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 Ly6Chi 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.
LCMV‐specific non‐neutralizing antibodies (
Chronic infections with noncytopathic viruses, such as HIV, hepatitis B or C viruses affect several hundred million people worldwide. The high incidence and mortality of these infections highlights the importance of studies devoted to better understand chronic diseases and of development of potent antiviral treatments. Viruses have evolved different strategies to evade the immune system and persist in the host. Together, the location, timing, and magnitude of the immune response combined with the speed of virus replication, its cytopathogenicity, and spread determine the fate of the viral infection.
Despite the fact that antiviral vaccines aim at producing primarily neutralizing antibodies (nAb), there is a growing body of evidence emphasizing the protective potential of non‐neutralizing antibodies (nnAbs) against infection with different viruses [
Usually, nnAbs mediate protection through the interaction of the Fc‐domain with different Fc‐receptors [
A few recent studies propose alternative protective mechanisms mediated by nnAb independent of the commonly described Fc gamma receptors (FcRγ). For example, FcRn is part of the endolysosomal system of most epithelial and hematopoietic cells where it regulates the intracellular fate of both IgG and IgG‐containing immune complexes [
Here, we show that LCMV‐specific nnAbs protect against establishment of chronic viral infection by binding predominantly Ly6Chi inflammatory monocytes (IMs). In this way, these nnAbs strongly promoted infection of IMs in an FcRγ‐independent manner and lead to acquisition of an activated APC phenotype by upregulating CD11c, MHC, and costimulatory molecules. IMs from infected mice treated with LCMV‐specific nnAbs served as additional APCs and boosted early activation of virus‐specific T cells, leading to improved effector phenotype differentiation and enhanced cytotoxicity, thereby preventing the establishment of chronic infection.
Considering the growing interest and importance of nnAbs as an alternative protection against persistent viral infections, defining their protective mechanisms contributes to the advance in the search of preventive and therapeutic treatments against chronic viral infections.
We used LCMV infection as a well‐established murine model for a chronic virus infection to study the mechanism by which nnAbs prevent viral persistence [
First, we confirmed that day 12 immune serum (IS) and the monoclonal Ab KL53 exhibited binding activity toward LCMV‐derived proteins, demonstrated by specific staining of LCMV infected cells (Supporting information Fig.
We hypothesized that opsonization of LCMV might influence its in vivo tropism. To get a broad overview on LCMV distribution in secondary lymphoid organs, we analyzed spleens from various time points after infection in presence or absence of IS by confocal microscopy. As previously described [
LCMV infects nonhematopoietic cells including fibroblastic reticular cells. Cytotoxic T‐cell‐mediated killing of infected cells results in destruction of the structure of secondary lymphoid organs, defined by distinct B‐ and T‐cell zones [
Next, we analyzed the splenic hematopoietic cell types that had bound the nnAb KL53 in vivo 1 day after infection. Mice were infected with LCMV in presence or absence of KL53 and 1 day later splenocytes were analyzed for cell‐surface bound KL53 using flow cytometry. Specifically, we evaluated the percentages of KL53+ cells among CD11chiMHCIIhi DC, Ly6ChiCD11b+ IMs, CD169+ metallophilic macrophages (MMf), and CD11b+Ly6Cint neutrophils (Nf). Interestingly, KL53 preferentially bound to Ly6Chi IMs and not to any other analyzed cell type (Fig.
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+MHCIIhi), IM (CD11b+Ly6Chi), MMf (CD169+), and Nf (CD11b+Ly6Cint). 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
Next, we asked whether and how the binding of nnAbs on Ly6Chi IMs affected their phenotype. We examined splenic CD11b+Ly6Chi monocytes 1dpi in the presence or absence of LCMV‐specific nnAb KL53. LCMV infection in absence of KL53 already increased CD11c, MHCII, and CD40 expression within CD11b+Ly6Chi monocytes to some extent, but treatment with KL53 boosted the expression of these markers significantly in Ly6Chi monocytes compared to infected mice in the absence of KL53 (Fig.
As we identified IMs to be particularly affected by treatment with LCMV‐specific antibodies early during infection, we further examined their phenotype in response to LCMV opsonized with virus‐specific KL53 mAb. We performed a cluster analysis based on an extended panel of myeloid markers on IM populations 1dpi from naïve, LCMV‐infected, and mice‐infected KL53‐opsonized LCMV. tSNE plots depict IMs from the three groups as distinct populations (Fig.
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+Ly6Chi 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.
NnAbs can shift the viral tropism towards hematopoietic cells and specifically professional APCs through the recognition by Fc‐receptors [
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+MHCIIhi dendritic cells, and (C) CD11b+Ly6Chi 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
Additionally, we analyzed the effects of IS on the infection of DCs. While IS did not affect the numbers of DCs in infected mice, it significantly increased the percentages of infected DCs in WT but not in FcRγ−/− mice (Fig.
