IgG4 subclass antibodies impair antitumor immunity in melanoma

Abstract

Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10-driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22+ B cells and IgG4(+)-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell-mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches.

Figures

Figure 1. B cells (CD22+) infiltrate melanoma lesions and produce IgG.

(A) Immunohistochemistry showing the presence of lymphocytes (CD45+), mature B cells (CD22+), activated lymphocytes (FoxP3+) (alkaline phosphatase [red], hematoxylin [blue]), and colocalization of all 3 within cutaneous metastases (scale bar: 100 μm; original magnification, ×10). CD22+ cells in melanoma are shown at higher magnification (original magnification, ×40). (B) Significantly increased CD22+ B cell infiltration was measured in primary (n = 6) and metastatic (n = 7) melanoma lesions compared with healthy skin (n = 8). HPF, high-powered microscope field. (C) Comparative real-time PCR showed significantly elevated CD22 expression in primary (n = 10) and metastatic (n = 10) melanomas compared with healthy skin (n = 9). (D) Increased expression of mature IgG mRNA in metastatic melanoma lesions (n = 10) compared with primary melanomas (n = 10) and healthy skin (n = 9) measured by comparative real-time PCR analysis. (E) IgG expression (by comparative real-time RT-PCR) is elevated in melanoma lesions of stage IV patients compared with lesions of stage I–III patients. (F) Immunofluorescent evaluations of IgG+ B cells in human metastatic melanoma lesions (CD22+ B cells in red; left) (IgG+ cells in green; middle) and CD22+IgG+ B cell infiltrates (right). Scale bar: 10 μm; original magnification, ×63. (B and E) *P < 0.01, **P < 0.01, ***P < 0.001, Mann-Whitney U test. (C and D) *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis 1-way ANOVA with Dunn’s post-hoc test. Horizontal lines in box plots represent the mean, and whiskers indicate minimum and maximum values

Figure 2. B cells in melanoma lesions are polarized to produce IgG4 antibodies with reactivity against tumor cells.

(A) Polarization of IgG subclasses in B cells derived from metastatic melanoma skin tumor lesions (n = 2), patient lymph nodes (n = 3), peripheral blood from patients with melanoma (n = 6), and from healthy volunteers (n = 4). Cells were cultured ex vivo, and IgG subclass expression profiles were analyzed from culture supernatants by ELISA (mean ± SD; each sample condition tested in triplicate). (B) Immunohistological evaluations confirm significantly increased IgG4+ cell infiltration in melanoma lesions (n = 10) but not healthy skin (n = 10) (***P < 0.001), and representative metastatic melanoma depicts IgG4+ cells (red; hematoxylin [blue]) (scale bar: 20 μm; original magnification, ×10). (C) Reactivity of patient B cell–derived IgG1 and IgG4 antibodies to A375 tumor cells evaluated using a cell-based ELISA. Dashed black and gray lines indicate cut-off points for IgG1 and IgG4 tumor-reactive antibodies, respectively (lines defined as 2 SDs above isotype control antibodies). (D) Immunofluorescent staining of IgG4+ (red) colocalized with S100+ (green) cells in metastatic melanoma (scale bar: 50 μm; original magnification, ×40). (E) Increased expression of IL4, IL10 and IFNG in metastatic melanomas (n = 10) compared with primary melanomas (n = 10) and healthy skin (n = 9) by comparative real-time PCR. Horizontal lines in box plots represent mean, and whiskers indicate minimum and maximum values. *P < 0.05, **P < 0.01, Kruskal-Wallis 1-way ANOVA with Dunn’s post-hoc test. (F) Cytokines IL-4, IL-10 and IFN-γ are secreted in metastatic melanoma lesion ex vivo cultures.

Figure 3. Ex vivo stimulation assays demonstrate that tumor cells polarize B cells to produce IgG4.

