ANTIBODIES Copyright © 2018 Enhancing Fc R …...Enhancing Fc R-mediated antibody effector function during persistent viral infection Andreas Wieland1*, Alice O. Kamphorst1†, Rajesh

Wieland et al., Sci. Immunol. 3, eaao3125 (2018) 21 September 2018

S C I E N C E I M M U N O L O G Y | R E S E A R C H A R T I C L E

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ANTIBODIES

Enhancing FcR-mediated antibody effector function during persistent viral infectionAndreas Wieland1*, Alice O. Kamphorst1†, Rajesh M. Valanparambil1, Jin-Hwan Han1‡, Xiaojin Xu1, Biswa P. Choudhury2, Rafi Ahmed1*

Persistent viral infections can interfere with FcR-mediated antibody effector functions by excessive immune complex (IC) formation, resulting in resistance to therapeutic FcR-dependent antibodies. We and others have previously demonstrated that mice persistently infected with lymphocytic choriomeningitis virus (LCMV) are resistant to a wide range of depleting antibodies due to excessive IC formation. Here, we dissect the mechanisms by which two depleting antibodies overcome the obstacle of endogenous ICs and achieve efficient target cell depletion in persistently infected mice. Efficient antibody-mediated depletion during persistent LCMV infection required increased levels of antibody bound to target cells or use of afucosylated antibodies with increased affinity for FcRs. Antibodies targeting the highly expressed CD90 antigen or overexpressed human CD20 efficiently depleted their target cells in naïve and persistently infected mice, whereas antibodies directed against less abundant antigens failed to deplete their target cells during persistent LCMV infection. In addition, we demonstrate the superior activity of afucosylated antibodies in the presence of endogenous ICs. We generated afucosylated anti-bodies directed against CD4 and CD8, which, in contrast to their parental fucosylated versions, efficiently depleted their respective target cells in persistently infected mice. Efficient antibody-mediated depletion can thus be achieved if therapeutic antibodies can outcompete endogenous ICs for access to FcRs either by targeting highly expressed antigens or by increased affinity for FcRs. Our findings have implications for the optimization of therapeutic antibodies and provide strategies to allow efficient FcR engagement in the presence of competing endogenous ICs in persistent viral infections, autoimmune diseases, and cancer.

INTRODUCTIONVarious immune cell dysfunctions have been documented during persistent viral infections such as hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Whereas the mechanisms and consequences of T cell dysfunction during persistent viral infections are well documented, little is known about the impact of antigen persistence on the humoral immune response and antibody- dependent effector functions. We and others have recently shown that excessive immune complex (IC) formation in mice persistently infected with lymphocytic choriomeningitis virus clone-13 (LCMV clone-13) interferes with various Fc receptor (FcR)–mediated anti-body effector functions (1, 2). IC formation during persistent LCMV infection was shown to interfere with FcR-mediated antibody effec-tor functions without negatively affecting the overall expression pat-tern of FcRs (1, 2). Furthermore, the inhibition of FcR-mediated effector functions during persistent LCMV infection was due to IC formation and not the persistent infection per se, as persistent LCMV infection in the absence of an antiviral B cell response did not inter-fere with FcR-mediated effector function.

The efficient engagement of FcRs on immune effector cells, such as macrophages and natural killer cells, by therapeutic immu-noglobulin Gs (IgGs) such as rituximab, an anti-CD20 monoclonal antibody (mAb) used for the treatment of B cell non-Hodgkin lym-

phoma (NHL), is crucial for their in vivo activity (3, 4). Further-more, broadly neutralizing antibodies against persistent viral in-fections such as HIV require Fc-FcR interactions for optimal therapeutic activity in vivo (5, 6). During persistent LCMV infec-tion, ICs inhibited FcR- mediated effector functions involved in controlling viral infections, including antibody-dependent cyto-toxicity (ADCC) of infected cells and antigen cross-presentation to CD8+ T cells, and required for the therapeutic activity of depleting and agonistic antibodies (1, 2).

Elevated levels of circulating ICs and deposition of ICs in the kidney resulting in glomerulonephritis have been reported to occur in patients persistently infected with viruses such as HBV, HCV, and HIV (7–9). Furthermore, circulating ICs have been detected in the sera of cancer patients, and epidemiological data show an association between membranous nephropathy and cancer (10, 11). In addition, excessive IC formation due to the presence of auto-antibodies is a hallmark of systemic lupus erythematosus (SLE), an autoimmune disease in which, upon rituximab treatment, consider-able variation in B cell depletion efficacy is observed (12). It was subsequently shown in the murine MRL/lpr lupus model that in-creased levels of circulating ICs could reduce or even completely abrogate B cell depletion by rituximab (13, 14). Together, these studies demonstrate that excessive IC formation and subsequent interference with FcR-mediated effector functions occur in many diseases, whereas the underlying mechanisms and therapeutic consequences are still ill-defined.

Whereas persistent LCMV infection abrogated the therapeutic activity of a wide range of depleting antibodies (Table 1), the deple-tion of T cells using an anti-CD90.2 antibody was not impaired, demonstrating the general feasibility to deplete cells in the presence of high levels of ICs (1). However, the exact mechanism underlying

1Emory Vaccine Center, Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA. 2Glycotechnology Core Resources, University of California at San Diego, La Jolla, CA 92093, USA.*Corresponding author. Email: [email protected] (A.W.); [email protected] (R.A.)†Present address: Department of Oncological Sciences and Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.‡Present address: Merck Research Laboratories, Palo Alto, CA 94304, USA.

Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

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the depletion efficiency of the anti-CD90.2 antibody during per-sistent LCMV infection remained unknown.

In this study, we thus wanted to explore why some antibodies efficiently deplete their target cells in the presence of excessive IC formation. We analyzed two antibodies that efficiently depleted their respective target cells in an FcR-dependent manner during persistent LCMV infection. These antibodies revealed two distinct approaches enabling antibody-mediated depletion in the presence of competing ICs: (i) targeting highly abundant surface antigens such as CD90 or (ii) using antibodies with altered Fc glycosylation known to increase affinity for FcRs. Our results demonstrate that therapeutic antibodies can outcompete endogenous ICs for FcRs, resulting in efficient depletion if their target antigen is highly ex-pressed or if they have increased affinity for FcRs.

