An immune evasion mechanism with IgG4 playing an essential role in cancer and implication for immunotherapy

Abstract

Background: Recent impressive advances in cancer immunotherapy have been largely derived from cellular immunity. The role of humoral immunity in carcinogenesis has been less understood. Based on our previous observations we hypothesize that an immunoglobulin subtype IgG4 plays an essential role in cancer immune evasion.

Methods: The distribution, abundance, actions, properties and possible mechanisms of IgG4 were investigated with human cancer samples and animal tumor models with an extensive array of techniques both in vitro and in vivo.

Results: In a cohort of patients with esophageal cancer we found that IgG4-containing B lymphocytes and IgG4 concentration were significantly increased in cancer tissue and IgG4 concentrations increased in serum of patients with cancer. Both were positively related to increased cancer malignancy and poor prognoses, that is, more IgG4 appeared to associate with more aggressive cancer growth. We further found that IgG4, regardless of its antigen specificity, inhibited the classic immune reactions of antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and complement-dependent cytotoxicity against cancer cells in vitro, and these effects were obtained through its Fc fragment reacting to the Fc fragments of cancer-specific IgG1 that has been bound to cancer antigens. We also found that IgG4 competed with IgG1 in reacting to Fc receptors of immune effector cells. Therefore, locally increased IgG4 in cancer microenvironment should inhibit antibody-mediated anticancer responses and help cancer to evade local immune attack and indirectly promote cancer growth. This hypothesis was verified in three different immune potent mouse models. We found that local application of IgG4 significantly accelerated growth of inoculated breast and colorectal cancers and carcinogen-induced skin papilloma. We also tested the antibody drug for cancer immunotherapy nivolumab, which was IgG4 in nature with a stabilizing S228P mutation, and found that it significantly promoted cancer growth in mice. This may provide an explanation to the newly appeared hyperprogressive disease sometimes associated with cancer immunotherapy.

Conclusion: There appears to be a previously unrecognized immune evasion mechanism with IgG4 playing an essential role in cancer microenvironment with implications in cancer diagnosis and immunotherapy.

Keywords: antibody specificity; immune evasion; immunotherapy; tumor microenvironment.

Figure 1

Significant increase of IgG4 and IgG4:IgG_total in serum and IgG4-positive B lymphocytes in esophageal cancer. (A) IgG4 in serum of esophageal squamous cell cancer (ESCC) (n=82) was significantly higher when compared with healthy controls (n=70) (p<0.0001). (B) IgG4:IgG_total ratio in ESCC (n=82) was significantly higher than that in matching healthy adults (n=70) (p<0.0001). (C) IgG4 in stage Ⅳ (n=18) was significantly higher than those in stages Ⅰ and Ⅱ (n=16) (p<0.01). (D) IgG4:IgG_total in serum of stage IV ESCC (n=18) was significantly higher than those in stages Ⅰ and Ⅱ (n=16) (p<0.05). (E) Scatter diagram of IgG4-positive cell numbers in cancer (cancer), adjacent normal tissue (adjacent) and normal tissues. IgG4-positive lymphocytes in and around the esophageal cancer mass (n=110) are significantly more abundant than those in the adjacent normal tissue (n=60) and normal lymphoid tissues (n=63) (***p<0.001 for both). Increases of IgG4-positive lymphocytes were most abundant in areas of cancer cell proliferation. (F) The increase of IgG4-positive cell numbers was related to the prognoses of the patients. More IgG4-positive cells were associated with worse outcome (p<0.05). (G) IgG4 concentration in cancer tissue (n=46) was significantly higher than that in adjacent normal tissue (n=46) (p<0.01). (H) Immunohistochemistry of IgG4 in esophageal cancer tissues. From left to right are IgG4 in cancer tissues, cancer-adjacent tissue and normal lymphoid tissue (tonsil). It clearly demonstrates that IgG4-positive lymphocytes (red) were markedly increased (left) in comparison with normal lymphoid tissue (right) and with tumor-adjacent normal tissue (middle). Scale bar: 100 µm. (I) Demonstration of four subpopulations of IgG-containing plasma cells with multiple immunostaining (SDS method) in cancer. Each subclass has its own distribution pattern and one plasma cell only produces one subclass of IgG. IgG1: yellow; IgG2: green; IgG3: purple; IgG4: red. (J) On the same tissue section, a triple immunostaining was performed with the SDS method to demonstrate the distribution and relationship among CD3-positive T cells (yellow), IgG1-positive B cells (greens) and IgG4-positive B cells (red). Each cell type has its own distribution, and no overlap between different cell types is observed. SDS, stain-decolorize-stain.