(A) IgG subclass production by patient peripheral blood B cells. Cells were cocultured with irradiated PBMCs with or without metastatic melanoma cells or primary melanocytes for 5 days. Culture supernatants were harvested, and IgG subclass expression profiles were analyzed by IgG subclass ELISA. IgG subclass fractions and total IgG concentrations were illustrated for each sample as mean ± SD of 3 independent experiments (all conditions performed in triplicates). *P < 0.01. Secretion of Th2 cytokines IL-10 and IL-4 from B cell cultures treated with or without (B) tumor cells or (C) primary melanocytes were analyzed by cytokine multiplex assay analysis. Both cytokines were significantly increased in cultures containing tumor cells but not in cocultures with melanocytes (*P < 0.01; **P < 0.01, Mann-Whitney U test; n = 9 replicates per coculture condition). Horizontal lines in box plots represent mean, and whiskers indicate minimum and maximum values. Comparative real-time PCR analysis of IL-4, IL-10, and VEGF expression by A375 melanoma cells isolated by flow cytometric sorting before (–) and after (+) cocultures with B cells and PBMCs shows (D) increased IL-10 expression by cocultured melanoma cells and (E) confirmation that A375 cells secrete IL-10 but not IL-4 in culture. ND, not detected. (F) Comparative real-time PCR analysis of IL-4, IL-10, and VEGF expression by B lymphocytes isolated by flow cytometric sorting from monocultures or from cultures with tumor cells, showing increased expression of VEGF expression by B cells when cocultured with tumor cells.

Figure 4. Biological properties of engineered IgG1 and IgG4 antibodies recognizing the melanoma-associated antigen CSPG4.

Anti-CSPG4 IgG1 and anti-CSPG4 IgG4 antibodies bind to receptors on the cell surface of (A) U937 monocytic cells, (B) primary monocytes, and (C) tumor cells. ImageStream and flow cytometric evaluations confirm IgG1 and IgG4 antibody binding to Fcγ receptors (top and middle panels) and also to the antigen CSPG4 (bottom panel) expressed on the surface of SK-MEL-28 melanoma cells. ImageStream images are shown on the left. IgG FITC, cell surface (area indicated by circle on top left of each panel) binding of antibodies on cells; BF, bright-field image of cells; IgG FITC/BF, composite image of antibody and bright-field images, confirming anti-CSPG4 antibody binding to the cell surface. Quantitative analyses are based on the acquisition of 20,000 cells. Control samples for U937 and monocytes were incubated with goat anti-human IgG (Fab′)2-FITC antibody. Control samples for antibody binding to tumor cells were human IgG1 and IgG4 isotype antibodies of irrelevant specificity. (D) Flow cytometric analysis of CSPG4 IgG1 or CSPG4 IgG4 antibody binding to a panel of tumor cell lines or primary human melanocytes. Antibody binding was detected with an anti-human IgG (Fab′)2-FITC, and specific binding was depicted by MFI values above isotype control antibody binding.

Figure 5. IgG4 irrespective of tumor specificity is ineffective in triggering anti-melanoma effector functions in vitro and prevents IgG1 from mediating tumor cell killing.

(A) Dot plots depicting CFSE+ tumor cells and CD89-PE+ monocytes (effector cells) were used to quantify CFSE+ tumor cells present within PE+ effector cells, indicating phagocytosis (ADCP) (CFSE+/PE+ cells) (top panel). CFSE+/DAPI+ double-positive tumor cell events indicate tumor targets killed by cytotoxicity (ADCC, bottom panel) by IgG1 but not IgG4. (B) Quantification of tumor antigen-specific IgG1 and IgG4 antibody induced ADCC/ADCP by monocytes. Tumor antigen-specific IgG1 mediated tumor cell killing compared with an unspecific control antibody or cells alone. The corresponding anti-CSPG4 IgG4 antibody induced no tumor cell killing above isotype control levels. (C) Titers of secreted IFN-γ, IL-1b, and TNF-a, but not of IL-10, were higher in culture supernatants of monocytes and tumor cells stimulated with anti-CSPG4 IgG1 antibodies compared with those stimulated with anti-CSPG4 IgG4 or controls. All samples were tested in duplicates; mean ± SD (n = 6). (D) Tumor antigen-specific IgG1 against the melanoma antigen CSPG4 can induce monocytes to kill tumor cells, but addition of anti-CSPG4 IgG4 blocks the antitumor functions of the same concentrations of IgG1. (E) Similar blocking of IgG1-mediated tumor cell killing can be observed when CSPG4 IgG1 is coincubated with a nonspecific human IgG4 antibody. (B, D, and E) All assays were performed in triplicates. Data represent percentage tumor cell killing (mean ± SD) (n = 6). *P < 0.05, **P < 0.01; ***P < 0.001.

