Fig 2: LILRB4 expression is upregulated by microglia and infiltrated myeloid cells into brains during ZIKV infection.(A) Diagram depicting the disease progression of ZIKV infection. (B) Heatmap for the fold changes in RNA expression of immune checkpoint receptors in the brains of ZIKV-infected mice at the indicated time points (n = 3–8, each time point). RNA expression was assessed by NanoString analysis using the nCounter mouse immunology panel. (C) Relative mRNA levels of Lilrb4 and Lilrb3 in the mouse brain during ZIKV infection compared with uninfected mouse brain. (D–F) Flow cytometry analysis of LILRB4 expression on cells isolated from the brains of ZIKV-infected mice at the indicated time points. The graph shows the percentage of LILRB4-expressing cells in total live cells (D). Live cells were separated based on CD45 and LILRB4 expression. The pie chart shows the percentage of macrophages (CD45hiCD11b+F4/80+), neutrophils (CD45hiCD11b+Ly6G+F4/80–), DCs (CD45hiCD11c+F4/80–Ly6G–), T cells (CD45hiCD3+NK1.1–), and NK cells (CD45hi NK1.1+CD3–) within CD45hiLILRB4+ cell lation (indicated by the dashed circle) at 15 dpi (E). The histogram shows LILRB4 expression on microglia (CD45loCD11b+), macrophages (CD45hiCD11b+F4/80+), neutrophils (CD45hiCD11b+Ly6G+F4/80–), and DCs (CD45hiCD11c+F4/80–Ly6G–). Gray histograms indicate staining with IgG isotype control antibodies (F). Data are representative of 2 independent experiments (n = 3–5, each time point) (E and F). Data were analyzed using 1-way ANOVA with Dunnett’s multiple comparisons, ***P < 0.001.

Fig 3: Histological validation of visium alteration. Analysis of T and B cells in the LG of the NOD.H-2b mice. (A–D) CD3+ cells in the LG of the 2 months old male penetrate throughout tissue and appeared to be attached to duct and acini (black arrows), whereas (B) in control BALB/c mice CD3+ cells are rare (B, black arrow). (E) Later at 3 months of age B cells (stained with the B220 or CD19 antibody) concentrate within an interductular space near the blood vessels (BV). (F) Double immunostaining of LG paraffin section with B220 (brown - B cell marker) and T3 (black – T cell marker, black arrows) showing that majority of cells within infiltrate are B cells. B and T cells are organized into an ectopic lymphoid structure.

Fig 4: Increase of B and T cells proportion in Male NOD.H-2b mice with age. CD45+ immune cells were analyzed by flow cytometry of LGs from BALB/c (B) and NOD.H.2b (N) mice. (A, A’) Comparison of B cells (CD19+) and T cells (CD3+) between LGs of NOD.H-2b male and female have been performed at 2 and 6M within immune population (CD45+) 2M old BALB/c have been used as control (empty bar). A representative plot at 2M is shown for male and female (A’). (B) Histogram showing proportion of CD4+ and CD8+ cells. A representative plot at 2M is shown for male and female (B’). (A, B) Values are shown as mean ± SD. Statistical significance assessed with ANOVA test. Adjusted P-value significance: *: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001; ns: non-significant. (C, D) H&E staining of LGs from NOD.H-2b female (C) and male (D) showed immune infiltrates (black arrows). (E) Histogram representing focus score of LGs between male and female at 4 months Plot shows mean ± SD. Statistical significance assessed with an unpaired t-test (**: p=0.0021).

Fig 5: LILRB4 deficiency leads to defects in NK cell activation.(A and B) Percentage of LILRB4-expressing (A) and CD11b-expressing (B) NK cells (CD45hiNK1.1+CD3–) in the brains of ZIKV-infected mice at 15 dpi. Data shown as the mean ± SD (n = 6 mice/group). (C) IFN-? expression in brain NK cells. Left: Representative flow data. Right: The percentage of IFN-?+ in NK cells. (D) IFN-? levels in the brain (Luminex; n = 3–6/group). (E) Gzmb mRNA levels in the brain. Mean ± SD (n = 3–4/group). (F) Granzyme B expression in NK cells from the brains of ZIKV-infected mice. (G and H) Granzyme B expression in naive splenic NK cells stimulated with poly(I:C) (100 µg/mL) for 24 hours (G) or IL-2 (50 ng/mL) for 48 hours (H). Data are representative of 3 independent experiments, each performed in triplicate or quadruplicate. Data were analyzed using 2-tailed unpaired Student’s t test. *P < 0.05, ***P < 0.001.