Figure 2

IgG4 extracted from a patient with cancer reacted to cancer-bound IgG1 and blocked antibody-mediated cancer immunity. (A) Upper panel: These photos serve as an example of the reactivity of IgG1 and IgG4 extracted from patients with cancer. IgG1 from the serum of a patient with breast cancer was labeled with biotin and stained a frozen cancer tissue section of the same patient. Cancer cells were positively stained by IgG1 (left). The cancer cells were confirmed by their characteristic histopathology of H&E staining (middle). IgG4 from serum of the same patient labeled with biotin and applied on the same cancer on a consecutive section was completely negative (right). Lower panel: Another breast cancer positively stained by IgG1 from the patient’s serum (left). The cancer cells were identified by positive immunostaining of cytokeratin (CK) on a consecutive section (middle). IgG4 from the same patient was not reactive to the same cancer on a consecutive section (right). (B) The upper panel illustrates the principle of the experimental reactions, and the middle and lower panels show staining results from two patients with breast cancer. Left: IgG1 from a patient with cancer positively reacted to frozen cancer tissue of the same patient (brown cells). Middle: IgG4 from the same patient with cancer applied to consecutive sections, but did not react to the same cancer. Right: However, when unlabeled IgG1 was applied to the same cancer tissue section followed by biotin-labeled IgG4, the cancer cells were positively stained (brown cells).

Figure 3

IgG4 reacted to IgG1 in western blot and tissue sections in an Fc-Fc fashion. (A) In western blot, non-cancer-specific IgG4 from a patient with breast cancer reacted to IgG1 and IgG4 from the same patient with cancer (right panel, arrows). However, when IgG1 and IgG4 were run on the gel and biotin-labeled IgG1 was used as the primary antibody, no band was seen (left panel). These are the same antibodies used in figure 2A, B, providing support to explain the reaction between IgG4 and IgG1 seen on cancer tissue. (B) Western blot demonstrated that IgG4 reacted with IgG1, IgG2, IgG3 and IgG4. (C) IgG4 reacted with IgG Fc fragment but not with Fab arm. (D) IgG4 reacted with IgG1 Fc fragment but not with Fab arm. (E) Biotin-labeled IgG4 Fc fragment reacted to IgG1 and IgG4 Fc fragments but not with IgG1 or IgG4 Fab. Biotin-labeled IgG4 Fab did not react to IgG1 or IgG4 Fc or Fab. These results demonstrate that it is the Fc region of IgG4 that reacted to Fc of IgG1.

