Inflammatory monocytes hinder antiviral B cell responses

Abstract

Antibodies are critical for protection against viral infections. However, several viruses, such as lymphocytic choriomeningitis virus (LCMV), avoid the induction of early protective antibody responses by poorly understood mechanisms. Here we analyzed the spatiotemporal dynamics of B cell activation to show that, upon subcutaneous infection, LCMV-specific B cells readily relocate to the interfollicular and T cell areas of the draining lymph node where they extensively interact with CD11b⁺Ly6C^hi inflammatory monocytes. These myeloid cells were recruited to lymph nodes draining LCMV infection sites in a type I interferon-, CCR2-dependent fashion and they suppressed antiviral B cell responses by virtue of their ability to produce nitric oxide. Depletion of inflammatory monocytes, inhibition of their lymph node recruitment or impairment of their nitric oxide-producing ability enhanced LCMV-specific B cell survival and led to robust neutralizing antibody production. In conclusion, our results identify inflammatory monocytes as critical gatekeepers that prevent antiviral B cell responses and suggest that certain viruses take advantage of these cells to prolong their persistence within the host.

Introduction

Antibodies (Abs) are critical for virus control and prevention of re-infection (1). Their production depends on B cells encountering viral antigen (Ag) in lymph nodes (LNs) draining infection sites, getting activated, interacting with different cells, proliferating and differentiating into Ab-secreting cells. Each of these events occur in distinct LN sub-compartments, requiring the migration of B cells from niche to niche in a fast and tightly coordinated fashion (2). Thanks to the recent advent of multiphoton intravital microscopy (MP-IVM), several cellular and molecular events by which LNs orchestrate the generation of humoral immune responses have been clarified (3–5). However, how viral infections affect the spatiotemporal dynamics of B cell activation is not well defined. Moreover, the mechanisms whereby some viruses (e.g. LCMV) interfere with the induction of early, potent neutralizing Ab responses remain largely unexplored.

Here we employed MP-IVM to study Ag-specific B cell behavior upon viral infection. We found that, upon LCMV infection, virus-specific B cells readily move from B cell follicles to the interfollicular and T cell areas of the draining LNs, where they engage in prolonged interactions with and are eventually killed by a population of inflammatory monocytes that is recruited in a type I interferon- and CCR2-dependent manner. Strategies aimed at preventing inflammatory monocyte accumulation within secondary lymphoid organs increased LCMV-specific B cell survival and caused robust neutralizing Ab production.

Results

Spatiotemporal dynamics of B cell activation in response to VSV and LCMV infection

To begin addressing these issues, we infected mice subcutaneously (s.c.) into the hind footpad with either vesicular stomatitis virus (VSV) or LCMV, two viruses that have been widely used to study adaptive immune responses (1). Consistent with previous results obtained with systemic routes of infection (1), early, potent neutralizing Ab responses were induced upon local infection with VSV, but not with LCMV (Fig. 1A). Since the co-evolution of the LCMV-mouse relationship might have resulted in the selection of a neutralizing epitope that is not readily recognized at a sufficiently high avidity by germline-encoded immunoglobulin V_H-V_L-region combinations in wild-type (WT) mice (1), we sought to correct for eventual disparities in the initial virus-specific B cell precursor frequency by making use of B cell receptor (BCR) transgenic mice. VSV-specific BCR transgenic mice (referred to as VI10YEN) have already been described (6); LCMV-specific BCR transgenic mice (referred to as KL25) were generated by crossing available LCMV-specific V_H-knock in mice (6) with newly generated LCMV-specific V_L-transgenic mice (Fig. S1A). The vast majority (~90%) of the resulting KL25 B cells bound to the LCMV glycoprotein (GP), and, upon in vitro incubation with LCMV, got readily activated and produced Abs to the same extent that VI10YEN B cells did in response to VSV (Fig. S1, B to D). Adoptive transfer of up to 10⁷ KL25 B cells into D_HLMP2A mice (which are devoid of surface-expressed and secreted Abs (7) but, in contrast to B cell-deficient mice, retain an intact LN architecture (8)) prior to s.c. LCMV infection, however, did not result in a detectable neutralizing Ab response (Fig. 1B and Fig. S2). By contrast, adoptive transfer of VI10YEN B cells using the same experimental setup – where Abs can be produced only by the transferred B cells – resulted in a readily detectable, potent neutralizing Ab response (Fig. 1B). Altogether, these results indicate that a low Ag-specific B cell precursor frequency is not the sole determinant of the impaired humoral immune response observed upon LCMV infection, and they suggest that events linked to LCMV replication in vivo actively interfere with the generation of a protective Ab response.

Figure 1. Spatiotemporal dynamics of B cell activation in response to VSV and LCMV infection.