Next, we evaluated the infection of Ly6Chi IMs in presence or absence of IS 3dpi (Fig.
Based on the observation that IMs acquired an activated APC phenotype after LCMV infection in presence of IS, we hypothesized that they might promote T‐cell priming and effector responses. To test this, we performed an in vitro experiment in which we supplemented DC‐CD8+ T‐cell cocultures with sorted Ly6Chi IMs from mice that had been infected with LCMV in presence or absence of IS. DCs were sorted from day 3 LCMV‐infected mice and cocultured with naïve CTV‐labeled LCMV gp33‐41‐specific TCR transgenic CD8+ T cells (P14). Of note, DCs were used directly ex vivo without additional antigen pulsing. We analyzed the extent of activation and proliferation of CTV+ P14 cells 3 days later by flow cytometry. While very few P14 cells diluted CTV and upregulated CD44 when primed with DCs from LCMV‐ infected mice in the presence of Ly6Chi IMs from naïve mice, a significant percentage of P14 cells proliferated and upregulated CD44 when cultures were supplemented by Ly6Chi IMs from LCMV‐infected mice (Fig.
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+Ly6Chi cells. Sorted IMs were cocultured with CTV‐labeled P14 cells and sorted endogenous CD11c+MHCIIhi 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+Ly6Chi 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
Next, we asked whether Ly6Chi IMs from IS‐treated LCMV‐infected mice would also promote CD8+ T‐cell priming in vivo. To address this question, we generated IMs in LCMV‐infected mice in the presence or absence of IS for 3 days. We then sorted and transferred these IMs into LCMV‐infected CCR2−/− mice that lack circulating IMs which had received P14 T cells. Seven days post‐transfer, we quantified virus‐specific P14 T cells in the blood of infected mice (Fig.
It was previously shown that nnAb‐supported control of chronic LCMV infection involves CD8+ T cells [
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
We also noted phenotypic differences in LCMV‐specific CD8+ T cells elicited in presence or absence of LCMV‐specific nnAbs. In IS‐treated mice, a higher proportion of total CD44hi CD8+ T cells, endogenous gp33‐specific as well as transgenic P14 T cells exhibited a phenotype compatible with short‐lived effector cells (CD127− KLRG1+) (Fig.
Finally and most importantly, we measured the in vivo killing activity of LCMV‐specific CD8+ T cells raised in presence or absence of IS. For this, we infected mice with LCMV opsonized with or without IS. Seven dpi we transferred congenically marked splenocytes (CD45.1 and CD90.1) pulsed with different concentrations (10−7, 10−8, and 10−9 M) of gp33 and np396 peptide. Three hours later, we determined the specific killing of target cells in spleen and blood. Transfer of target cells into naïve B6 mice served as control. Both groups of infected mice were able to kill gp33‐ and np396‐loaded target cells. However, mice that had received IS at the time of infection exhibited a significantly stronger killing capacity of all target cell populations both in spleen (Fig.
In contrast to virus‐neutralizing antibodies, virus‐specific nnAbs have not been extensively studied in the context of antiviral therapies, despite their broad range of physiological effects.
Deposition of nnAbs on virus particles or infected cells and subsequent recognition by FcRγ+ cells was demonstrated for various viruses [
The currently described protective mechanisms of nnAbs involve FcR on target cells and include antibody‐dependent cellular cytotoxicity (ADCC), ADCP, complement‐dependent cytolysis (CDC), or steric inhibition of viral proteins important for viral replication, assembly, and release [
Previous studies have described a role of pre‐existing humoral immunity in shaping the size and quality of antiviral T‐ cell responses in the context of chronic infection [
In the context of chronic viral infections, virus‐specific CD8+ T cells are often described as “exhausted,” indicative of a differentiation status that is characterized by increased expression of coinhibitory receptors, such as PD1, Lag3, and Tim3, reduced effector functions like IFN‐γ, TNF‐α or IL2 secretion, or cytotoxicity [
APCs play a decisive role in the control of chronic viral infections. LCMV infection disrupts DC function and homeostasis to facilitate persistent infection. Some of the described mechanisms include downregulation of MHC, costimulatory molecules, and proinflammatory cytokines, and the induction of immunosuppressive cytokines [
Conversely, we identified Ly6Chi IMs being infected at markedly increased frequencies in presence of virus‐specific nnAbs. In addition, these IMs acquired an activated, DC‐like phenotype and served as additional APCs in T‐cell priming. IMs are usually associated with detrimental effects in chronic diseases [
The function of recruited monocytes is highly dependent on the type of infection, the tissue, and microenvironment [
Monocytes can contribute to viral clearance or exacerbate pathological damage depending on the context of the infection [
Mice were kept under specific pathogen‐free conditions and animal experiments were performed according to the guidelines of the animal experimentation law (SR 455.163; TVV) of the Swiss Federal Government. The protocol was approved by the Cantonal Veterinary Office (animal experimentation number 127/2011 and 117/2017). C57BL/6J (B6; Janvier Elevage, France), FcRγ−/− mice (deficient for the Fc receptor gamma chain shared by all activating FcγRs), congenic CD45.1, Thy1a.Igha/J, CCR2−/− (deficient for circulating monocytes, kindly provided by Burkhard Becher), and gp33‐specific TCR transgenic P14 mice expressing the congenic marker CD45.1 were bred and kept under at the ETH EPIC facility.
