High incidence of AFN in PV+LCMV double immune mice following PV re-challenge.

<p>(A) Naïve, PV-immune, and (PV+LCMV WT) double immune mice were re-challenged with PV, sacrificed 3 days PI, and the severity of AFN in the visceral fat pads was assessed. (*) indicates <i>p</i>&lt;.05 in frequency of AFN using the Kruskai-Wallis test (one-way ANOVA non-parametric). (B), (C), and (D) represent experiments performed using the LCMV clone 13 system and its naturally derived V207A mutant. (B) Domination of NP205-specific CD8 T cells in PV+Clone 13 LCMV WT double immune mice. PBL were collected from double-immune mice, before the final challenge with PV, and stimulated with peptides <i>ex vivo</i> in a standard ICS assay. These are representative frequencies of the IFNγ positive CD8α+ T cells from 4 independent experiments using 5 mice per group. (C) Incidence of AFN after PV challenge. Naïve, (PV+Clone 13 LCMV WT), and (PV+Clone 13 LCMV NP-V207A) double immune mice re-challenged with PV were sacrificed 4 days PI, and the severity of AFN in the visceral fat pads was assessed. Compilation of data from 4 independent experiments. (*) and (***) indicate <i>p</i>&lt;.05 and <i>p</i>&lt;.0001, respectively. (D) Domination of cross-reactive NP205-specific CD8 T cells isolated from the visceral fat pad of (PV+Clone 13 LCMV WT) double immune mice following PV re-challenge. Standard ICS and FACs analyses were performed. Numbers are representative frequencies of IFNγ+, CD8α+ T cells from two similar experiments.</p

Lack of MHC stabilization by LCMV NP L212A.

<p>MHC stabilization assays for LCMV WT (NP205) and mutant (L212A) peptides. RMA-S cells were incubated with different concentrations of peptides and stained against H2K^b to detect its stabilization on the cell surface.</p

Analysis and comparisons of NP205-K^b structures.

<p>(A), (B) and (C): superposition of LCMV (pink) with PV (blue) structures, with the peptide in stick representation and the MHC H2K^b in grey cartoon. The tip of the α2-helix is colored accordingly to the peptides bound by the H2K^b molecules, representing the section from residue 150 to 156 of the α2-helix (B &amp; C). (C) shows, with a different orientation, the residues that change conformation between the peptide-MHC complexes, namely Serine-99, Glutamine-114, Leucine-156, Glutamate-152 as well as Glycine-151, for which the Cα atom is represented by a sphere. (D) superposition of the LCMV (pink) with LCMV-V207A (green) structures, with peptide in stick representation and MHC in grey cartoon. (E) and (F): comparison of LCMV (pink) and LCMV-V207A (green) mutant peptide, both bound to the H2K^b molecule (grey cartoon) in the same orientation. The P3 residues are colored in yellow. Arginine-155, Glutamate-152 and Alanine-151 of the H2K^b molecule are represented as grey stick to show the different interaction of their side chains between both structures. The red dashed lines represent the hydrogen bond made between the residues.</p

Analysis of immune response and immunopathology with the LCMV-Armstrong rL212A anchoring amino acid mutant.

<p>(A) Diminished cross-reactive NP205 CD8 T cell responses in the (PV+rV207A) double immune mice. PV-immune mice were immunized with either rWT or rV207A variant Armstrong strain LCMV. After six weeks, PBL were collected and stimulated with LCMV-specific CD8 T cell peptides. The data represent average frequencies of the IFNγ-positive, CD8α+ T cells. This is representative of 3 experiments, with n = 5/group. (B) Complete elimination of cross-reactive NP205 CD8 T cell responses in (PV+rL212A) double immune mice. PV-immune mice were immunized with either rWT or rL212A LCMV Armstrong. After six weeks, PBL were collected and stimulated with LCMV-specific CD8 T cell peptides. Data represent average frequencies of IFNγ-positive, CD8α+ T cells. This is representative of two experiments, with n = 5/group. (C) Prevention of AFN by the rL212A anchoring mutant. Naïve, PV-immune, (PV+rWT) and (PV+rL212A) double immune groups were re-challenged with PV. Four days later fat pads were harvested and AFN scores evaluated. This is a compilation of two similar experiments. (***) indicates <i>p</i>&lt;.0001. (D) Photographs of abdominal fat pads and tissue histology sections. Abdominal fat pads were harvested, photographed (top), and then fixed in 10% neutral buffered formaldehyde and embedded in paraffin at the UMMS histology core facility. Thin tissue sections (5 µm) were stained with hemotoxylin and eosin (bottom). The digital photographs of the sections were taken using a Nikon Eclipse E300 microscope system.</p

Correlation of frequencies of CD8 T cells in double-immune mice with pathology after PV re-challenge.