RESULTSFcR-dependent anti-CD90.2 efficiently depletes T cells during persistent LCMV infectionWe and others have previously shown that persistent LCMV infec-tion impairs antibody-mediated depletion and other FcR-mediated antibody effector functions due to excessive IC formation (1, 2). This defect was observed with a wide range of therapeutic anti-bodies differing in antigen specificity, isotype, or species of origin (Table 1). Injection of 500 g of anti-CD4 (clone GK1.5) and anti-

CD8 (clone 2.43) on two consecutive days resulted in efficient depletion of CD4+ and CD8+ T cells, respectively, from the periph-eral blood of naïve mice within 2 days (Fig. 1A). In contrast, the same treatment regimen failed to deplete CD4+ or CD8+ T cells from the peripheral blood of persistently infected mice. However, two injections of 250 g of anti-CD90.2 (clone 30H12) were suffi-cient to efficiently deplete T cells (>94%) from the peripheral blood of naïve and persistently infected animals within 2 days (Fig. 1, B and C). Similar results were obtained when we analyzed the T cell depletion efficiency in the spleen (Fig. 1D). The superior depletion activity of the anti-CD90.2 antibody in persistently infected mice could not simply be explained by its isotype or species of origin, because all antibodies (anti-CD4, anti-CD8, and anti-CD90.2) were confirmed to be rat IgG2b isotype.

FcR-mediated phagocytosis by macrophages is the main mech-anism of depleting antibodies targeting circulating cells in vivo (3, 15). However, FcR-independent mechanisms of action, such as complement-dependent cytotoxicity and direct induction of cell death, have also been proposed (16, 17). We thus sought to deter-mine whether the anti-CD90 antibody was potentially circum-venting the obstacle of competing ICs during persistent infection by depleting T cells via FcR-independent mechanisms. To address this question, we used mice deficient for the common chain sub-unit required for the surface expression and function of activating FcR I, III, and IV (FcR−/−). In contrast to wild-type mice, treat-ment of FcR−/− mice with anti-CD90.2 did not result in a measur-able reduction in T cell numbers, suggesting that anti-CD90.2 mediates its depletion activity in vivo solely through the engage-ment of activating FcRs (Fig. 1E). In addition, the depletion activity of anti-CD90.2 was dependent on the presence of phago-cytes because mice depleted of phagocytes using clodronate lipo-somes were resistant to T cell depletion mediated by anti-CD90.2 (Fig. 1F). Injection of clodronate liposomes resulted in the effi-cient depletion of phagocytic cells, such as red pulp macrophages (>85%), without affecting T cell numbers (fig. S1, A and B). These data demonstrate that the CD90.2-specific antibody used in this study can efficiently deplete T cells during persistent LCMV in-fection despite its dependence on FcRs, raising the question about the mechanism allowing it to bypass the obstacle of competing endogenous ICs.

High expression of CD90 allows efficient T cell depletion during persistent LCMV infectionWe next sought to determine how the FcR-dependent anti-CD90.2 antibody could efficiently mediate T cell depletion during persistent LCMV infection. CD90 is one of the most abundantly expressed proteins on the surface of T cells with about 200,000 copies per cell (18). Using quantitative flow cytometry, we confirmed that CD90.2 is highly expressed on splenic T cells of persistently infected mice and compared its expression with CD4 and CD8 (Fig. 2A), both targeted by rat IgG2b antibodies unable to deplete their targets during persistent LCMV infection (Fig. 1A). Using calibration beads with known numbers of fluorophores and antibodies with known fluorophore-to-protein ratios under saturating staining conditions, we determined the number of surface molecules per cell assuming a 1:1 binding interaction between antibodies and surface antigens. The number of CD90.2 molecules on splenic T cells was about four- to sixfold higher compared with the number of CD4 and CD8 molecules on the respective splenic T cell subset (Fig. 2A). We thus

Table 1. Antibodies with severely impaired activity during persistent LCMV infection and murine SLE model. tg, transgenic; Treg cells, regulatory T cells; DCs, dendritic cells.

Specificity (clone) Species and isotype Target Reference

Anti-hCD20 (rituximab)

Human IgG1 hCD20 tg B cells

(1, 2, 13)

Anti-hCD20 (2H7) Mouse IgG2b hCD20 tg B cells

(1, 13)

Anti-hCD20 (8B9) Mouse IgG2a/c hCD20 tg B cells

(13)

Anti-CD20 (MB20)

Mouse IgG2a/c B cells (2)

Anti-CD20 (18B12)

Mouse IgG1 B cells (13)

Anti-CD4 (GK1.5) Rat IgG2b CD4 T cells (1, 2)

Anti-CD4 (YTS191)

Rat IgG2b CD4 T cells (2)

Anti-CD25 (PC61) Rat IgG1 Treg cells (1)

Anti-CD8 (2.43) Rat IgG2b CD8 T cells (1, 2)

Anti-CD8 (53.6.72)

Rat IgG2a CD8 T cells (2)

Anti-platelets (6A6)

Mouse IgG2a/c Platelets (2)

Anti-LCMV-GP (KL25)

Mouse IgG1 LCMV-infected cells

(2)

Anti-CD40 (FGK4.5)

Rat IgG2a DCs, B cells (1)


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hypothesized that the high abundance of CD90 on the surface of T cells and thus increased amount of bound antibody surpass the threshold required for efficient FcR engagement in the presence of competing ICs.

To determine whether increased amounts of bound antibody are required to promote efficient depletion during persistent LCMV infection, we performed in vivo depletion assays using fluorescently labeled T cells coated with antibody (1). We cotransferred differ-entially labeled T cells coated with titrated amounts of anti-CD90.2 antibody into naïve or infected hosts (Fig. 2B). T cells coated with high amounts of anti-CD90.2 (0.8 to 4 g/107 T cells) were effi-ciently depleted in naïve and persistently infected mice, whereas low to intermediate amounts of anti-CD90.2 (3 to 50 ng/107 T cells) only resulted in measurable depletion in naïve but not infected hosts (Fig. 2, C and D). Achieving 50% depletion of transferred cells in persistently infected recipients required about 10-fold more anti- CD90.2 compared with naïve controls (Fig. 2E). Similar results were obtained upon transfer of T cells coated with saturating amounts of a pan-CD90 specific antibody (clone T24/31, rat IgG2b) into naïve or persistently infected mice (fig. S2, A and B). Together, these data demonstrate that, in the presence of competing ICs, increased levels of antibody are required to bind to the target cells for their efficient depletion.