(A) Neutralizing Ab titers in the sera of C57BL/6 mice that were infected s.c. with 10⁵ pfu of VSV (gray) or 10⁵ ffu of LCMV (black). n = 5; results are representative of at least three independent experiments. (B) Neutralizing Ab titers in the sera of D_HLMP2A mice that were transferred with 5 x 10⁶ purified VI10YEN (gray) or KL25 (black) B cells 18h prior to s.c. infection with VSV or LCMV, respectively. n = 5; results are representative of at least two independent experiments. (C) Multiphoton intravital (left and middle panels) or confocal (right panel) micrographs in the popliteal LN of a D_HLMP2A mouse that was injected with VI10YEN (red) and WT (cyan) B cells 18h prior to s.c. VSVeGFP (green) infection. Snapshots were acquired at the indicated time points after infection. Dotted white line denotes B cell follicles. Scale bars represent 50μm (left and middle panels) or 100μm (right panel). Results are representative of at least five independent experiments. See also Movie S2. (D) Multiphoton intravital (left and middle panels) or confocal (right panel) micrographs in the popliteal LN of a D_HLMP2A mouse that was injected with KL25 (red) and WT (cyan) B cells 18h prior to s.c. LCMVeGFP (green) infection. Snapshots were acquired at the indicated time points after infection. Dotted white line denotes B cell follicles. Scale bars represent 50μm (left and middle panels) or 100μm (right panel). Results are representative of at least five independent experiments. See also Movie S3. (E) Percentage of WT (blue) and VI10YEN (red) B cells within the indicated LN compartments 72h after VSV infection. IFA, Interfollicular area. n = 2; results are representative of at least three independent experiments. See also Movie S2. (F) Percentage of WT (blue) and KL25 (red) B cells within the indicated LN compartments 72h after LCMV infection. IFA, Interfollicular area. n = 2; results are representative of at least three independent experiments. See also Movie S3. (G) Meandering index of WT (blue) or KL25 (red) B cells in the interfollicular area (total, left panel) or in interfollicular areas that were > 15 μm distant from GFP⁺ cells (GFP^- areas, right panel), 72h after LCMV infection. n = 2. Results are representative of at least two independent experiments. See also Movie S3.

Results are expressed as mean ± SEM. **p < 0.01, ***p < 0.001.

To gain insight into the mechanism whereby LCMV inhibits Ab responses, we next set out to directly visualize the spatiotemporal dynamics of B cell activation by performing MP-IVM in LNs draining the infection site. To this end, we infected mice s.c. into the right hind footpad with recombinant VSVeGFP (9, 10) or LCMV-M1eGFP (11, 12), both of which drive green fluorescent protein (GFP) expression selectively within infected cells and show replication kinetics in footpad-draining popliteal LNs that are very similar to the respective parental strains (Fig. S3A). As previously reported (10), VSV replication in LNs was restricted to CD169⁺ subcapsular sinus (SCS) macrophages, it reached peak levels at 8-12 hours post infection (p.i.) and ended within 24-48 hours (Fig. S3, A and B); by contrast, LCMV replicated at early time points (12 hours p.i.) in both SCS and medullary macrophages (12), it peaked at 72 hours p.i. where replication extended to stromal cells and myeloid cells in the interfollicular areas (IFAs), and it was cleared in about a week (Fig. S3, A and B). To visualize virus-specific B cells, we then adoptively transferred fluorescently-labelled VI10YEN or KL25 B cells – along with wild type (WT) polyclonal B cells – into D_HLMP2A mice prior to s.c. infection (note that in uninfected mice WT, VI10YEN and KL25 B cells showed virtually identical motility parameters [i.e. speed, meandering index and track direction], Fig. S4, A and B, Movie S1). We first analyzed the dynamics of B cell activation in response to VSV. Consistently with what has been previously reported with non-viral Ags or non-replicating VSV (13–15), as early as 2 hours p.i. Ag-specific B cells accumulated within the SCS, where they interacted extensively with infected CD169⁺ macrophages (Fig. 1C, Fig. S4, C and D, and Movie S2). By 8 hours p.i., despite residual viral replication within the SCS, VI10YEN B cells redistributed to the T/B border where they remained until ~24 hours p.i. (Fig. 1C, Fig. S4, E and F, Movie S2). This redistribution was associated with robust VI10YEN B cell activation, as assessed by significant upregulation of CD25 and CD69 by 24 hours p.i. (Fig. S4G). By 72 hours p.i., VI10YEN B cells had undergone vigorous proliferation and relocated back to the follicle (Fig. 1, C and E, Movie S2). We next analyzed the spatiotemporal dynamics of B cell activation in response to LCMV. LCMV replication in popliteal LNs was undetectable at 2h p.i. and, accordingly, no redistribution of Ag-specific B cells to the SCS occurred (Fig. 1D, Fig. S5, A and B, and Movie S3). At 8 hours p.i., we started to detect some LCMV-infected cells but no substantial changes in the behavior of KL25 B cells happened (Fig. S5, C and D, and Movie S3). By contrast, at 24 hours p.i., there was an extensive redistribution of KL25 B cell to the IFA, with a fraction of Ag-specific B cells engaging in interactions with virus-replicating (GFP⁺) cells (Fig. 1D, Fig. S5E, and Movie S3). While the meandering index of the few remaining follicular KL25 B cells was indistinguishable from those of control WT B cells, KL25 B cells that relocated to the IFA showed a confined motility, characterized by a low meandering index and a restricted directionality (Fig. S5, F to H, and Movie S3). By contrast, the few control WT B cells that ended up in the IFA readily moved back to the follicle (Fig. S5H). Of note, the abovementioned changes in motility parameters were associated with robust KL25 B cell activation, as assessed by significant upregulation of CD25 and CD69 by 24 hours p.i. (Fig. S5I). Altogether, these results indicate that LCMV-specific B cells recognize LCMV and get activated in vivo, yet they fail to produce Abs (Fig. 1B). At 72 hours p.i., when VI10YEN B cells in VSV-infected mice have already returned to B cell follicles (Fig. 1, C and E, and Movie S2), the vast majority of KL25 B cells in LCMV-infected mice were still confined to the IFA (Fig. 1, D and F, and Movie S3). The different localization of virus-specific B cells in VSV- and LCMV-infected mice correlated with a differential expression of the B cell follicle-homing chemokine receptor CXCR5 (Fig. S5J). Because KL25 B cells at the IFA showed a confined motility, particularly in areas where LCMV-replicating cells (GFP⁺) were absent (Fig. 1G), we reasoned that LCMV-specific B cells actively engaged with a population of cells that impaired their differentiation into Ab-secreting cells.