LCMV clone 13 was propagated on baby hamster kidney 21 cells, and viral titers were determined as described previously [
LCMV NP‐specific mAb was derived from mouse IgG2a secreting hybridoma KL53 [
Splenocyte suspensions were prepared by passing the spleens through a metal mesh using syringe plungers or by digesting them in RPMI containing 1 mg/mL Liberase (SigmaAldrich, Switzerland) and 0.2 mg/mL DNase I (Roche Diagnostics, Switzerland).
Splenic CD8+ T cells were isolated from CD45.1+ P14 mice by immunomagnetic sorting according to the manufacturer's instructions (Miltenyi Biotec, Germany) and cultured either in vitro 5.104 cells/mL or transferred i.v. 1.104 cells/mouse into recipients 1 day prior to virus infection.
The antibodies used for flow cytometry are listed in Supporting information Table
IMs were defined as CD11b+Ly6C+Ly6G− and DCs as CD11chiMHC‐IIhi. For sorting IMs and DCs, spleens were digested with Liberase and DNase I, depleted of CD19+ cells using MojoSort nanobeads (BioLegend), stained for CD11b, CD11c, Ly6C, IAb, and Ly6G and sorted by FACS Aria (BD Biosciences). Sorted IMs were cocultured with MACS‐purified P14 T cells and sorted DCs from LCMV‐infected mice for 3 days in ratio IM:DC:P14 10:1:1.
For analysis of CTL activity of LCMV‐infected mice, we transferred LCMV‐peptide loaded splenocytes into B6 mice infected with LCMV for 7 days. We used splenocytes from CD45.1+ mice loaded with 10−7, 10−8, 10−9 M of gp33‐peptide and labeled them with 1, 0.5, and 0.1 μM of CellTrace Violet (CTV; ThermoFischer), respectively. Additionally, we used splenocytes from Thy1.1+ mice loaded with 10−7, 10−8, 10−9 M of np396‐peptide and labeled them again with 1, 0.5, and 0.1 μM of CTV, respectively. We transferred a mixture of unloaded and unlabeled and gp33‐ or np396‐loaded CTV‐labeled CD45.1+ and Thy1.1+ splenocytes into naïve or infected mice and calculated the cytotoxic abilities of the recipient mice according to the recovered target cells 3 h after transfer.
Cell populations were pregated (single live, CD45+, Lin‐ (CD3, CD19, NK1.1, Ly6G)) for DCs or IMs. Afterwards scaled marker expression (VL4, MHCII, CD209, CD24, CD11b, CD64, Ly6G, CD169, XCR1, Ly6C, CD40, CX3CR1, F480, CD11c) was used to compute tSNE (perplexity = 50) [
Cells gated on IMs from infected mice (treated or untreated with KL53 1dpi) were pooled and clustered based on their marker profile (VL4, MHCII, D209, CD24, CD11b, CD64, Ly6G, CD8, CD169, XCR1, Ly6C, CD40, CX3CR1, F480, CD11c) using FlowSOM [
Values of marker medians (or frequency of KL53+ cells) were used to compute heatmaps in R with MADE4 package [
The two‐tailed unpaired Student's
Conceptualization: Annette Oxenius and Diana Stoycheva; Methodology: Diana Stoycheva, Fabienne Gräbnitz, Ioana Sandu, Ana Amorim, Mariana Borsa, Stefan Weber; Investigation: Diana Stoycheva; Resources: Annette Oxenius, Burkhard Becher; Writing: Diana Stoycheva; Visualization: Diana Stoycheva, Ioana Sandu; Supervision: Annette Oxenius; Funding Acquisition: Annette Oxenius.
The authors declare no commercial or financial conflict of interest.
The peer review history for this article is available at
antibody‐dependent cellular phagocytosi
days postinfection
Lymphocytic choriomeningitis virus
metallophilic macrophages
non‐neutralizing antibodies
immune serum
inflammatory monocyte
Fc gamma receptor
Supporting Information
Click here for additional data file.
We thank Nathalie Oetiker and Franziska Wagen for excellent technical assistance and the members of the Oxenius group for fruitful discussions. This work was supported by the ETH Zurich and the Swiss National Science Foundation (Grant No. 310030_166078 to AO).
The data that support the findings of this study are available from the corresponding author upon request.