Increased surface antigen expression facilitates efficient antibody-mediated depletion during persistent LCMV infectionWe next sought to confirm our findings and exclude that antigen- specific features of CD90 such as clustering patterns, cell surface half-life, or reinternalization rate contributed to the high in vivo depletion efficiency of anti-CD90 antibodies. We thus developed a model allowing us to investigate the impact of differential ex-pression of an additional model antigen, human CD20 (hCD20), on a standardized target cell (EL4) on antibody-mediated depletion in vivo. We generated three hCD20-expressing EL4 cell lines differing in their surface expression of hCD20, ranging from ~2700 mole-cules (low) to ~80,000 molecules per cell (high; Fig. 3A). To assess in vivo depletion, we cotransferred differentially labeled EL4 hCD20 cell lines together with the parental EL4 cell line to naïve or per-sistently infected hosts that received the hCD20-specific murine mAb 2H7 before cell transfer (Fig. 3B). In naïve mice, the depletion efficiency coincided with the expression level of hCD20 on EL4 cells—with low hCD20 expression resulting in ~50%, intermediate expression in ~70%, and high expression in ~90% depletion (Fig. 3, B and C). In persistently infected mice, EL4 cells expressing high levels of hCD20 were efficiently depleted to an extent that was slightly lower but overall comparable with naïve mice, whereas low

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Fig. 1. FcR-dependent anti-CD90.2 efficiently depletes T cells during persistent LCMV infection. (A) Naïve and LCMV clone-13–infected mice (28 dpi) were injected twice intraperitoneally with PBS, 500 g of anti-CD4, or 500 g of anti-CD8. Representative flow plots show the frequency of CD8+ and CD4+ T cells among peripheral blood mononuclear cells (PBMCs) 2 days after treatment start. Graphs show the depletion efficiency of anti-CD4 and anti-CD8. (B to D) Naïve and LCMV clone-13–infected mice (28 dpi) were injected twice intraperitoneally with PBS or 250 g of anti-CD90.2. (B) Representative flow plots showing the frequency of CD3+ T cells among PBMCs 2 days after treatment start. (C and D) T cell depletion efficiency in peripheral blood and spleen. (E) Wild-type (wt) and FcR−/− mice were injected twice intraperitoneally with PBS or 250 g of anti-CD90.2, and the number of splenic CD3+ T cells was assessed after 2 days. (F) Wt mice were injected with 200 l of clodronate liposomes or PBS, followed by anti-CD90.2 treatment and analysis as described in (E). Data (mean and SEM) from one representative experiment of at least two are shown (n = 3 to 4 mice per group). Unpaired two-sided Student’s t test was used for analyses shown in (A) and (E). ***P < 0.001. Ordinary one-way ANOVA with post hoc Tukey’s test for multiple comparisons was used for analysis shown in (F). ns, not significant. ****P < 0.0001. by guest on June 8, 2020

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and intermediate expression levels failed to efficiently deplete cells (Fig. 3, B and C). Despite expressing ~7-fold less antigen, EL4 hCD20 low cells were more efficiently depleted in naïve mice than EL4 hCD20 medium cells in infected mice. Overall, these data demonstrate that, although in naïve mice depletion efficiency is also dependent on the surface antigen expression level, increased anti-gen expression on the cell surface is required to promote efficient depletion in the presence of competing ICs.

FcR-dependent anti-CD8 efficiently depletes CD8+ T cells during persistent LCMV infectionDuring our study, we also tested an anti-CD8 antibody (clone H35-17.2, rat IgG2b) for its ability to deplete CD8+ T cells in naïve and LCMV-infected mice. Unexpectedly, two injections of 250 g of this antibody were sufficient to achieve efficient depletion in the peripheral blood and spleen of naïve and persistently infected animals (Fig. 4, A to C). The depletion activity of this anti-CD8 antibody, which was confirmed to be of the rat IgG2b isotype, cru-cially depended on the engagement of activating FcRs, because FcR−/− mice showed no reduction in splenic CD8+ T cells upon treatment with anti-CD8 (Fig. 4D). We also show that CD8+ T cell depletion by anti-CD8 requires phagocytosis, because mice lack-ing phagocytes due to injection of clodronate liposomes failed to deplete CD8+ T cells upon anti-CD8 treatment (Fig. 4E). Our above experiments with CD90-specific antibodies showed that high surface

expression allows efficient target cell depletion during persistent infection. We thus quantified the expression of CD8 on splenic CD8+ T cells and compared its expression with CD90.2 and CD8, an antigen also targeted by rat IgG2b antibodies, which failed to deplete during persistent LCMV infection (Table 1). The expression level of CD8 was about five- to sixfold lower compared with that of CD90.2 and was even slightly lower, although not statistically sig-nificant, compared with that of CD8 (Fig. 4E). These data demon-strate that the FcR- dependent anti-CD8 antibody can efficiently deplete CD8+ T cells during persistent LCMV infection despite anti-gen expression levels being similar to other “unresponsive” target antigens such as CD8 and CD4.

The anti-CD8 antibody exhibits increased glycosylation heterogeneityThe above data suggested that the anti-CD8 antibody more efficient-ly engaged FcRs in the presence of competing ICs than anti- CD4 and anti-CD8 antibodies, despite identical isotype and species of origin of the antibodies (rat IgG2b isotype) and comparable expres-sion levels of target antigens. IgGs of all species and subclasses have a conserved N-linked glycosylation site N297 in the Fc por-tion, which is crucial for the binding to FcRs. Fc glycans share a common biantennary heptasaccharide core with varying extensions modulating the affinity to FcRs and rendering this region tradi-tionally considered as invariant highly variable (19). We thus sought

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Fig. 2. High expression of CD90 allows efficient T cell depletion during persistent LCMV infection. (A) Surface expression of CD90.2, CD4, and CD8 on CD3+ T cells, CD4+ T cells, and CD8+ T cells, respectively, in the spleen of LCMV clone-13–infected mice (28 dpi). Data (mean and SEM) from one representative experiment (n = 5 mice) of two are shown. Ordinary one-way ANOVA with post hoc Tukey’s test for multiple comparisons was used for analysis. (B) Experimental design. iv, intravenous. (C) Rep-resentative flow plots of transferred cell populations (CD3+ TCR+ CellTrace Violet/CFSE+) before and 3 to 4 hours after transfer to naïve and LCMV clone-13–infected (28 dpi) recipients. Numbers indicate the percentages of the individual populations among transferred cells. (D) Graph shows dose-dependent depletion efficiency of T cells in naïve and LCMV clone-13–infected mice. (E) EC50 values (anti-CD90.2 coating concentration required to achieve 50% depletion) were calculated using data shown in (D). Data (mean and SEM) from one representative experiment (n = 5 recipients per group) of three are shown. Unpaired two-sided Student’s t test was used. ****P < 0.0001.