Inflammatory monocytes, recruited to LNs in a type I interferon- and CCR2-dependent fashion, suppress LCMV-specific Ab responses

Among potential immune suppressive cells, we focused on CD11b⁺ myeloid cells as they are known to be recruited to LNs draining sites of infection (16) and subsets of these cells have been shown to suppress adaptive (i.e. T cell-mediated) immune responses in various pathophysiological settings (16–19). We therefore set out to characterize the CD11b⁺ cell population that is recruited to popliteal LNs upon footpad LCMV infection. We found that a distinct population of CD11b⁺Ly6C^hiF4/80⁺MHC-II^intCD11c^lo/int inflammatory (or “classical”) monocytes (20) was recruited to draining LNs as early as 24 hours after LCMV infection and persisted for at least seven days p.i. (Fig. 2, A and B, and Fig. S6A). To determine whether these inflammatory monocytes played a role in regulating LCMV-specific B cells responses, we designed experimental conditions to inhibit their LN recruitment or to deplete them. As type I interferon and CCR2 have been shown to regulate inflammatory monocyte recruitment to infected/inflamed tissues (21–23), we reasoned that these molecules might also be implicated in the LN recruitment of CD11b⁺Ly6C^hi cells upon LCMV infection. Indeed, inflammatory monocytes were not recruited in the LNs of LCMV-infected type I interferon (IFNAR)^-/- and CCR2^-/- mice and they were absent in the LNs of inflammatory monocyte-depleted animals treated with anti-Gr-1 Abs (Fig. 2C and Fig. S6B). Remarkably, KL25 B cells that were transferred into IFNAR^-/-, CCR2^-/- or anti-Gr-1-treated mice produced large quantities of LCMV-GP-binding and LCMV-neutralizing Abs (Fig. 2, D and E). Of note, LCMV-specific Ab responses in the absence of inflammatory monocytes were also detected (albeit at substantially lower levels) in a more physiological setting that did not entail the adoptive transfer of KL25 B cells (Fig. 2F). These results, based on three mechanistically distinct approaches, indicate that inflammatory monocytes prevent the induction of LCMV-specific Ab responses. One potential caveat of the experimental strategies we utilized to prevent inflammatory monocyte accumulation within LNs is that they might have increased LCMV titers and therefore the amount of Ag available to LCMV-specific B cells. Indeed, and consistently with previously published literature (24), IFNAR^-/- mice were characterized by higher viral titers in draining LNs at the peak (72 hours p.i., see Fig. S3A) of LCMV replication (Fig. S6C). However, we did not detect increased viral titers at 72h p.i. in LNs of CCR2^-/- or anti-Gr-1-treated mice (Fig. S6C). Moreover, infection of WT mice with up to 100-fold higher doses of LCMV did not lead to LCMV-specific Ab responses (Fig. S7). Altogether, these results suggest that the mechanism whereby inflammatory monocytes hinder Ab responses upon LCMV infection of WT mice does not rely on limiting Ag availability to B cells. To further increase the specificity of our approaches targeting inflammatory monocytes and to rule out potential off-target effects, we next reconstituted CCR2^-/- mice with WT monocytes. Adoptive transfer of 5 x 10⁶ bone marrow-derived monocytes from WT mice into CCR2^-/- animals 6 hours after LCMV infection resulted in the LN recruitment of inflammatory monocytes and in the inhibition of LCMV-specific Ab responses (Fig. 2G). Of note, the induction of neutralizing Abs we observed in the absence of inflammatory monocytes had a significant impact on viral clearance, as CCR2^-/- mice had lower LCMV titers than WT mice at day 7 p.i. (Fig. 2H), despite T cell responses that were, if anything, lower in CCR2^-/- than WT mice (25).

