Grafting subdominant SM9 at GRA6_II C-terminus enhances its presentation, overturns the dominance hierarchy and provides efficient cyst control.

<p>(A) Schematic description of the GRA6_II-SM9 chimeric constructs. For GRA6_II-SM9_Cter, SM9 was introduced at the C-terminus, downstream of HF10. For GRA6_II-SM9_internal, SM9 was introduced internally within GRA6_II after residue 153, before the putative transmembrane domain. In both cases, SM9 was preceded by a leucine to mimic the endogenous flanking sequence of HF10. (B) Western blot analysis of GRA6 (upper panels) and SAG1 (loading control, lower panels) of the indicated parasite clones. (C,D) SM9 and HF10 presentation by BMDMs infected for 24 h with the indicated parasites, assessed with the BDSM9Z (C) and CTgEZ.4 (D) hybridomas, respectively. (E,F,G) <i>Ex vivo</i> evaluation of the CD8 responses and the parasite load 3 weeks post-infection with the indicated parasites. (E) IFN-γ intracellular staining of spleen cells restimulated with the SM9 peptide (left panel) or the HF10 peptide (right panel). (F) Tetramer staining of brain cells with SM9:L^d (left panel) and HF10:L^d (right panel). Bars represent the mean +/− SEM. Data pooled from 2 independent experiments. (G) Parasite burden in the brains of mice infected with control CEP (white bar), CEP+GRA6_II-SM9_internal (black bar) or CEP+GRA6_II-SM9_Cter (hatched bar), evaluated microscopically by enumerating the cysts. Histograms represent the mean +/− SEM of 14 mice per group, pooled from 3 independent experiments. *: <i>P</i><0.05 ; **: <i>P</i><0.005.</p

Presence of the GRA6_II-derived HF10 peptide correlates with better parasite control during acute and chronic <i>T. gondii</i> infection.

<p>(A,B) BALB/c mice were infected intraperitoneally with 10⁵ tachyzoites of the indicated parasite strains. Parasite clearance was followed from day 7 to 13 by bioluminescence imaging on the ventral side. (A) Raw images of luminescence in pseudocolor scale. (B) Quantification of total flux (photons/s) over time. Dots show the mean + s.d. with 3 mice per group. The differences between CEP+GRA6_I and CEP+GRA6_II did not achieve statistical significance. Data representative of 2 independent experiments with at least 3 mice per condition. (C,D) B10.D2 mice were infected intraperitoneally with 10⁵ tachyzoites of the indicated parasite strains. (C) Parasite load in the spleen 3 weeks post-infection, as measured by the number of cells harboring a GFP⁺ parasite. Bars show the mean +/− SEM for at least 4 mice per group. The dotted line shows the background in uninfected mice. Data pooled from 2 independent experiments. (D) Parasite burden in the brains of B10.D2 mice 4 weeks post-infection, evaluated microscopically by enumerating the cysts. Histograms represent the mean +/− SEM of at least 5 mice per group. Data pooled from 3 independent experiments. ns: <i>P</i>>0.05 ; *: <i>P</i><0.05 ; **: <i>P</i><0.005.</p

Type III transgenic <i>T. gondii</i> to study parasite immunogenicity.

<p>(A) Schematics of GRA6 protein from type I/III and type II <i>T. gondii</i>. C-terminal amino acid sequence is shown. (B) Surface labeling of peptide-loaded L^d analyzed by flow cytometry on TAP-deficient RMA-S.L^d cells left unpulsed or pulsed with increasing concentrations of HF10, HY10 or a control D^d-restricted AI9 peptide. Shown is the mean fluorescence intensity (MFI) in arbitrary units (a.u.). Data representative of 2 independent assays. (C) Western blot analysis of GRA6 (upper panel) and SAG1 (lower panel) in type II Prugnaud (Pru), control CEP (a resistant HXGPRT+ clone which did not integrate the transgene), CEP+GRA6_II and CEP+GRA6_I clones. Data representative of 4 independent experiments. (D) <i>Ex vivo</i> IFN-γ intracellular staining of splenocytes from B10.D2 mice 3 weeks post-infection with CEP+GRA6_II (upper plots) or CEP+GRA6_I (lower plots), restimulated <i>in vitro</i> with HY10 or HF10. Numbers represent the percentage of IFN-γ⁺ out of CD8⁺ cells. Plots show one representative mouse out of 3 per group.</p

Immunodominance, but not immunodomination, of the GRA6_II-derived HF10 peptide during chronic stage.

<p>For all panels, B10.D2 mice were infected with the indicated parasite strains and analyzed 4 weeks post-infection. (A) Spleen cells stained <i>ex vivo</i> with the L^d DimerX loaded with the GRA6_II-derived HF10, ROP7-derived IF9 or GRA4-derived SM9 peptide. Each symbol represents one mouse, horizontal lines represent the mean +/− SEM. Data pooled from 3 independent experiments. (B) <i>Ex vivo</i> IFN-γ intracellular staining of spleen cells restimulated with the HF10, IF9 or SM9 peptide. Bars represent the mean +/− SEM. Data pooled from 3 independent experiments. (C) <i>Ex vivo</i> L^d DimerX staining of brain-infiltrating leukocytes. Bars represent the mean +/− SEM. Data pooled from 2 independent experiments. (D) <i>Ex vivo</i> IFN-γ intracellular staining of spleen cells restimulated with J774 macrophages infected <i>in vitro</i> with the indicated parasites. Upper panel: mice infected with CEP+GRA6_I, lower panel: mice infected with CEP+GRA6_II. Bars represent the mean +/− SEM. Data representative from 2 independent experiments. ns: <i>P</i>>0.05 ; *: <i>P</i><0.05 ; **: <i>P</i><0.005.</p

Immunodominance of GRA6_II-derived HF10 cannot be explained by peptide affinity for L^d or naïve T cell frequency.