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to determine whether the anti-CD8 antibody used in this study displayed a differential N-glycosylation pattern that could explain its superior activity during persistent LCMV infection. Compared with anti-CD90.2 and anti-CD4, which only contained three glycan structures (G0F, G1F, and G2F), anti-CD8 showed an increased heterogeneity in its N-linked glycans with five glycan structures (G0, G0F, G1F, G0FB, and G1FB) present (Fig. 5, A and B). Galac-tosylation levels (G1 + G2) varied substantially between the ana-lyzed mAbs ranging from 42% down to 5% for anti-CD90.2 and anti-CD8, respectively (Fig. 5C). In contrast to anti-CD90.2 and anti-CD4, which exclusively contained glycans with a core fucose, ~15% of anti- CD8–derived glycans were afucosylated (Fig. 5D). Afucosylation has been shown to increase the affinity of IgG for mouse FcRIV and human FcRIII by at least one order of magnitude (4, 20). In addition, the anti-CD8 antibody contained a measurable fraction of bisecting glycans (~7%; Fig. 5E), which have been suggested to result in a modest increase in affinity for FcRs (21). These data demonstrate that, compared with other antibodies used in this study, anti- CD8 was differentially glyco-sylated containing glycans commonly associated with increased

FcR affinity. These data thus suggest that the anti- CD8 antibody might be able to outcompete endogenous ICs and efficiently engage FcRs during persistent LCMV infection due to in-creased affinity for FcRs. However, we cannot exclude that other antibody- intrinsic features contribute to the ob-served efficacy.

Afucosylated anti-CD4 mediates CD4+ T cell depletion during persistent LCMV infectionThe glycosylation analysis of anti-CD8 suggested that afucosylation and bisec-tion, two glycosylation patterns associ-ated with increased FcR affinity, could be beneficial for depleting antibodies during persistent LCMV infection. Of all tested Fc glycan variants, afucosyla-tion has so far shown the greatest im-pact on the affinity of IgG for FcRs, increasing the affinity for mouse FcRIV and human FcRIIIA 10- to 50-fold (4, 20). We thus sought to determine whether a glycoengineered antibody lacking the core fucose would be more efficiently depleting its target cells during persistent LCMV infection. We chose to generate an afucosylated version of the anti-CD4 antibody (clone GK1.5) because its parental fucosylated ver-sion failed to reduce the number of CD4+ T cells during persistent infection (Fig. 1A). The addition of the core fucose to the Fc glycan is mediated by the -(1,6)- fucosyltransferase (FUT8), which can be inhibited by 2F-peracetyl- fucose (2F) (22). As previously described

for Chinese hamster ovary cells, culturing of the GK1.5 hybri-doma in the presence of increasing concentrations of 2F resulted in a dose-dependent reduction of surface fucosylation, as detected by Aurelia aurantia lectin (AAL) binding (Fig. 6A) (22). Twenty- five micromolar 2F resulted in a surface fucosylation level comparable with a FUT8-deficient GK1.5 hybridoma cell line we generated (GK1.5FUT8). We confirmed the absence of the core fucose on anti-bodies purified from the supernatant of 2F-treated and FUT8- deficient GK1.5 hybridoma cultures by AAL blot analysis and mass spectrometry (Fig. 6, B and C). Besides the lack of fucosylation, the chemical inhibition of FUT8 by 2F or ablation of its expression (GK1.5FUT8) did not change the overall glycan profile of anti- CD4, which was dominated by agalactosylated structures G0/G0F (~85%; Fig. 6C).

We next performed in vivo depletion assays using fluorescently labeled CD4+ T cells coated with antibody to directly compare the ability of fucosylated and afucosylated anti-CD4 antibodies to de-plete their target cells within the same environment (Fig. 6D). CD4+ T cells coated with fucosylated and afucosylated anti-CD4 were efficiently depleted upon transfer into naïve mice, whereas only

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Fig. 3. Increased surface antigen expression facilitates efficient antibody-mediated depletion. (A) Histogram shows surface expression of hCD20 on parental EL4 and hCD20 transgenic EL4 cell lines. Graph shows calculated number of hCD20 molecules per cell. (B) Representative, concatenated flow plots of transferred EL4 cells 3 to 4 hours after transfer to naïve and LCMV clone-13–infected (31 dpi) recipients that received 250 g of anti-hCD20 (2H7) 3 hours before cell transfer or untreated naïve mice. Numbers indicate the percentages of the individual populations among transferred cells. (C) Graph shows depletion efficiency of EL4 hCD20 cells in naïve and LCMV clone-13–infected mice. Data (mean and SEM) from one representative experiment (n = 4 to 8 recipients per group) of two are shown. Ordinary one-way ANOVA with Sidak’s test for multiple comparisons was used. ns, not significant. *P < 0.05, ****P < 0.0001.


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CD4+ T cells coated with afucosylated anti-CD4 were removed upon transfer into LCMV clone-13–infected mice (Fig. 6, D and E). We next sought to determine whether the administration of afucosylated anti-CD4 would result in efficient CD4+ T cell depletion in mice persistently infected with LCMV clone-13. We treated persistently infected animals on two consecutive days with either fucosylated or afucosylated anti-CD4 and assessed CD4+ T cell depletion in the peripheral blood 2 days after treatment initiation (Fig. 6F). Admin-istration of afucosylated, but not fucosylated, anti-CD4 resulted in efficient depletion of CD4+ T cells in the peripheral blood (>97%; Fig. 6G). Moreover, although not as notable as in the peripheral blood, we observed a significant reduction in the number of splenic CD4+ T cells (~75%) within 2 days of treatment initiation with afucosylated anti-CD4 (Fig. 6H). Overall, these data demonstrate the superior acti vity of afucosylated anti-CD4 to deplete its target cells during persistent LCMV infection and suggest that it is possible to outcompete endogenous ICs for access to FcRs if afucosylated antibodies are used.

Afucosylated anti-CD8 mediates CD8+ T cell depletion during persistent LCMV infectionTo further confirm our findings with the afuco-sylated anti-CD4 antibody, we generated an afucosylated version of the anti-CD8 antibody (clone 2.43) because its parental fucosylated version failed to efficiently deplete CD8+ T cells during persistent infection (Fig. 1A). In accor-dance with our observations using the GK1.5 hybridoma, culturing the 2.43 hybridoma in the presence of 25 M of 2F resulted in the produc-tion of a completely afucosylated antibody as demonstrated by AAL blot analysis of purified anti-CD8 (fig. S3A). We first evaluated the depletion efficiency of afucosylated anti-CD8 in peripheral blood, spleen, liver, and the epithelium of the small intestine [intraepithelial lymphocyte (IEL)] of naïve mice. Whereas the afucosylated and the parental fucosylated anti-CD8 antibody caused efficient depletion of CD8+ T cells in both lymphoid and nonlymphoid tissues, afucosylated anti-CD8 exhibited a significantly higher deple-tion activity in all examined tissues (fig. S3, B to F). These data are in accordance with previous results and demonstrate the superior activity of afucosylated antibodies (4, 20). We next sought to determine whether afucosylated anti-CD8 would also result in efficient CD8+ T cell deple-tion in mice persistently infected with LCMV clone-13. Administration of afucosylated, but not fucosylated, anti-CD8 resulted in efficient de-pletion of CD8+ T cells from the peripheral blood and spleen with >90% depletion efficiency in peripheral blood and ~80% reduction in splenic CD8+ T cell numbers (Fig. 7, A to C). Of note, both afucosylated and fucosylated anti-CD8 resulted in a notable reduction of CD8+ T cells in the liver, although afucosylated anti-CD8 was more efficient compared with the parental fucosylated variant, demonstrating ~97 and ~70% deple-tion efficiency, respectively (Fig. 7, A and D).