Figure 2. Inflammatory monocytes, recruited to LNs in a type I interferon- and CCR2-dependent fashion, suppress LCMV-specific Ab responses.

(A) FACS plots showing CD11b and Ly6C expression in the popliteal LNs of mice injected in the footpad with PBS (left) or LCMV (right) 72h earlier. Numbers show the percentage of cells within the gates. Plots are representative of at least ten independent experiments. (B) Number of CD11b⁺Ly6C^hi cells within the popliteal LNs of WT mice, at the indicated time points after s.c. LCMV infection. Results are representative of at least three independent experiments. (C) Number of CD11b⁺Ly6C^hi cells within the popliteal LNs of WT (black), IFNAR^-/- (red), CCR2^-/- (blue) or anti-Gr-1-treated WT mice (green), 72h after s.c. LCMV infection. (D-E) 5 x 10⁶ KL25 B cells were transferred into WT (black), IFNAR^-/- (red), CCR2^-/- (blue) or into WT mice injected with anti-Gr-1 (green) or isotype control (black) Abs prior to s.c. LCMV infection. LCMV-GP-binding (D) or neutralizing (E) Abs were measured in the sera seven days after LCMV infection. LCMV-GP-binding Abs are expressed as fold induction over uninfected controls. Results are representative of at least five independent experiments. (F) LCMV-GP-binding Abs in the sera of WT (black), IFNAR^-/- (red), CCR2^-/- (blue) mice or anti-Gr-1-treated WT mice (green), 7 days after s.c. LCMV infection. Data are expressed as fold induction over uninfected controls. Results are representative of two independent experiments. (G) KL25 B cells were transferred into WT (black), CCR2^-/- (blue) or CCR2^-/- mice reconstituted with 5 x 10⁶ WT monocytes (yellow). LCMV-GP-binding Abs (fold induction over uninfected controls) were measured 7 days after s.c. LCMV infection. Results are representative of at least two independent experiments. (H) LCMV titers 7 days p.i. in popliteal LNs of WT (black) or CCR2^-/- (blue) mice that were transferred with 5 x 10⁶ KL25 B cells prior to s.c. LCMV infection. Results are representative of at least two independent experiments.

Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Inflammatory monocytes recruited to LCMV-infected LNs interact with Ag-specific B cells and impair their survival

We next sought to characterize how inflammatory monocytes recruited to LNs draining LCMV infection sites hindered Ab responses. Because inflammatory monocytes are characterized by the concomitant expression of the chemokine receptors CX3CR1 and CCR2 (20, 26), we crossed CX3CR1^GFP/+ (27) to CCR2^RFP/+ (28) reporter mice to visualize this cell population in LCMV-draining LNs. While uninfected CX3CR1^GFP/+;CCR2^RFP/+ mice had very few LN cells that expressed both GFP and RFP, LCMV infection induced a prominent accumulation of GFP⁺RFP⁺ in the LN IFAs (Fig. 3A and Fig. S8, A to C). Note that the majority (~51%) of GFP⁺RFP⁺ cells within LNs of infected CX3CR1^GFP/+;CCR2^RFP/+ mice were indeed CD11b⁺Ly6C^hi monocytes and, conversely, virtually all CD11b⁺Ly6C^hi cells expressed both GFP and RFP (Fig. S8, A and B).

Figure 3. Inflammatory monocytes recruited to LCMV-infected LNs interact with Ag-specific B cells and impair their survival.

(A) Confocal micrographs in the popliteal LNs of CX3CR1^GFP/+;CCR2^RFP/+ mice that were transferred with fluorescent WT (cyan) and KL25 (red) B cells, injected s.c. with either PBS (left panel) or LCMV (right panel) and sacrificed 72h after infection. Scale bars represent 100μm. Dotted white line denotes B cell follicle. Results are representative of at least three independent experiments. (B) Multiphoton intravital micrograph in the popliteal LNs of a LCMV-infected CX3CR1^GFP/+;CCR2^RFP/+ mouse that was transferred with fluorescent WT (cyan) and KL25 (red) B cells and sacrificed 72h after infection. Scale bar represents 20 μm. Result is representative of at least three independent experiments. See also Movie S4. (C) Quantification of the contact time (minutes) between CXCR1⁺ CCR2⁺ cells and WT (blue) or KL25 (red) B cells in the popliteal LNs of CX3CR1^GFP/+;CCR2^RFP/+ mice that were infected s.c. with LCMV 72h earlier. See also Movie S4. n = 4. Results are representative of at least 2 independent experiments. (D-E) 5 x 10⁶ KL25 B cells were transferred into WT (black), IFNAR^-/- (red), CCR2^-/- (blue) or WT mice injected with anti-Gr-1 (green) or isotype control (black) Abs and infected s.c. with LCMV. Frequency of annexin V⁺ apoptotic KL25 B cells (D) and number of KL25 B cells (E) were quantified in the popliteal LNs 3 days after infection. Results are representative of at least five independent experiments. (F) 5 x 10⁶ VI10YEN B cells were transferred into WT mice prior to s.c. immunization with PFA-inactivated VSV, and either simultaneously infected (red) or not (black) with LCMV. VI10YEN B cells were quantified in the popliteal LN 72h after immunization. Results are representative of at least two independent experiments.

Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Because inflammatory monocytes within LNs of LCMV-infected mice were found to almost completely co-localize with LCMV-specific B cells (Fig. 3A), we hypothesized that inflammatory monocytes might physically interact with Ag-specific B cells to suppress Ab production. To test this hypothesis, we performed MP-IVM in popliteal LNs of CX3CR1^GFP/+;CCR2^RFP/+ mice that were adoptively transferred with fluorescent WT and KL25 B cells prior to LCMV infection. As shown in Movie S4 and Fig. 3, B and C, inflammatory monocytes engaged in prolonged interaction with LCMV-specific B cells but not with control WT B cells. That such interactions are responsible for the confined motility of LCMV-specific B cells within LN IFAs of WT mice is further demonstrated by the observation that LCMV-specific B cells had a higher meandering index when transferred to CCR2^-/- mice than to WT mice (Fig. S9). We then asked whether the physical interaction of inflammatory monocytes with LCMV-specific B cells affected B cell survival. Indeed, a lower frequency of apoptotic cells was detected within KL25 B cells isolated from the LNs of IFNAR^-/-, CCR2^-/- or anti-Gr-1-treated mice as compared to KL25 B cells isolated from the LNs of WT mice (Fig. 3D); accordingly, depletion of inflammatory monocytes or inhibition of their LN recruitment led to a higher recovery of KL25 B cells in the popliteal LN of LCMV-infected mice (Fig. 3E). In contrast to the striking effect on KL25 B cell survival, inflammatory monocytes did not significantly affect KL25 B cell proliferation or early activation phenotype upon LCMV infection (Fig. S10). We next asked whether the detrimental effect that inflammatory monocytes recruited to LCMV-infected LNs exert on B cells was dependent on the Ag specificity of B cells. To this end, we sought to analyze VSV-specific B cell responses in LCMV-infected mice. Because in co-infection experiments the IFN-I produced in response to LCMV might affect VSV titers (and hence the amount of viral Ags available for B cells), we decided to challenge LCMV-infected mice with paraformaldehyde (PFA)-inactivated VSV. As shown in Fig. 3F, fewer VI10YEN B cells were recovered in LCMV-infected mice concomitantly challenged with paraformaldehyde (PFA)-inactivated VSV 3 days earlier when compared to mice immunized with inactivated VSV alone. Together, these results indicate that inflammatory monocytes recruited to LNs draining an LCMV infection site interact with activated B cells regardless of their Ag-specificity, and induce B cell apoptosis.

We next asked whether VSV induced LN recruitment of inflammatory monocytes and, if so, whether these cells suppressed Ag-specific B cell responses in that setting as well. CD11b⁺Ly6C^hi monocytes were indeed recruited to VSV-infected LNs within 24 h p.i. but they disappeared by 48-72 hours p.i. (Fig. S11A), likely because CCL-2 expression within VSV-infected LNs was not sustained (Fig. S11B). Possibly due to this rapid clearance, CD11b⁺Ly6C^hi cells recruited to VSV-infected LNs did not suppress VSV-specific B cell responses, as VSV-infected D_HLMP2A mice injected with VI10YEN showed no differences in VSV-specific B cell recovery or in neutralizing Ab titers when treated with anti-Gr-1 or isotype-matched Abs (Fig. S11, C to E).