Figure 4

Non-cancer-specific IgG4 inhibited classic ADCC, ADCP and CDC reactions against cancer but had no direct effect on cancer cell growth. (A) On western blot, the chimeric antibody cetuximab (IgG1 against EGFR) was run on the gel, and IgG4 and IgG1 at concentrations of 3, 5 and 10 µg/mL were used as the primary antibodies. IgG4 reacted to cetuximab at a concentration-dependent manner, but IgG1 did not react. (B) Left: In a classic ADCC experiment, cetuximab (IgG1) was incubated with an EGFR-expressing lung cancer cell line (A549) and then with PBMC from normal healthy adult. Cancer cell activity was significantly reduced (n=12). Non-cancer-specific IgG1 and HSA were used as controls showing that they had no direct effect on the cancer cells (n=12). Middle: When non-cancer-specific IgG4 was added to the mixture, the effect of cetuximab was significantly reversed demonstrating an inhibitory effect of IgG4 in ADCC (n=12). Non-cancer-specific IgG1 had a much smaller, but also significant, effect in inhibiting ADCC action (n=12). Right: When Fc receptor blocker was incubated with PBMC, the effect of cytotoxicity was blocked. (C–E) ADCP was performed with a lung cancer cell line A549 (expressing EGFR) as the targets, human peripheral monocyte-derived macrophages as the effector cells and the antibody cetuximab (IgG1) against EGFR as the mediating antibody. The tumor cells were stained with CFDA-SE fluorescence probes (green). Macrophages derived from PBMC were stained with DiI fluorescent probes (orange). Blue fluorescence is the nuclei stained with DAPI. (D) Higher magnification of (C). The orange-colored macrophages were in close contact with green tumor cells. Tumor debris ingested by macrophages appeared yellow in the cytoplasm of macrophages. (E) Bar chart showing the effect of ADCP and its inhibition by IgG4. (F) Left: In 10 µg/mL rituximab-mediated ADCP model, Giemsa staining results of phagocytosis of Raji cells by macrophages after the addition of 100 µg/mL IgG1 and IgG4, respectively. Right: IgG4 significantly inhibited rituximab-mediated ADCP in phagocytosis by macrophage, but IgG1 could not inhibit the ADCP effect. Scale bar=30 µm (*p<0.05, **p<0.01, ***p<0.001). (G) In a classic CDC experiment, cetuximab anti-EGFR antibody was incubated with an EGFR-expressing lung cancer cell line (A549) and then with complement (Co) from serum of a normal healthy adult. The cancer cell activity was significantly reduced. (H) When non-cancer-specific IgG4 was added to the mixture in the above CDC experiment, the effect of cetuximab was significantly reversed. (I) IgG4 and IgG1 were incubated with KYSE150 for 24 hours and no effect on cell growth was found. ADCC, antibody-dependent cell-mediated cytotoxicity; ADCP, antibody-dependent cellular phagocytosis; CDC, complement-dependent cytotoxicity; CFSE-DA, carboxyfluorescein diacetate succinimidyl ester; DAPI, 4',6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; HSA, human serum albumin; PBMC, peripheral blood mononuclear cell.

Figure 5

IgG4 accelerated cancer cell growth in three immune potent mouse models. (A, B) Non-cancer-specific IgG4 accelerated breast cancer cell growth. Local inoculation of mouse breast cancer cells (4T1, 1×10⁵ cells per mouse) into immune competent mice (BALB/c, body weight 20 g, n=5) resulted in sizeable tumor masses in 21 days. Local injections of non-cancer-specific IgG4 resulted in tumor masses that doubled the size of those similarly injected with ‘IgG without IgG4’ or with PBS. As there is no direct effect of IgG4 on cancer cell growth (figure 4I), these results clearly demonstrate that IgG4 can effectively promote tumor growth by inhibiting local immunity. (C, D) IgG4, anti-PD-1 (nivolumab) and anti-PD-1-Fc induced significant tumor (CT26 mouse colon cancer) progression in mice. Five groups of mice were tested by injecting IgG4, nivolumab (IgG4 subtype), nivolumab-Fc, IgG1 and PBS. By 19 days, IgG4, nivolumab and nivolumab-Fc induced significant progression of tumor size in comparison with the other groups. (E–G) IgG4 significantly accelerated skin papilloma formation in a carcinogen-induced skin tumor model in immune potent mice. IgG4 significantly accelerated tumor development and growth in comparison with control (PBS), which in turn had more tumor formation than the group treated with IVIG without IgG4. By 12 weeks, the total tumor volume in IgG4-treated group was more than 12 times larger than the group that was treated with IVIG without IgG4, and was six times larger than the group treated with PBS. (E) The removed back skin of mice from the three groups (at 15 weeks). The tumors are shown in dark-brown color. (F) Tumors at higher magnification. (G) Changes of tumor numbers and volume over time of the three groups. (H) Total tumor volumes of the three groups at 12 weeks after treatment. *p<0.05, **p<0.01, ***p<0.001. IVIG, intravenous IgG; PBS, phosphate buffered saline; PD-1, programmed cell death-1.

Figure 6

Diagrammatic illustration of proposed mechanism of cancer-initiated B lymphocyte-derived IgG4-mediated immune evasion. Chronic stimulation by cancer antigens induces class switch of B lymphocytes to produce IgG4. Such increased IgG4 can react to cancer-bound IgG with its Fc-Fc binding property and also to Fc receptors of immune effector cells. With its unique structural and biological property, increased IgG4 in cancer microenvironment mediates an effective immune escape for cancer. ADCC, antibody-dependent cell-mediated cytotoxicity; ADCP, antibody-dependent cellular phagocytosis; CDC, complement-dependent cytotoxicity; NK, natural killer cells.