<p>Linear regression analyses comparing the frequencies of antigen-specific CD8 T cells in the (PV+WT LCMV) double immune mice with the severity of AFN following PV re-challenge. These represent data compiled from four independent experiments using LCMV Clone 13 virus. (A) LCMV NP205-specific CD8 T cell response. (B) PV NP38-specific CD8 T cell response. (C) Ratio of LCMV NP205 to PV NP38. (D) LCMV GP33 and the ratio of GP33/PV NP38.</p

Abrogation of heterologous immunity by point mutation in NP205.

<p>Immunologically naïve control or LCMV-immune mice were challenged with 2×10⁷ PFU of PV and tested for PV PFU in spleens or abdominal fat pads 4 days post-infection. Exp. 1 is representative of three experiments using WT LCMV Clone 13 and its naturally derived V207A mutant. Exp. 2 is representative of two experiments using rescued recombinant LCMV Armstrong and its rV207A mutant. Exp. 3 is representative of two experiments using rescued recombinant LCMV Armstrong and its rL212A mutant. n = 5 per group. All comparisons of WT LCMV-immune to naïve mice are <i>p</i>&lt;0.05 as indicated by one-way ANOVA analysis and <i>p</i>≤0.02 by Students t-test. There was no statistically significant difference in PFU in PV-challenged naïve mice vs. challenged NP205 mutant LCMV-immune mice.</p

Increased immunopathology and reduced survival of <i>Zbtb32</i>^<i>-/-</i> mice in response to chronic LCMV infection.

<p>WT or <i>Zbtb32</i>^<i>-/-</i> mice were infected intravenously with high dose LCMV-clone 13. (a) The percent survival (top) and percentage of original body weight (bottom) of mice were recorded as indicated. The graphs include compilations of three independent experiments; error bars represent the SEM (bottom). (b,c) At days 0 and 10 post-infection, lung sections were stained with hematoxylin and eosin and displayed at 4x (top) or 20x (below) (b) and histology of lung sections at day 10 was scored (c) on a five-point scale (0–5) in a blind study. The criteria used for scoring include pulmonary oedema (pink material in air spaces), hemorrhage, necrotizing bronchiolitis (NB), interstitial mononuclear infiltration, and presence of bronchus-associated lymphoid tissue (BALT). Data are compilation of two independent experiments with eight mice for WT and seven for <i>Zbtb32</i>^-/-. (d) At day 10 post-infection, lymphocytes from spleen and lungs were stimulated with NP396 or GP33 peptides followed by intracellular cytokine staining. The percentages ± SEM of IFNγ⁺ CD8⁺ T cells are depicted. Data are a representative of two independent experiments with three mice per genotype per experiment. (e) LCMV-clone 13 titers in spleen and liver were determined by plaque assay at day 10 post-infection. Data are representative of two independent experiments with five mice per genotype. (f,g) Congenically-marked WT and <i>Zbtb32</i>^<i>-/-</i> P14 cells mixed 1:1 were co-transferred into recipients, which were infected with LCMV-clone 13. At day 14, P14 donor cells from surviving recipients were analyzed for their frequencies (f) and analyzed for expression of exhaustion markers as indicated (g). Plots show percentages ± SEM (g, upper panels) and the MFI of exhaustion marker expression (g, lower graphs) as indicated. Data are compilations from two independent experiments with five surviving of eight recipient mice.</p

ZBTB32 is required intrinsically in CD8⁺ T cells to regulate memory cell development during acute virus infection.