Inflammatory monocytes inhibit B cell responses via nitric oxide

Next, we sought to characterize the mechanism whereby inflammatory monocytes recruited to LNs draining LCMV infection sites hinder B cell responses. We first checked whether T cells were required for monocyte-induced B cell suppression. In contrast to the well-established role that both CD4⁺ and CD8⁺ T cell play in influencing LCMV-specific B cell responses at late time points after infection (29–33), depletion of either CD4⁺ or CD8⁺ cells did not alter KL25 B cell recovery 3 days p.i. (Fig. S12, A and B), suggesting that inflammatory monocyte-mediated B cell suppression occurs independently of CD4⁺ and CD8⁺ T cells. We then set out to identify the molecular mediator of monocyte-mediated B cell inhibition. We focused on nitric oxide, a well-established myeloid cell-derived immune suppressive factor (18, 34). In order to create a system in which CCR2⁺ inflammatory monocytes that accumulate within LCMV-infected LNs (but not CCR2^- cells) lacked the capacity to produce nitric oxide, we reconstituted irradiated WT recipients with a 1:1 mixture of iNOS^-/- and CCR2^-/- BM. Irradiated WT recipients that were reconstituted with 100% WT BM, 100% CCR2^-/- BM or a 1:1 mixture of WT and CCR2^-/- BM served as controls. Notably, nitric oxide did not affect the capacity of inflammatory monocytes to accumulate within LCMV-infected LNs, but it severely impaired their B cell suppressive ability (Fig. 4, A to C). Together, these results indicate that inflammatory monocytes hinder B cell responses via nitric oxide.

Figure 4. Inflammatory monocytes inhibit B cell responses via nitric oxide.

Irradiated WT recipients were reconstituted with WT (black), CCR2^-/- (blue), a 1:1 mixture of WT and CCR2^-/- (gray) and a 1:1 mixture of iNOS^-/- and CCR2^-/- (orange) bone marrow 8 weeks prior to injection with 5 x 10⁶ KL25 B cells followed by s.c. LCMV infection. (A) Number of CD11b⁺Ly6C^hi cells recovered in the popliteal LNs 3 days after infection. (B) Number of KL25 B cells recovered in the popliteal LNs 3 days after infection. (C) Serum LCMV-GP-binding Abs (fold induction over uninfected controls) measured 7 days after infection. Data are representative of at least two independent experiments.

Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

In this study, we have analyzed the spatial and temporal constrains whereby virus-specific naïve B cells respond to infections with live lymph-borne viruses that either do (VSV) or don’t (LCMV) elicit early, robust neutralizing Ab responses. We found that, whereas the spatiotemporal dynamics of early B cell activation in response to live VSV follows what has been previously described with non-replicating Ags (13–15, 35), upon LCMV infection Ag-specific B cells readily relocate to the IFA and T cell area of the draining LN where they extensively interact with CD11b⁺Ly6C^hi inflammatory monocytes that are recruited in a type I interferon-, CCR2-dependent manner. The main consequence of such interaction is impaired B cell survival, caused by nitric oxide produced by inflammatory monocytes. Indeed, depletion of inflammatory monocytes, inhibition of their LN recruitment or deficiency in their nitric oxide production improves LCMV-specific B cell survival and leads to robust neutralizing Ab production.

Our results shed light on a long-standing question in viral immunobiology, which is how non-cytopathic viruses such as LCMV avoid the induction of early, effective neutralizing Ab responses (1). The mechanism constraining the early B cell response to LCMV described here has significant functional consequences in terms of viral clearance, as mice devoid of inflammatory monocytes have > 50-fold lower LCMV titers than WT mice 7 days after infection. It is important to point out that type I interferon receptor blockade had a larger effect on B cell responses than CCR2 blockade or Gr-1⁺ cell depletion, suggesting that LN recruitment of inflammatory monocytes is only one of the mechanisms whereby type I interferon impairs antiviral Ab responses. The identification of additional type I interferon-dependent mechanisms limiting humoral immune responses requires further investigation.

Although the capacity of myeloid-derived cells to suppress T cell responses is being increasingly recognized (17–19), very little is known on myeloid cell-mediated B cell inhibition. The results reported here unravel a key role for inflammatory monocytes in limiting humoral responses and pave the road to further investigations aimed at exploring whether similar suppressive mechanisms are operative in other infections or in the tumor microenvironment. The mechanism whereby inflammatory monocytes engage in prolonged interactions with activated B cells is still unresolved. One possibility is that these cells capture and display surface-bound viral particles (in a way reminiscent of what has been previously described for subcapsular sinus macrophages (15)).

Of note, experimental needs pertaining to intravital microscopy studies required the adoptive transfer of supra-physiological numbers of high affinity virus-specific BCR transgenic cells, which raises the question of whether the immune suppressive mechanism described here is operative for B cells expressing BCRs with lower affinity. Although the notion that inflammatory monocytes suppress LCMV-specific Ab responses in WT mice as well – where, presumably, BCRs with a wide range of affinities for LCMV are present – suggests that this is a general phenomenon that occurs independently of BCR signal strength, further investigation is needed to definitively settle this issue.

Whether the suppressive effect of monocyte-derived nitric oxide on B cells is direct or indirect remains to be clarified. Indeed, nitric oxide produced by myeloid-derived suppressor cells has been shown, in certain experimental conditions, to inhibit T cell responses (17, 18). Of note, we ruled out a role for CD4⁺ or CD8⁺ T cells in the B cell response to LCMV at day 3 post infection (time point in which the effect of inflammatory monocytes on B cell responses is already evident), suggesting that inflammatory monocyte-mediated B cell suppression is T cell-independent. Although these data, together with the reported extensive interactions between monocytes and activated B cells, argues in favor of monocytes directly inhibiting B cells, further investigation is warranted.