LCMV clone-13–infected mice exhibited a significant infiltration of CD8+ T cells in the IEL with ~7-fold higher cell numbers compared with naïve controls (fig. S3G). Administration of neither afucosylated nor fucosylated anti-CD8 resulted in a significant depletion of CD8+ T cells in the IEL of infected animals despite the depleting antibody being bound as demonstrated by the blockade of the flow cytometric detection of CD8 (Fig. 7, A and F, and fig. S3H). These data sug-gest that, compared with naïve mice, CD8+ T cells in the IEL of persistently infected mice exhibit a higher degree of tissue residen-cy, do not readily enter circulation, and are thus more resistant to antibody-mediated depletion because intravascular access has been previously shown to be a major determinant of susceptibility to antibody-mediated depletion (15). Overall, our data demonstrate that afucosylated antibodies exhibit superior depletion activity irre-spective of the infection status. However, whereas in naïve mice both fucosylated and afucosylated antibodies efficiently deplete their tar-get cells, during persistent LCMV clone-13 infection only afuco-sylated antibodies can efficiently deplete their target cells. Our data

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thus suggest that it is possible to outcompete endogenous ICs for access to FcRs if antibodies with enhanced affinity for FcRs such as afucosylated antibodies are used.

DISCUSSIONIn this study, we present two distinct strategies to improve FcR engagement of depleting antibodies in the presence of competing endogenous ICs. Efficient antibody-mediated depletion can be achieved by targeting highly abundant antigens such as CD90 or by modifying the Fc glycosylation of the depleting antibody to increase FcR effector functions.

Impairment of FcR functions by excessive IC formation has been reported during persistent LCMV infection and in the murine MLR/lpr lupus model (1, 2, 13, 14). The efficient depletion of target cells during persistent LCMV infection reported in this study supports a model in which endogenous ICs do not simply abrogate

the engagement of FcRs by depleting antibodies but rather compete with them for binding of available FcRs. In this study, we observed that two antibodies specific for the highly expressed CD90 antigen efficiently depleted T cells in persistently infected mice; how-ever, efficient depletion required increased amounts of surface- bound antibody compared with naïve mice. These data suggest that increased levels of bound antibodies on the cell surface can outcom-pete endogenous ICs for access to FcRs and mediate efficient de-pletion. Our experiments with EL4 cell lines differentially expressing hCD20 further support this hypothesis. Two studies showed a posi-tive correlation between CD20 expression levels and the response to rituximab treatment in B cell lymphoma patients (23, 24). Surface antigen expression levels might thus be an important factor to take into consideration when selecting a therapeutic antibody for a given patient because the expression level of CD20 and CD52, targeted by the therapeutic antibodies rituximab and alemtuzumab, respectively, can vary up to 20-fold between patients (25).

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The Fc region of IgG and its interaction with FcRs are crucial for the in vivo activity of several antitumor antibodies and neutral-izing antibodies against influenza and HIV (5, 26, 27). Enhancing the affinity of the Fc region of IgG for activating FcRs is thus an

attractive approach to improve the therapeutic activity of anti-bodies and is intensively pursued with multiple antibodies in clinical trials (28–31). Obinutuzumab, a glycoengineered anti- CD20 antibody, was recently approved for treatment of chronic

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Fig. 6. Afucosylated anti-CD4 (GK1.5) can deplete CD4+ T cells during persistent LCMV infection. (A) Surface fucosylation of GK1.5 hybridoma cells cultured in the presence of 0 to 50 M 2F analyzed by flow cytometry using biotinylated AAL and APC-labeled streptavidin. Streptavidin- only stained parental GK1.5 hybridoma is shown in gray. Graph shows percentage surface fucosylation of 2F-treated and FUT8-deficient (FUT8) hybridoma compared with untreated parental GK1.5 hybridoma. (B and C) Anti-CD4 antibodies were purified from supernatant of untreated, 25 M 2F-treated, and FUT8 hybridoma. (B) Immunoblot (anti-rat IgG) and lectin blot (AAL) of IgG to assess fucosylation. (C) Composition (relative abundance) of N-linked IgG glycans was analyzed by mass spectrometry. MW, molecular weight. (D and E) In vivo depletion assay using CD4+ T cells coated with fucosylated and afucosylated anti-CD4 antibody. (D) Representative plots of transferred cell populations (CD3+ CD4+ CFSE+) before and 3 to 4 hours after transfer to naïve and LCMV clone-13–infected (28 dpi) recipients. Numbers indicate the percentage among transferred cells. (E) De-pletion efficiency of CD4+ T cells coated with fucosylated and afucosylated anti-CD4 in naïve and LCMV clone-13–infected mice. (F to H) LCMV clone-13– infected mice (28 dpi) were injected twice intraperitoneally with PBS or 300 g of fucosylated (fucos.) or afucosylated (afucos.) anti-CD4 and analyzed 2 days after treatment start. (F) Representative flow plots show frequency of CD4+ T cells among CD3+ T cells in peripheral blood. (G) CD4+ T cell depletion efficiency in peripheral blood. (H) Number of CD4+ T cells in the spleen. Data (mean and SEM) from one representative experiment of at least two are shown (n = 4 to 5 mice per group). Two-way ANOVA with post hoc Tukey’s test for multiple comparisons was used for analysis shown in (E). Unpaired two-sided Student’s t test was used for analyses shown in (G). Ordinary one-way ANOVA with post-hoc Tukey’s test for multiple comparisons was used for analysis shown in (H). ns, not significant. ***P < 0.001, ****P < 0.0001.


