Research ArticleANTIBODIES

Enhancing FcγR-mediated antibody effector function during persistent viral infection

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Science Immunology  21 Sep 2018:
Vol. 3, Issue 27, eaao3125
DOI: 10.1126/sciimmunol.aao3125
  • Fig. 1 FcγR-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 μg of anti-CD4, or 500 μg of anti-CD8α. Representative flow plots show the frequency of CD8β+ 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-CD8α. (B to D) Naïve and LCMV clone-13–infected mice (28 dpi) were injected twice intraperitoneally with PBS or 250 μg 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 FcRγ−/− mice were injected twice intraperitoneally with PBS or 250 μg of anti-CD90.2, and the number of splenic CD3+ T cells was assessed after 2 days. (F) Wt mice were injected with 200 μl 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.

  • Fig. 2 High expression of CD90 allows efficient T cell depletion during persistent LCMV infection.

    (A) Surface expression of CD90.2, CD4, and CD8α 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) Representative flow plots of transferred cell populations (CD3+ TCRβ+ 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.

  • 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 μg 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.

  • Fig. 4 FcγR-dependent anti-CD8β efficiently depletes CD8+ T cells during persistent LCMV infection.

    (A) Naïve and LCMV clone-13–infected mice (28 dpi) were injected twice intraperitoneally with PBS or 250 μg of anti-CD8β. Representative flow plots show the frequency of CD8α+ and CD4+ T cells among PBMCs 2 days after treatment initiation. (B and C) CD8α+ T cell depletion efficiency in peripheral blood and spleen. (D) Wt and FcRγ −/− mice were injected twice intraperitoneally with PBS or 250 μg of anti-CD8β, and the number of splenic CD8α+ T cells was assessed after 2 days. (E) Wt mice were injected with 200 μl of clodronate liposomes or PBS, followed by anti-CD8β treatment and analysis as described in (D). (F) Surface expression of CD8α, CD8β, and CD90.2 on CD8+ T cells and CD3+ T cells, respectively, in the spleen of LCMV clone-13–infected mice (28 dpi). Data (mean and SEM) from one representative experiment of at least two are shown (n = 3 to 5 mice per group). Unpaired two-sided Student’s t test was used for analyses shown in (D). Ordinary one-way ANOVA with post hoc Tukey’s test for multiple comparisons was used for analysis shown in (E) and (F). ns, not significant. ****P < 0.0001.

  • Fig. 5 Glycosylation analysis of monoclonal antibodies.

    N-linked glycans released from purified anti-CD90.2, anti-CD4, and anti-CD8β by PNGase F were analyzed by mass spectrometry. (A) List of identified glycans and annotated mass spectra. a.u., arbitrary units. m/z, mass/charge ratio. (B) Summary of N-linked glycan composition (relative abundance). Relative abundance of (C) galactosylated, (D) fucosylated, and (E) bisecting glycans.

  • 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 μM 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 (ΔFUT8) hybridoma compared with untreated parental GK1.5 hybridoma. (B and C) Anti-CD4 antibodies were purified from supernatant of untreated, 25 μM 2F-treated, and ΔFUT8 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) Depletion 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 μg 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.

  • Fig. 7 Afucosylated anti-CD8α (2.43) can deplete CD8+ T cells during persistent LCMV infection.

    LCMV clone-13–infected mice (28 dpi) were injected twice intraperitoneally with PBS or 300 μg of fucosylated or afucosylated anti-CD8α and analyzed 2 days after treatment start. (A) Representative flow plots show frequency of CD8β+ T cells among total lymphocytes in peripheral blood, spleen, liver, and the epithelium of the small intestine (IEL). CD8 T cell depletion efficiency in (B) blood, (C) spleen, (D) liver, and (E) IEL. Unpaired two-sided Student’s t test was used for analysis. ns, not significant. *P < 0.05, ***P < 0.001.

  • 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 isotypeTargetReference
    Anti-hCD20
    (rituximab)
    Human IgG1hCD20 tg
    B cells
    (1, 2, 13)
    Anti-hCD20 (2H7)Mouse IgG2bhCD20 tg
    B cells
    (1, 13)
    Anti-hCD20 (8B9)Mouse IgG2a/chCD20 tg
    B cells
    (13)
    Anti-CD20
    (MB20)
    Mouse IgG2a/cB cells(2)
    Anti-CD20
    (18B12)
    Mouse IgG1B cells(13)
    Anti-CD4 (GK1.5)Rat IgG2bCD4 T cells(1, 2)
    Anti-CD4
    (YTS191)
    Rat IgG2bCD4 T cells(2)
    Anti-CD25 (PC61)Rat IgG1Treg cells(1)
    Anti-CD8α (2.43)Rat IgG2bCD8 T cells(1, 2)
    Anti-CD8α
    (53.6.72)
    Rat IgG2aCD8 T cells(2)
    Anti-platelets
    (6A6)
    Mouse IgG2a/cPlatelets(2)
    Anti-LCMV-GP
    (KL25)
    Mouse IgG1LCMV-infected cells(2)
    Anti-CD40
    (FGK4.5)
    Rat IgG2aDCs, B cells(1)

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/27/eaao3125/DC1

    Fig. 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-CD8α antibody exhibits superior depletion activity in naïve mice.

    Table S1. Raw data sets.

  • Supplementary Materials

    The PDF file includes:

    • Fig. 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-CD8α antibody exhibits superior depletion activity in naïve mice.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data sets.

    Files in this Data Supplement:

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