The results presented here establish inflammatory monocytes as critical gatekeepers that prevent antiviral B cell responses. From an evolutionary perspective, it is tempting to speculate that viruses such as LCMV might have evolved strategies to exploit the recruitment of these cells to LNs draining the infection site in order to prolong their persistence within the host and maximize the chance of being transmitted to other susceptible individuals.

Materials and methods

Mice

C57BL/6 and CD45.1 (inbred C57BL/6) mice were purchased from Charles River. D_HLMP2A mice (7) (inbred Balb/c) were originally provided by K. Rajewsky (Harvard Medical School) and bred more than 10 generations against C57BL/6 mice. Heavy chain knock-in and light chain BCR transgenic mice specific for VSV Indiana (VI10YEN (6)) and heavy chain knock-in BCR-transgenic mice specific for LCMV WE (KL25 (6)) were obtained through the European Virus Archive. Light chain BCR transgenic mice specific for LCMV WE (inbred C57BL/6) were generated as described in the legend to Fig. S1A and bred against heavy chain BCR transgenic mice specific for LCMV WE prior to subsequent use (except in selected experiments where Ab titers against LCMV were measured). β-actin-DsRed (B6.Cg-Tg(CAG-DsRed*MST)1Nagy/J) and β-actin-CFP (B6.129(ICR)-Tg(CAG-ECFP)CK6Nagy/J) transgenic mice were purchased from The Jackson Laboratory. For imaging experiments VI10YEN and KL25 mice were bred against both β-actin-DsRed and β-actin-CFP mice. Mice lacking the type I interferon receptor (IFNAR1^-/- mice) were obtained through the Swiss Immunological Mutant Mouse Repository (Zurich, Switzerland). CCR2^-/- (B6.129S4-Ccr2tm1Ifc/J) mice were purchased from The Jackson Laboratory. CX3CR1^GFP/+ (27) and CCR2^RFP/+ (28) mice were provided by I. Charo (UCSF) by way of B. Engelhardt (University of Bern). Bone marrow from iNOS^-/- mice was provided by E. Clementi (University of Milan). Bone marrow chimeras were generated by irradiation of C57BL/6 mice with ~730 rad and reconstitution with the indicated bone marrow; mice were allowed to reconstitute for at least 8 weeks prior to use. Mice were housed under specific pathogen-free conditions and used at 8-10 weeks of age, unless otherwise indicated. In all experiments, mice were matched for age and sex before experimental manipulation. All experimental animal procedures were approved by the Institutional Animal Committee of the San Raffaele Scientific Institute.

Infections and immunizations

Mice were infected intrafootpad with 1 x 10⁵ plaque-forming units (pfu) of VSV serotype Indiana, 1 x 10⁵ pfu of VSVeGFP (9, 10), 1 x 10⁵, 1 x 10⁶ or 1 x 10⁷ focus-forming units (ffu) of LCMV-M1 (a variant of LCMV Clone 13 that is recognized by KL25 B cells) (11) or 1 x 10⁵ ffu of LCMV-M1eGFP. Alternatively, mice were immunized intrafootpad with 10 μg of recombinant LCMV WE glycoprotein (GP)-1-human IgG fusion protein (36) mixed at 1:1 ratio with a squalene-based oil-in-water nano-emulsion (AddaVax, InvivoGen) or with Alum (Sigma). Viruses were propagated and quantified as described (37, 38), and dissolved in 20 μl of PBS prior to footpad injection.

Mice were retro-orbitally bled at the indicated time points for VSV- or LCMV-specific Abs measured by VSV neutralization assay (37), LCMV focus reduction assay (39) or LCMV GP-1-binding ELISA (40). For determination of VSV- or LCMV-neutralizing IgG titers, sera were incubated with equal volumes of 0.1 M 2-mercaptoethanol in DMEM 2% FCS for 1 hour at room temperature before dilution (41). Viral titers from sera, LNs and spleens of VSV- or LCMV-infected mice were measured by plaque (8) or focus assay (39), respectively. All infectious work was performed in designated BSL-2 and BSL-3 workspaces in accordance with institutional guidelines.

B cell activation in vitro

Naïve B cells from spleens of VI10YEN and KL25 mice were negatively selected by magnetic isolation with CD43 beads (Miltenyi Biotec), as described (37). The purity was ~98% as determined by B220 staining. Purified B cells (10⁶ cells / ml) were cultured in the presence of PFA-inactivated VSV or LCMV (0.5 m.o.i. equivalent). In some experiments, B cells were harvested after 24 hours and analyzed by flow cytometry for the surface expression of activation markers (see below). In other experiments, B cells were cultured for 72 hours and the supernatants were tested for the presence of total mouse IgM by ELISA.

