1. Volume 21, Issue 1, Pages 35-46 (January 2017)
Mapping and Role of the CD8+ T Cell Response During Primary Zika Virus Infection in Mice 
Annie Elong Ngono, Edward A. Vizcarra, William W. Tang, Nicholas Sheets, Yunichel Joo, Kenneth Kim, Matthew J. Gorman, Michael S. Diamond, Sujan Shresta 
Cell Host & Microbe 
Volume 21, Issue 1, Pages (January 2017)
DOI: /j.chom
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2. Cell Host & Microbe 2017 21, 35-46DOI: (10.1016/j.chom.2016.12.010)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
Characterization of ZIKV Infection and Identification of Epitopes Recognized by CD8+ T Cells in WT C57BL/6 Mice Treated with IFNAR-Blocking Antibody
WT C57BL/6 mice were administered IFNAR-blocking antibody (MAR1-5A3) 1 day prior to infection with 104 FFU ZIKV strains MR766 (n = 4) or FSS13025 (n = 4). MOCK represents control mice injected with 10% FBS/PBS.
(A) Levels of infectious ZIKV in serum and spleen on day 1 or 3 post-infection were measured by BHK-21 cell-based FFA.
(B) The expansion of total CD8+ T cells and CD44+CD62L− CD8+ T cells in MOCK group (n = 3) or ZIKV-infected mice (MR766 and FSS13025, n = 4) was determined on day 7 post-infection.
(C) The percentage of granzyme B produced by infected (n = 4) and MOCK mice (n = 4) is represented. Kruskall-Wallis test was used first to compare all groups, followed by Mann-Whitney test to compare MOCK versus each ZIKV-infected mouse group. See also Figure S1.
Cell Host & Microbe  , 35-46DOI: ( /j.chom )
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4. Figure 2 The LysMCre+IFNARfl/fl Mouse Model of ZIKV Infection
WT and LysMCre+IFNARfl/fl C57BL/6 mice at 5 weeks of age were infected with 106 FFU MR766 or FSS Serum, liver, spleen, and brain were harvested at day 1 and 3 post-infection, and the levels of infectious ZIKV were determined using BHK-21 cell-based FFA.
(A and B) The quantities of infectious (A) MR766 or (B) FSS13025 virus at day 1 (black circles) and day 3 (white squares) post-infection are shown. Four mice were included in each group.
(C) Weight and clinical scores of infected WT and LysMCre+IFNARfl/fl mice were monitored and unpaired t test with Welch’s correction was used to compare the two groups at each time point.
(D and E) A representative density plot (D) showing CD44 and CD62L expression and (E) the frequency of CD3+CD8+ T cells and CD44+CD62L− CD8+ T cells from LysMCre+IFNARfl/fl mice infected with 104 FFU ZIKV or MOCK are shown. Kruskall-Wallis test was used first to compare all groups, and the Mann-Whitney test was used to compare MOCK and each ZIKV-infected group. All error bars correspond to SEM.
Cell Host & Microbe  , 35-46DOI: ( /j.chom )
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5. Figure 3
Identification of ZIKV Epitopes Recognized by CD8+ T Cells in LysMCre+IFNARfl/fl Mice
Five-week-old LysMCre+IFNARfl/fl were infected retroorbitally with 104 FFU ZIKV MR766 or FSS13025.
(A) IFNγ-ELISPOT was performed using CD8+ T cells isolated from infected mice. A total of 244 peptides from ZIKV strains predicted to bind H-2Kb and H-2Db with high affinity were screened. Two independent experiments for each ZIKV strain (n = 5 mice for each experiment) were performed in triplicate per peptide. The data are expressed as the mean of spot-forming cells (SFCs) per 106 CD8+ T cells, and error bars represent SEM. One-way ANOVA was used to compare the mean of each peptide with the control (DMSO) (p > 0.05).
(B) To confirm ZIKV-derived epitopes recognized by CD8+ T cells in LysMCre+IFNARfl/fl mice and IFNγ, production via ICS was determined 7 days post-infection with positive peptides. The dotted line corresponds to the average amount of IFNγ produced by MOCK mice when stimulated with positive ZIKV-derived peptides (0.19% of IFNγ+CD8+ T cells).