<p>(A) Flow cytometry surface labeling (MFI) of L^d on TAP-deficient RMA-S.L^d cells left unpulsed or pulsed with increasing concentrations of the indicated L^d-restricted peptides or a control D^d-restricted AI9 peptide. Data representative of 3 independent assays. (B,C) Estimation of frequencies of naïve <i>T. gondii</i>-specific CD8α⁺ T cell populations in naive B10.D2 mice. (B) Representative flow plots of T cells isolated from spleen and lymph nodes of uninfected mice. Shown are CD62L and tetramer (SM9:L^d, IF9:L^d or HF10:L^d) stainings after gating on live dump⁻ (dump = B220, F4/80, MHC II) CD3⁺ CD8α⁺ T cells. One million events were collected for each sample. (C) Summary of total tetramer⁺ T cells per mouse. Each dot represents one mouse. Pooled data from 3 independent experiments. Mean +/− SEM are represented by horizontal lines and values are noted on the graph. *: <i>P</i><0.05 ; **: <i>P</i><0.005.</p

Modeling Host Genetic Regulation of Influenza Pathogenesis in the Collaborative Cross

<div><p>Genetic variation contributes to host responses and outcomes following infection by influenza A virus or other viral infections. Yet narrow windows of disease symptoms and confounding environmental factors have made it difficult to identify polymorphic genes that contribute to differential disease outcomes in human populations. Therefore, to control for these confounding environmental variables in a system that models the levels of genetic diversity found in outbred populations such as humans, we used incipient lines of the highly genetically diverse Collaborative Cross (CC) recombinant inbred (RI) panel (the pre-CC population) to study how genetic variation impacts influenza associated disease across a genetically diverse population. A wide range of variation in influenza disease related phenotypes including virus replication, virus-induced inflammation, and weight loss was observed. Many of the disease associated phenotypes were correlated, with viral replication and virus-induced inflammation being predictors of virus-induced weight loss. Despite these correlations, pre-CC mice with unique and novel disease phenotype combinations were observed. We also identified sets of transcripts (modules) that were correlated with aspects of disease. In order to identify how host genetic polymorphisms contribute to the observed variation in disease, we conducted quantitative trait loci (QTL) mapping. We identified several QTL contributing to specific aspects of the host response including virus-induced weight loss, titer, pulmonary edema, neutrophil recruitment to the airways, and transcriptional expression. Existing whole-genome sequence data was applied to identify high priority candidate genes within QTL regions. A key host response QTL was located at the site of the known anti-influenza <i>Mx1</i> gene. We sequenced the coding regions of <i>Mx1</i> in the eight CC founder strains, and identified a novel <i>Mx1</i> allele that showed reduced ability to inhibit viral replication, while maintaining protection from weight loss.</p> </div

QTL identified in the pre-CC population.

<p>QTL identified in the pre-CC population.</p

Candidate genes within QTL regions.

<p>For <i>HrI2</i>, (A) refers to genes with a private A/J SNP, (C) refers to genes with a private 129S1/SvImJ SNP. Unmarked genes had both A/J and 129S1/SvImJ SNPs.</p

A novel <i>Mx1</i> allele differentially impacts host response to influenza.

<p>The founder strain alleles at <i>Mx1</i> were grouped based on their phenotypic effects into three functionally distinct classes corresponding to <i>domesticus</i> (<i>dom</i>: A/J, C57BL6/J, 129s1/SvImJ, NOD/HiLtJ and WSB/EiJ), <i>castaneus</i> (<i>cast</i>: CAST/EiJ) and <i>musculus</i> (<i>mus</i>: PWK/PhJ and NZO/ShILtJ). Points shown are individual pre-CC animals with these haplotypes, mean bars are shown for each class. These functionally distinct classes were separable based upon differences in (A) D4 weight and (B) Log titer, with the heterozygous classes showing intermediate phenotypes. Across the pre-CC population, homozygous <i>dom</i> animals had severe weight loss and high titers. Homozygous <i>mus</i> animals showed little weight loss and low titers. Homozygous <i>cast</i> animals showed little weight loss, but had intermediate viral titers. Brackets between groups represent significant differences (* = p<0.05, ** = p<0.003) based on Tukey's HSD. We found no difference by qPCR (C) in expression of <i>Mx1</i> at 2 days post-infection following influenza infection in a strain from each of these three functional classes. By sequencing <i>Mx1</i> (D), we were able to identify five haplotypes across the eight founder strains (Haplotype 1 = A/J, C57BL/6J, 129S1/SvImJ, NOD/HiLtJ; Haplotype 2 = WSB/EiJ; Haplotype 3 = PWK/PhJ; Haplotype 4 = NZO/HiLtJ; Haplotype 5 = CAST/EiJ). Arrows indicate locations of polymorphisms, with small arrows indicating non-coding changes, and large arrows indicating coding changes. Colors correspond to the founder strains having those polymorphisms (brown = multiple strains possess mutation). Grey exons indicate those not transcribed due to either deletion and frameshift, or insertion and early stop codon.</p

Genetic variation at <i>HrI3</i> contributes to variation in pulmonary edema in an independent set of Collaborative Cross lines.

<p>Following the identification of <i>HrI3</i>, we infected animals from fully inbred Collaborative Cross lines, where each line was homozygous for a single founder allele at <i>HrI3</i>. (A) We found a significant effect of genotype at <i>HrI3</i> on the extent and severity of pulmonary edema at four days post infection. Mild (B) and Severe (C) pulmonary edema can be seen at 200× magnification in animals from this experiment. Pulmonary edema was scored on the basis of evidence of transudates accumulating in the alveolar spaces (denoted by star marks in panel C).</p