Figure 6. IgG4 blocks IgG1 antibody-dependent tumor cell killing by inhibiting IgG1 binding and activation through FcγRI.

(A) Competition assay of IgG1 binding on the surface of monocytic cells displaced by addition of increasing concentrations of IgG4 antibody. Proportion of cells binding IgG1 is decreased with increasing concentrations of IgG4, demonstrated by flow cytometric evaluations and representative confocal images (yellow, by ImageStream). (B) Anti-CSPG-4 IgG1-mediated tumor cell killing (by flow cytometry) is inhibited by addition of an antibody known to block IgG Fc binding to FcγRI but not with addition of blocking antibodies to FcγRII or to FcγRIII. (C) Inhibitory functions of anti-CSPG-4 IgG4 are not lost by blocking FcγRII or FcγRIII with previously described specific FcγR blocking antibodies in flow cytometric antibody-dependent tumor cell killing assays. (D) Protein extracts of primary human monocytes isolated by flow cytometric sorting at different times during the antibody-mediated tumor cell killing assay were examined for phosphorylated products of the FcγR signaling pathway. Western blots of phospho-proteins and band density quantifications relative to freshly isolated monocytes demonstrate that IgG4 inhibits the activatory signaling cascades of FcγR (Src, AKT, MEK), while lack of pSHIP implies that FcγRII signaling is not involved in the IgG4 blockade. (B and C) Data are representative figures of 3 independent experiments.

Figure 7. IgG4 has no antitumoral effector functions in vivo and blocks tumor-specific IgG1 function.

(A) Anti-CSPG4 IgG1 antibody is capable of restricting the growth of subcutaneous melanoma lesions in NSG mice engrafted with human immune effector cells, while tumors in mice treated with an anti-CSPG-4 IgG4 antibody or coadministered with anti-CSPG4 IgG1 and IgG4 antibodies grow similarly to those from mice treated with nonspecific antibody or vehicle alone (n = 7 mice per group; mean ± SEM tumor volume in mm3). *P < 0.05, ***P < 0.001, 2-way ANOVA with Bonferroni post-hoc test. Data are representative of 2 experiments. (B) Representative NanoSPECT/CT images of Indium-111–labeled anti-CSPG4 IgG4 antibody (red), demonstrating accumulation of antibody in subcutaneous melanoma lesions at 20 minutes, 8 hours, and 24 hours following intravenous administration (n = 3). (C) Representative immunohistological images and (D) quantitative analyses of sections from human melanoma tumors grown in NSG mice treated with vehicle alone, anti-CSPG4 IgG1, anti-CSPG4 IgG4, or a combination of anti-CSPG4 IgG1 plus anti-CSPG4 IgG4 antibody, demonstrating elevated levels of human CD68+ (brown) immune cell infiltration in human xenograft tumors in animals treated with anti-CSPG4 IgG1 antibody. Horizontal bar indicate the mean, and individual symbols indicate individual tumors. Scale bar: 50 μm; original magnification ×20. ***P < 0.001.

Figure 8. Elevated levels of IgG4 antibodies in patient sera correlate with patient survival.

(A) IgG subclass ELISA evaluations of sera from 33 patients with stage III and IV melanoma were evaluated using Spearman correlation analyses for IgGsubclass/IgGtotal, fractions in relation to patient survival (months), showing a statistically significant negative correlation between IgG4 fraction only (r = –0.32; P = 0.00353). (B) Kaplan-Meier cumulative survival analysis of overall patient survival was compared with respect to IgG4/IgGtotal fractions (stratified according to fractions above (blue) or below (black) the 75 percentile of the IgG4/IgGtotal fractions measured from the 33 metastatic melanoma patient cohort), indicating that high IgG4/IgGtotal ratios in sera are associated with a significantly lower overall survival (P = 0.003; hazard ratio 0.19; 95% CI 0.0635–0.5685).