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lymphocytic leukemia. Increased FcR binding of IgG can be achieved by introducing mutations into the backbone of the Fc region or by modifying the glycan moiety at the conserved N-glycosylation site N297 in the Fc region (20, 28). Human IgG1 lacking the core fucose shows about 50-fold higher affinity for the activating human FcRIIIA, and a comparable albeit slightly smaller effect of afucosylation has been shown for the interaction of mouse IgG and murine FcRIV (4, 20). However, previous studies demon-strating the superior in vivo activity of afucosylated antibodies were almost exclusively performed in animal models lacking excessive IC formation. The efficacy of an afucosylated antibody to deplete B cells in a murine hCD19 transgenic lupus model has been recently shown, although without directly comparing it with a fucosylated antibody and carefully examining the expression level of the hCD19 transgene (32). In this study, we show that in naïve mice, efficient depletion of CD4+ and CD8+ T cells can be achieved by admin-istration of fucosylated antibodies, whereas during persistent LCMV infection, only afucosylated anti-CD4 and anti-CD8 antibodies efficiently deplete CD4+ and CD8+ T cells, respectively. Afucosylated IgG thus seems to outcompete endogenous ICs for access to activating FcRs due to its higher affinity, resulting in FcR-mediated effector functions in the presence of competing ICs. Thus, the use of Fc-

engineered antibodies might allow efficient antibody-mediated depletion even if the target antigen is not highly expressed.

The alternative Fc glycosylation pattern and superior depletion activity of the anti-CD8 antibody during persistent LCMV in-fection led us to investigate whether afucosylated antibodies can efficiently deplete their targets during persistent LCMV infection. Although afucosylated antibodies directed against CD4 or CD8 showed superior depletion activity, these completely afucosylated antibodies were still inferior in their depletion activity (~75 to 80% in spleen) compared with the partially afucosylated anti-CD8 anti-body (~90% in spleen) during persistent LCMV infection. These data suggest that additional antibody-intrinsic features, such as the location of the targeted epitope, could contribute to the superior activity of anti-CD8. Of note, a recent study showed that the distance of an antibody-targeted epitope to the membrane is a cru-cial determinant for the efficiency with which an antibody can trigger FcR-mediated effector functions, with ADCC and antibody- dependent cellular phagocytosis demonstrating divergent pref-erences (33).

Persistent LCMV infection and the MLR/lpr lupus model are widely used model systems to elucidate immunological mechanisms contributing to disease pathogenesis, and antibody-mediated depletion

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is a commonly used strategy to determine the role of specific cell populations. Of note, we initially observed impaired FcR functions due to excessive IC formation during persistent LCMV infection by serendipity while attempting to deplete CD4+ T cells to study their role during persistent viral infection (1). Here, we demonstrate that afucosylated antibodies are superior in their in vivo activity in the presence of competing endogenous ICs and that afucosylated anti-bodies can be easily generated by both genetic and chemical ap-proaches. Addition of the chemical FUT8 inhibitor 2F allows for the fast production of afucosylated antibodies without detectable cellular cytotoxicity or time-consuming genetic manipulations and selection. Achieving efficient antibody- mediated depletion by using afucosylated antibodies produced by the above-mentioned techniques will thus allow studying the role of various cell populations in model systems characterized by excessive IC formation.

In accordance with previous studies (4, 34), afucosylated anti-bodies exhibited increased depletion activity in naïve mice. However, the observed difference between afucosylated and fucosylated anti-bodies was more pronounced in persistently infected animals, in which fucosylated variants failed to induce significant depletion. Among activating murine FcRs, only FcRIV shows significantly increased affinity for afucosylated IgG (10-fold), suggesting that FcRIV is cru-cially involved in the successful depletion of target cells mediated by afucosylated antibodies during persistent LCMV infection (4, 34). It is interesting that efficient antibody-mediated depletion during per-sistent infection required about 10-fold higher levels of opsonized antibody (quantity) or antibodies with a 10-fold higher affinity for FcRIV (quality) and thus tempting to speculate that both approaches act independently but with the common goal to increase the chances of efficient FcR engagement in the presence of competing ICs.

Afucosylation is the most widely used strategy to improve the activity of therapeutic antibodies depending on the engagement of activating FcRs without inducing potential immunogenic mutations in the Fc region. Fc-engineered human IgG1 antibodies with amino acid substitutions in the Fc region aimed at enhancing affinity for activating human FcRs have been developed and showed increased activity in a number of models including in vitro assays, nonhuman primate studies, and human FcR transgenic mice (28, 31, 35). The increased activity of afucosylated antibodies during persistent viral infection is most likely due to increased affinity for FcRIV, and thus Fc-engineered mAbs with increased affinity for activating FcRs are expected to also show increased activity under these conditions. How-ever, the experimental validation of Fc-engineered mAbs would re-quire the use of human FcR transgenic mice because modifications to the human IgG1 Fc region are aimed at increasing affinity for activating human FcRs.

Prolonged treatment with extremely high doses of depleting an-tibody (10 mg weekly for 10 weeks) has been previously shown to efficiently deplete resistant B cells in the MLR/lpr lupus model (13). Although such an approach might theoretically prove successful in LCMV-infected animals due to the same underlying cause for resis-tance, that is, ICs, the relatively rapid kinetics of LCMV infection compared with the treatment cycle renders such an approach im-practical for studying the role of certain subsets during the chronic phase of the infection. Furthermore, most depleting antibodies originated from rats and will thus induce potent “antidrug” antibody responses preventing efficient long-term treatments.

An additional approach to achieve efficient antibody-mediated de-pletion in the presence of excessive ICs, which might potentially syn-

ergize with the strategies of targeting highly abundant antigens and the use of Fc-engineered antibodies mentioned above, could be the simulta-neous administration of a blocking anti-CD47 antibody. Blockade of CD47, a transmembrane protein overexpressed on many lymphomas and interacting with the inhibitory receptor signal-regulatory- protein (SIRP) on macrophages, has been shown to synergize with rituximab and promote phagocytosis of NHL (36–38). However, it is not known whether CD47 can reduce the critical threshold required for FcR- mediated phagocytosis. Future studies should therefore address the question of whether blockade of the CD47-SIRP pathway can promote FcR- mediated phagocytosis in situations of suboptimal FcR engagement such as persistent viral infections, autoimmune diseases, and cancer.

Our study using depleting antibodies for various lymphocyte subsets serves as a proof of concept demonstrating that, during persistent viral infection, FcR-mediated effector functions can be efficiently triggered, although increased surface antigen expression or antibodies with increased affinity for activating FcRs are required. However, one limitation of our study is that we assessed the FcR- mediated depletion of T cell subsets with a limited number of monoclonal antibodies. Further experiments are thus required to confirm that depleting antibodies against other lymphocyte mark-ers and antibodies directed against nonlymphoid target cells behave comparably. Furthermore, our experiments were predominantly focused on circulating lymphocytes in lymphoid and nonlymphoid tissues. The failure of even the afucosylated anti-CD8 antibody to deplete CD8 T cells in the intestinal epithelium of LCMV clone-13–infected animals suggests that resident cells in certain compartments of the body may be more resistant to FcR-mediated depletion. Thus, additional experiments targeting various surface antigens and cellular targets are required to precisely assess the impact of tissue residency and differences between various compartments. These experiments should provide important insights, especially for sessile target popu-lations such as solid tumors.