<p>(a-b) WT or <i>Zbtb32</i>^<i>-/-</i> P14 splenocytes (CD90.1⁺) were adoptively transferred into CD90.2⁺ recipient mice followed by LCMV-Armstrong infection. (a) The percentages (top panels) and total numbers (graph below) of splenic P14 cells were enumerated at days 6, 9 and 15 post-infection. (b) At days 9 and 15, P14 cells were analyzed for memory markers as indicated. (c) P14 cells were cultured with GP33 peptide followed by intracellular cytokine staining. Plots show percentages ± SEM of TNF⁺ IL-2⁺ cells gated on P14 IFNγ⁺ cells. (d) Plots show percentages ± SEM of Eomes-positive splenic P14 cells and the MFI ± SEM of EOMES expression at day 9 (top) and day 15 (bottom) post infection. Data are representative of two independent experiments with three mice per genotype per experiment for day 6, and three independent experiments with three mice per genotype per experiment for days 9 and 15 post-infection. (e-g) Congenically-marked WT P14 cells stimulated <i>in vitro</i> with αCD3/CD28 for 24 hours were transduced with ZBTB32-expressing retrovirus (Zbtb32 RV) or mock retrovirus (mock RV) and then the two populations (mixed 1:1) were co-transferred into recipients, which were infected with LCMV-Armstrong. (e) A subset of transduced P14 cells was cultured <i>in vitro</i> for additional 2 days, and the transduction efficiency assessed by GFP fluorescence. At days 14 and 45 post-transfer and LCMV infection, P14 cells were analyzed for their (f) frequencies and for the (g) percentages ± SEM and MFI of IL-7R expression on each population. All data are compiled from two independent experiments with nine recipient mice for day 14 and six recipient mice for day 45.</p

ZBTB32 represses <i>Eomes</i> and <i>Cd27</i> gene expression in CD8⁺ T cells by recruiting histone deacetylases 1 and 2.

<p>WT or <i>Zbtb32</i>^<i>-/-</i> P14 splenocytes were transferred into recipients, which were then infected with LCMV-Armstrong. At days 6, 8 and 10 post-infection, P14 cells were isolated and pooled from three mice per genotype for RNA isolation; chromatin was prepared at day 7 post-infection. (a) <i>Eomes</i> and <i>Cd27</i> mRNA levels were examined by quantitative RT-PCR relative to <i>Actb</i> mRNA. (b) Schematic of <i>Eomes</i> and <i>Cd27</i> gene loci showing position of specific (Amplicon 1; Amp1) and non-specific (Amplicon 2; Amp2) primers. In each case, Amp1 corresponds to putative ZBTB32 binding site. (c) The enrichment of ZBTB32 on <i>Eomes</i> and <i>Cd27</i> genes by chromatin immunoprecipitation (ChIP). (d) The enrichment of HDAC1 and HDAC2 on <i>Eomes</i> and <i>Cd27</i> genes by ChIP. (e) ChIP for Pol II, p300 or modified histone H3 at the <i>Eomes</i> and <i>Cd27</i> loci. Data are a compilation of three independent experiments; error bars represent the SEM. Iso; isotype control antibody.</p

Non-redundant roles for ZBTB32 and Blimp-1 in anti-viral CD8⁺ T cell responses.

<p>(a,b) WT P14 cells were isolated from three recipient mice at the indicated days post-LCMV-Armstrong infection. (a) <i>Prdm1</i> mRNA was quantified. (b) Lysates were immunoblotted for Blimp-1 protein. Data are from three independent experiments; error bars represent SEM. (c,d) WT or <i>Prdm1</i>^<i>-/-</i> P14 cells were isolated from three recipient mice at days 8 and 14 post-LCMV-Armstrong infection. <i>Zbtb32</i> mRNA was quantified (c) and chromatin was prepared for ChIP assays with antibodies to Blimp1 or mouse IgG (Iso). ChIP eluates were amplified by Q-PCR for <i>Zbtb32</i> gene. In each case amplicon 1 (Amp1) corresponds to putative Blimp-1 binding site and amplicon 2 (Amp2) to a negative control region. All graphs shown are from a compilation of three independent experiments and error bars represent the SEM. (e-h) LCMV-specific CD8⁺ T cells in splenocytes from WT, <i>Zbtb32</i>^<i>-/-</i>, <i>Prdm1</i>^<i>-/-</i> and <i>Zbtb32</i>^<i>-/-</i> <i>Prdm1</i>^<i>-/-</i> mice were analyzed at day 9 post-LCMV-Armstrong infection. Percentages ± SEM (left panels) and total numbers (graph at right) of LCMV-specific CD8⁺ T cells are depicted (e,f). LCMV-specific CD8⁺ T cells (g) and total CD8⁺CD44^hi T cells (h) were analyzed for memory markers as indicated. Data are representative of two independent experiments with three mice per genotype per experiment.</p