Cell Host & Microbe  , 35-46DOI: ( /j.chom )
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6. Figure 4
Polyfunctional Phenotype of ZIKV Epitope-Specific CD8+ T Cells in LysMCre+IFNARfl/fl Mice
Splenocytes from LysMCre+IFNARfl/fl mice that were infected with 104 FFU ZIKV strain MR766 (n = 4) or FSS13025 (n = 5).
(A) Frequency of CD8+ T cells producing IFNγ and TNFα upon stimulation with MR766-derived peptides (top panel) and FSS13025-derived peptides (bottom panel) is shown. The background obtained in MOCK mice was 0.07%. All experiments were performed twice and error bars represent SEM.
(B) The gating strategy used to select cells expressing both CD107a and IFNγ in MOCK and infected mice upon stimulation with relevant ZIKV peptide (E294–302) or with irrelevant ZIKV peptide (E710–718) is represented.
(C) Frequency of CD107a+IFNγ+ CD8+ T cells obtained after stimulation with MR766- or FSS13025-derived peptides is shown. The background obtained in MOCK mice is represented by dotted lines and corresponds to 2.36% for CD107a+IFNγ+ double-positive CD8+ T cells.
(D) The percentage of granzyme B produced by CD8+ T cells from mice infected with MR766 (n = 8) or FSS13025 ZIKV(n = 8) and MOCK (n = 4) is represented.
(E) A representation of in vivo cytotoxicity of target cells in ZIKV-infected mice is shown.
(F) The percentage of killing was obtained in mice infected with ZIKV (n = 4) for 7 days or in MOCK (n = 4).
Cell Host & Microbe  , 35-46DOI: ( /j.chom )
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7. Figure 5
Kinetics of the ZIKV-Specific CD8+ T Cell Response in LysMCre+IFNARfl/fl Mice
LysMCre+IFNARfl/fl mice were infected with 104 FFU ZIKV strain MR766 or FSS Splenocytes were harvested at 3, 7, and 14 days post-infection and stimulated with immunodominant ZIKV-derived peptides to assess cytokine production by ICS. (A) The frequency of IFNγ-producing CD8+ T cells and (B) the frequency of CD44+CD62L− CD8+ T cells at day 3 (white), 7 (black), and 14 (gray) post-infection are shown. The background production of IFNγ obtained in MOCK was subtracted from all values. Two-way ANOVA test was used to compare the time points for each peptide (p > 0.05). The error bars correspond to SEM.
Cell Host & Microbe  , 35-46DOI: ( /j.chom )
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8. Figure 6
Protective Role of CD8+ T Cells against ZIKV Infection in Mice
LysMCre+IFNARfl/fl mice were treated with depleting anti-CD8 or isotype control antibody on days 3 and 1 before infection with 105 FFU MR766 or FSS Mice were sacrificed and tissues harvested at 6, 8, and 10 days post-infection.
(A–D) The levels of infectious virus in the (A) serum, (B) spleen, (C) brain, and (D) sciatic nerve were quantified using BHK-21 cell-based FFA. A two-way ANOVA test was used to compare the levels of infectious ZIKV between the isotype and the anti-CD8 antibody-administered groups for all time points and tissues.
(E) On day 120 after infection with MR766 or FSS13025, 7.5 × 106 CD8+ T cells were transferred into 5-week-old naive mice 1 day before challenge with 105 FFU MR766 or FSS For controls, CD8+ T cells were isolated from naive LysMCre+IFNARfl/fl mice. Infectious ZIKV was quantified. Mann-Whitney test was used to compare naive CD8+ T cells versus ZIKV-immune CD8+ T cells.
(F) Seven-week-old WT and CD8α−/− were treated with 2 mg IFNAR-blocking antibody at day −1, and then inoculated subcutaneously with 105 plaque-forming units (PFU) mouse adapted Dakar ZIKV strain at day 0. Survival was monitored for 21 days in both groups and reported for WT (n = 15, black square) and CD8−/− (n = 11, red circle) mice. Pooled data from three independent experiments are represented and the log-rank (Mantel-Cox) test was used to compare groups.
Cell Host & Microbe  , 35-46DOI: ( /j.chom )
Copyright © 2017 Elsevier Inc. Terms and Conditions