Future studies using LCMV coinfection models could provide novel insights into how persistent viral infections can modulate FcR-mediated antibody effector functions involved in antimicro-bial protection and provide a platform to evaluate Fc-engineered antibodies in a stringent experimental setting. Overall, our study presents two distinct and potentially synergistic strategies to achieve efficient antibody-mediated depletion during persistent LCMV in-fection, which is characterized by excessive IC formation resulting in compromised FcR-mediated antibody effector functions. The quantity of the target antigen and the quality of the depleting anti-body, that is, its affinity for activating FcRs, might thus be both important factors to consider before treatment of autoimmune diseases, cancer, and persistent viral infections with antibodies rely-ing on the engagement of FcRs.

MATERIALS AND METHODSStudy designThe present study was designed to investigate strategies allowing the efficient depletion of target cells during persistent LCMV clone-13 infection, which is characterized by excessive IC formation and resistance to FcR-mediated depletion. We performed depletion experiments by directly injecting depleting antibodies into mice or by adoptively transferring fluorescently labeled and antibody-coated cells, followed by fluorescence-activated cell sorting (FACS) analyses to assess depletion efficiency. The number of mice per experimental


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group ranged from three to eight, and experiments were repeated at least once, as indicated in the figure legends.

Mice and virusesFemale C57BL/6J mice were purchased from Jackson Laboratory. FcR−/− mice were a gift from S. R. Stowell, Emory University. All mice were housed at the Emory University School of Medicine animal facility under standard pathogen-free conditions. All experiments were performed in accordance with approved Institutional Animal Care and Use Committee protocols. For persistent LCMV infection, 6- to 8-week-old mice were infected intravenously with 2 × 106 plaque- forming units of the LCMV clone-13 strain. Depletion experiments were performed around 24 to 31 days postinfection (dpi).

Cell preparationLymphocytes from anticoagulated peripheral blood were isolated using Histopaque-1077 (Sigma-Aldrich). Splenocytes were isolated by mechanical disruption, followed by digestion with collagenase D (0.4 U/ml; Roche) for 30 min at 37°C with gentle shaking. EDTA was added to a final concentration of 5 mM, and spleens were homoge-nized by passing through a 70-m cell strainer (BD Biosciences). Red blood cells were lysed using ACK Lysing buffer (Lonza). Cells were washed and resuspended in RPMI 1640 supplemented with 5% fetal bovine serum (FBS).

Flow cytometry and antibodiesSingle-cell suspensions were stained for 30 min at 4°C in FACS staining buffer [2% FBS and 2 mM EDTA in phosphate-buffered saline (PBS)] with the following fluorochrome-conjugated antibodies (purchased from BioLegend): CD3 (145-2C11), CD4 (RM4-4), CD8 (53-6.7), CD8 (53-5.8), CD11b (M1/70), CD19 (6D5), CD44 (IM7), CD90.2 (30H12), and F4/80 (BM8). We used the anti-CD4 clone RM4-4 to ensure detection of CD4 expression on the cell surface in the presence of the depleting anti-CD4 antibody (GK1.5). Dead cells were excluded by staining with the LIVE/DEAD Fixable Near-IR Dead Cell Stain (Invitrogen). After staining, cells were fixed in 1% paraformaldehyde and acquired on a Canto II flow cytometer (BD Biosciences). Quantification of surface molecules was performed using Rainbow Calibration Particles (BioLegend). Data were analyzed using FlowJo (TreeStar).

Depletion of lymphocytes and phagocytesDepletion of various lymphocyte populations and phagocytes was performed as described previously (1). Mice were injected intraperi-toneally on two consecutive days with depleting antibody. Deple-tion efficiency in the peripheral blood and spleen was determined 2 days after treatment initiation. The following antibodies were pur-chased from BioXCell and used at the indicated doses: anti-CD4 (GK1.5; 300/500 g), anti-CD8 (2.43; 500 g), anti-CD90.2 (30H12; 250 g), and anti-CD90 (T24/31; 250 g). The anti-CD8 (H35-17.2; 250 g) was produced in-house. The hybridoma H35-17.2 was a gift from A. E. Lukacher. The hybridoma was cultured in Hybridoma- SFM (Gibco) in CELLine bioreactors (INTEGRA Biosciences). Super-natants were purified using protein G sepharose, concentrated, and sterile-filtered. All antibodies were tested using a rat mAb isotyping test kit (Bio-Rad) and confirmed to be of the rat IgG2b isotype. Phagocytic active cells were depleted 24 hours before initiation of anti-body treatment by intraperitoneal injection of 200 l of clodronate liposomes (Chlophosome-A, FormuMax).

Generation of afucosylated antibodiesThe GK1.5 and 2.43 hybridoma cell lines were obtained from American Type Culture Collection and cultured in Hybridoma- SFM. To produce afucosylated antibodies by chemical inhibition of FUT8, we cultured the cells in the presence of 25 M 2F (EMD Mil-lipore). To generate a FUT8-deficient GK1.5 hybridoma, we used the CRISPR-Cas9 system. Briefly, GK1.5 hybridoma cells were trans-fected using the Avalanche Sp2/0-Ag14 Cell Transfection Reagent (EZ Biosystems) with the plasmid pSp-Cas9(BB)-2A-GFP containing a FUT8 guide RNA specific for mouse and rat FUT8 (5′-CAGAATTG-GCGCTATGCTAC-3′; GenScript). Two days after transfection, we single cell–sorted green fluorescent protein–positive (GFP+) cells into 96-well flat bottom plates using a FACSAria II, followed by clonal expansion in Hybridoma-SFM. We screened for the absence of FUT8 activity by analyzing surface fucosylation and by lectin blot analysis of purified antibodies.

