Non-neutralizing antibodies protect against chronic LCMV infection by promoting infection of inflammatory monocytes in mice

Abstract

Antibodies play an important role in host defense against microorganisms. Besides direct microbicidal activities, antibodies can also provide indirect protection via crosstalk to constituents of the adaptive immune system. Similar to many human chronic viral infections, persistence of Lymphocytic choriomeningitis virus (LCMV) is associated with compromised T- and B-cell responses. The administration of virus-specific non-neutralizing antibodies (nnAbs) prior to LCMV infection protects against the establishment of chronic infection. Here, we show that LCMV-specific nnAbs bind preferentially Ly6C^hi inflammatory monocytes (IMs), promote their infection in an Fc-receptor independent way, and support acquisition of APC properties. By constituting additional T-cell priming opportunities, IMs promote early activation of virus-specific CD8 T cells, eventually tipping the balance between T-cell exhaustion and effector cell differentiation, preventing establishment of viral persistence without causing lethal immunopathology. These results document a beneficial role of IMs in avoiding T-cell exhaustion and an Fc-receptor independent protective mechanism provided by LCMV-specific nnAbs against the establishment of chronic infection.

Keywords: Chronic viral infection; Inflammatory monocytes; LCMV; Non-neutralizing antibodies; T-cell activation.

Figure 1

nnAbs bind predominantly inflammatory monocytes. WT mice were infected with LCMV with or without 0.5 mg KL53. (A) KL53⁺ cells detected by surface staining of KL53 with anti‐IgG2a, (B) LCMV⁺ (VL4⁺) cells detected by intracellular staining of LCMV NP by VL4 antibody, or (C) MFI of CD11c, MHCII, or CD40 in IMs or DCs in the spleens of naïve (grey), LCMV‐infected (open circles), or LCMV‐infected mice with KL53 (black circles) were determined 1dpi by flow cytometry, gated on live single cells, DCs (CD11c⁺MHCII^hi), IM (CD11b⁺Ly6C^hi), MMf (CD169⁺), and Nf (CD11b⁺Ly6C^int). Data of three independent experiments were pooled with n = 3 mice per group. Horizontal line represents the mean. Statistical analysis was performed using two‐tailed unpaired Student's t‐test, ****p < 0.0001.

Figure 2

LCMV‐specific nnAbs alter the IM phenotype. WT mice were infected with LCMV or opsonized LCMV with 0.5 mg KL53. Splenocytes were analyzed 1dpi by flow cytometry. (A) tSNE plots of pooled cells, gated on single, live, CD45⁺, Lin⁻(CD3, NK1.1, CD19, Ly6G) CD11b⁺Ly6C^hi inflammatory monocytes (gray). Color gradient represents distribution of cells isolated from infected mice (+LCMV), mice infected with KL53‐opsonized virus (+LCMV+KL53), naïve (naïve) or VL4+ cells. (B) Heatmap showing medians of measured markers or percentages of KL53⁺ inflammatory monocytes in mice infected with LCMV or KL53‐opsonized LCMV 1dpi. Dendograms show hierarchical clustering of mice (columns) or markers (rows). (C) Clustering of inflammatory monocytes isolated from mice infected with LCMV or KL53‐opsonized LCMV 1dpi. Heatmap of marker medians of the clusters generated by FlowSOM. Dendograms show hierarchical clustering of clusters (columns) or markers (rows). Cluster frequency ±SEM in mice infected with LCMV or KL53‐opsonized LCMV. Data are representative of two independent experiments with n = 3 per group.

Figure 3

LCMV‐specific nnAbs alter the tropism of virus‐infected cells to IMs in an FcRγ‐independent manner. WT and FcRγ‐KO (FcRg) mice were infected with LCMV or LCMV opsonized with IS. Splenocytes were analyzed 3dpi by flow cytometry. Representative dot blots and histograms of VL4⁺ single live (A) total splenocytes, (B) CD11c⁺MHCII^hi dendritic cells, and (C) CD11b⁺Ly6C^hi inflammatory monocytes are presented. Data are representative or pooled of two to four independent experiments with n = 3 per group. Horizontal line represents the mean. Statistical analysis was performed using two‐tailed unpaired Student's t‐test, *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001, ns = not significant.

Figure 4

nnAb‐primed IM from LCMV‐infected mice enhance virus‐specific T‐cell proliferation. (A) IMs from WT mice infected with LCMV opsonized with or without IS for 3 days were sorted by FACS,gated on single live Ly6G⁻CD11b⁺Ly6C^hi cells. Sorted IMs were cocultured with CTV‐labeled P14 cells and sorted endogenous CD11c⁺MHCII^hi DCs from LCMV‐infected mice for 3 days. Shown are representative dot blots of P14 cells, percentages of CTV‐negative P14 cells, and calculated numbers of recovered P14 cells of two independent experiments performed in duplicates. (B) IMs from WT mice infected with LCMV with or without IS for 3 days were sorted by FACS, gated on single live Ly6G⁻CD11b⁺Ly6C^hi cells. Sorted IMs were retransferred in IM‐deficient CCR2^−/− mice together with naïve P14 cells and mice were infected with LCMV. Shown are representative blots and percentages of P14 cells gated on CD8⁺ single cells in the blood of recipient mice 7dpi. Data of two independent experiments were pooled with n = 3 mice per group. Horizontal line represents the mean. Statistical analysis was performed using two‐tailed unpaired Student's t‐test, *p < 0.05, **p < 0.01.

Figure 5

LCMV‐specific nnAb increase virus‐specific T‐cell responses. (A, B, D, E) WT mice were infected with LCMV opsonized with or without IS. (C, F, G) WT mice received purified 10⁴ P14 T cells 1 day prior infection with LCMV with or without IS. Splenocytes were analyzed 7dpi by flow cytometry. Data are representative of two to four independent experiments with n = 3‐4 per group. (H) WT CD45.2 CD90.2 mice were infected with LCMV with or without IS. Seven dpi mice received a mixture of CTV‐labeled CD90.1⁺ np₃₉₆‐ or CD45.1⁺ gp₃₃‐loaded splenocytes. The percentage of killed target cells in infected mice was evaluated 3 h after transfer normalized to the percentage of target cells in naïve mice. Shown are representative data from two independent experiments with n = 3‐4 per group. Statistical analysis was performed using two‐tailed unpaired Student's t‐test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.