and are averaged over three animals. (D) PtprcL3X mice received 2.5×106 CD4+ T cells from IFN-γ−/− mice. PtprcL3X/+ and PtprcL3X mice that received only PBS respectively correspond to the positive and negative controls. After two hours, all mice were infected i.p. with 1×104 pfu of HSV-1 and survival was monitored for two weeks (n≥5).