Lectin blot and cytometryEqual amounts of purified antibody were separated in reducing SDS– polyacrylamide gel electrophoresis, followed by transfer to polyvi-nylidene difluoride membranes. After blocking with tris-buffered saline with 0.1% Tween 20 + 3% bovine serum albumin (BSA), we incubated the membranes with either biotinylated AAL (200 ng/ml; Vector Laboratories) overnight at 4°C, followed by a 1-hour incuba-tion with horseradish peroxidase (HRP)–conjugated streptavidin (20 ng/ml; Jackson ImmunoResearch), or as control overnight at 4°C with an HRP-conjugated goat anti-rat IgG (20 ng/ml; Jackson ImmunoResearch). Bound HRP was detected using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

The surface fucosylation of the GK1.5 hybridoma was analyzed by staining in Hanks’ balanced salt solution + 0.5% BSA with biotinylated AAL for 30 min at 4°C, followed by a 30-min incubation with allo-phycocyanin (APC)–labeled streptavidin (Thermo Fisher Scientific). Samples were then acquired on a Canto II flow cytometer, and mean fluorescence intensities were determined.

In vivo depletion assayThis assay was performed as described previously (1). We purified total T cells and CD4+ T cells from spleens of naïve C57BL/6J mice using the EasySep Mouse T Cell or EasySep Mouse CD4+ T Cell Isolation Kit (STEMCELL), respectively. Cells were labeled with different concentrations of CellTrace Violet and carboxyfluorescein diacetate succinimidyl ester (CFSE; both from Invitrogen), followed by coating with the indicated depleting antibodies. Labeled and coated cells were mixed to equal proportions and adoptively trans-ferred into naïve or LCMV clone-13–infected mice by intravenous injection. Depletion was assessed 3 to 4 hours after transfer using flow cytometric analysis of the spleen. Half-maximal effective con-centration (EC50) values for anti-CD90.2 experiments were calcu-lated by fitting the data using a five-parameter logistic equation.

EL4 cell lines with differential expression of hCD20 were estab-lished by lentiviral transduction (pLVX-hCD20-IRES-ZsGreen1), followed by single-cell sorting of cells expressing low, medium, and high levels of hCD20 into 96-well flat bottom plates using a FACSAria II. After clonal expansion, three EL4 cell lines with differ-ential hCD20 surface expression were selected, and the number of hCD20 molecules per cell was determined by using the BD Quantibrite PE system. To assess in vivo depletion, we labeled EL4 cell lines with different concentrations of CellTrace Violet and CellTrace


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FarRed (Invitrogen), mixed them to equal proportions, and intra-venously transferred them into naïve or LCMV clone-13–infected mice, which received 250 g of anti-hCD20 (2H7) intraperitoneally 4 hours earlier. Production and purification of the murine anti- hCD20 (2H7) have been described previously (1). Depletion was assessed 3 to 4 hours after transfer using flow cytometric analysis of the spleen.

Glycan analysisN-linked glycans were released from purified antibodies under dena-turing conditions with peptide-N-glycosidase (PNGase F), purified by solid phase extraction chromatography, and permethylated using NaOH–dimethyl sulfoxide slurry and methyl iodide, as described previ-ously (39). Mass spectrometry data were acquired in positive mode using a 4800 Plus MALDI-TOF/TOF (Applied Biosystems) mass spectrom-eter, and data were analyzed using GlycoWorkBench software (40).

Statistical analysisAll statistical tests were performed using GraphPadPrism 6 (GraphPad). Values are expressed as means and SEM. As indicated in the figure leg-ends, unpaired two-sided Student’s t test was used to compare two groups and ordinary one- or two-way analysis of variance (ANOVA) with post hoc Tukey’s test or Sidak’s test for multiple comparisons. *P < 0.05 was considered significant.

SUPPLEMENTARY MATERIALSimmunology.sciencemag.org/cgi/content/full/3/27/eaao3125/DC1Fig. S1. Clodronate liposomes efficiently deplete phagocytic cells.Fig. S2. CD90-specific antibodies can efficiently deplete T cells in naïve and persistently infected mice.Fig. S3. Afucosylated anti-CD8 antibody exhibits superior depletion activity in naïve mice.Table S1. Raw data sets.

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Acknowledgments: We thank members of the Ahmed laboratory for the insightful discussion and J. Gensheimer for the proofreading. We thank S. R. Stowell for providing us FcR−/− mice and A. E. Lukacher for providing the H35-17.2 hybridoma. Funding: This work was supported by NIH grant R01AI030048 to R.A. This study was also supported in part by the Emory Flow Cytometry Core, one of the Emory Integrated Core Facilities, and is subsidized by the Emory University School of Medicine. Additional support was provided by the National Center for Advancing Translational Sciences of the NIH under award number UL1TR000454. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the NIH. Author contributions: A.W. conceived the study and designed, performed, and analyzed the experiments. A.O.K., R.M.V., J.-H.H., and X.X. helped with experiments and data analysis. B.P.C. performed the glycan analysis. R.A. supervised the study. A.W. and R.A. wrote the manuscript. All authors participated in editing the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The anti-CD8 hybridoma (H35-17.2) and the FUT8-deficient GK1.5 hybridoma cell line are available from A.W. All data needed to evaluate the conclusions are in the paper or in the Supplementary Materials.

Submitted 6 July 2017Resubmitted 28 May 2018Accepted 1 August 2018Published 21 September 201810.1126/sciimmunol.aao3125

Citation: A. Wieland, A. O. Kamphorst, R. M. Valanparambil, J.-H. Han, X. Xu, B. P. Choudhury, R. Ahmed, Enhancing FcR-mediated antibody effector function during persistent viral infection. Sci. Immunol. 3, eaao3125 (2018).


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R-mediated antibody effector function during persistent viral infectionγEnhancing Fc

AhmedAndreas Wieland, Alice O. Kamphorst, Rajesh M. Valanparambil, Jin-Hwan Han, Xiaojin Xu, Biswa P. Choudhury and Rafi

DOI: 10.1126/sciimmunol.aao3125, eaao3125.3Sci. Immunol.

infections remains to be seen.infected with LCMV. Whether afucosylation can be universally used to enhance antibody functions during chronic

T cells in mice persistently+ and CD8+ enhanced the ability of these antibodies to deplete CD4αagainst CD4 and CD8target cells is dependent on antigen expression levels. Further, they also found that afucosylation of antibodies directedhave examined how to enhance antibody functions in this setting. They found that the ability of antibodies to deplete

.et alwith chronic infections. Using lymphocytic choriomeningitis virus (LCMV) as a model of chronic infection, Wieland deposition of immune complexes. The actions of antibody-based drugs can therefore be severely impaired in individuals

Persistent immune activation during chronic infections is often associated with increased generation andGiving antibodies a boost

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MATERIALSSUPPLEMENTARY http://immunology.sciencemag.org/content/suppl/2018/09/17/3.27.eaao3125.DC1

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ANTIBODIES Copyright © 2018 Enhancing Fc R …...Enhancing Fc R-mediated antibody effector function during persistent viral infection Andreas Wieland1*, Alice O. Kamphorst1†, Rajesh