Adoptive transfer experiments

Naïve B cells from spleens of C57BL/6, VI10YEN, VI10YEN x β-actin-DsRed, VI10YEN x β-actin-CFP, KL25, KL25 x β-actin-DsRed and KL25 x β-actin-CFP mice were negatively selected by magnetic isolation with CD43 beads (Miltenyi Biotec), as described (37). The purity was ~98% as determined by B220 staining. 1 x 10⁶, 5 x 10⁶ or 1 x 10⁷ B cells were injected i.v. into the indicated recipient animals 18 hours prior to footpad virus infection. In selected imaging experiments, WT, VI10YEN and KL25 B cells were labeled with 10 μM CMAC, 10 μM CMTMR, 2.5 μM BODIPY 650/665 or 1 μM Deep Red (Life Technologies) for 20 min at 37°C in plain RPMI prior to adoptive transfer, as described (42). In some experiments, inflammatory monocytes were depleted by intravenous injection of 200 μg of anti-Gr-1 (RB6-8C5, Bioxcell) Abs every 48 hours, beginning 1 day prior to footpad virus infection. In other experiments, CD4⁺ T cells were depleted by intravenous injection of 200 μg of anti-CD4 (GK1.5, Bioxcell) Abs three days and one day prior to infection. In other experiments CD8⁺ T cells were depleted by intravenous injection of 200 μg of anti-CD8 (YTS 169.4, Bioxcell) Abs one day prior to and three days after infection. For IL10R-blocking experiments, 500 μg of anti-IL-10R (1B1-3a, Bioxcell) Abs were injected i.v. six hours after footpad infection. For monocyte reconstitution experiments, monocytes from the bone marrow of C57BL/6 or CX3CR1-GFP; CCR2 RFP mice were negatively selected by magnetic isolation using the Monocyte Isolation Kit (BM) (Miltenyi Biotec), according to the manufacturer instructions. The purity was > 95% as determined by CD11b and Ly6C staining. 5 x 10⁶ monocytes were injected i.v. into CCR2^-/- mice six hours after footpad infection.

Cell isolation and flow cytometry

Single-cell suspensions of spleens and LNs were generated as described (37). All flow cytometry stainings of surface-expressed and intracellular molecules were performed as described (37, 42). Fluorochrome-conjugated Abs to B220 (RA3-6B2), CD45.1 (A20), CD45.2 (104), CD69 (H1.2F3), CD25 (PC61), CD11b (M1/70), Ly6G (IA8), Ly6C (HK1.4), F4/80 (BM8), CD11c (N418), MHCII (I-Ab, AF6-120.1), CD4 (RM4-5), CD8 (53-6.7), CXCR5 (2G8), IgD (11-26c.2a), CD80 (16-10A1), IFN-γ (XMG1.2) and TNF-α (MP6-XT22) were purchased from BioLegend, eBioscience or BD Pharmingen. B cell proliferation was assessed by CFSE dilution, as described (10). Intracellular IFN-γ staining was performed in the presence or absence of GP61 or GP33, as described (43). The proportion of LCMV-binding B cells in WT or KL25 mice was assessed by virtue of their binding ability to the recombinant LCMV WE glycoprotein (GP)-1-human IgG fusion protein (36), using the Strep-tag®/Strep-Tactin® system (IBA), according to the manufacturer’s instructions. All flow cytometry analyses were performed in FACS buffer containing PBS with 2 mM EDTA and 2% FBS on either a FACS CANTO or a FACS FORTESSA (BD Pharmingen) and analyzed with FlowJo software (Treestar).

Confocal immunofluorescence histology

Confocal microscopy analysis of popliteal LNs was performed as previously described (37). The following primary Abs were used for staining: rat anti-B220 (RA3-6B2), rat anti-CD169 (AbD Serotec), rabbit anti-GFP (Invitrogen), rat anti-LCMV Nucleoprotein (VL-4, Bioxcell), rat anti-TCRβ (H57-597, BioLegend). The following secondary Abs were used for staining: Alexa Fluor 488-, 514-, 568- or 647-conjugated goat anti-rabbit or anti-rat IgG (Life Technologies), Alexa Fluor 647-conjugated chicken anti-rat IgG (Life Technologies). Images were acquired on an inverted Leica microscope (SP8, Leica Microsystems) with a motorized stage for tiled imaging. To minimize fluorophore spectral spillover, we used the Leica sequential laser excitation and detection modality. B cell follicles, interfollicular areas (IFA) and T cell areas were defined based on the positioning of naïve B cells and, in certain experiments, by B220 and TCRβ stainings, as described (44).

Statistical analyses

Results are expressed as mean ± SEM. All statistical analyses were performed in Prism 5 (GraphPad Software). Means between two groups were compared with unpaired two-tailed t test. Means among three or more groups were compared with one-way or two-way ANOVA with Bonferroni post-test.

One sentence summary.

LCMV evades B cell responses by recruiting inflammatory monocytes to draining lymph nodes