Antigen Specificity and Clinical Significance of IgG and IgA Autoantibodies Produced in situ by Tumor-Infiltrating B Cells in Breast Cancer

Abstract

An important role for tumor infiltrating B lymphocytes (TIL-B) in the immune response to cancer is emerging; however, very little is known about the antigen specificity of antibodies produced in situ. The presence of IgA antibodies in the tumor microenvironment has been noted although their biological functions and clinical significance are unknown. This study used a 91-antigen microarray to examine the IgG and IgA autoantibody repertoires in breast cancer (BC). Tumor and adjacent breast tissue supernatants and plasma from BC patients together with normal breast tissue supernatants and plasma from healthy controls (patients undergoing mammary reduction and healthy blood donors) were analyzed to investigate relationships between autoantibodies and the clinical, histological and immunological features of tumors. Our data show that >84% of the BC samples tested contain autoantibodies to one or more antigens on the array, with ANKRD30BL, COPS4, and CTAG1B being most frequently reactive. Ex vivo TIL-B responses were uncoupled from systemic humoral responses in the majority of cases. A comparison of autoantibody frequencies in supernatants and plasma from patients and controls identified eight antigens that elicit BC-associated autoantibody responses. The overall prevalence of IgG and IgA autoantibodies was similar and while IgG and IgA responses were not linked they did correlate with distinct clinical, pathological and immunological features. Higher levels of ex vivo IgG responses to BC-associated antigens were associated with shorter recurrence-free survival (RFS), HER2 overexpression and lower tumor-infiltrating CD8⁺ T cell counts. Higher IgA levels were associated with estrogen and progesterone receptor-negative cancers but were not significantly associated with RFS. Furthermore, ex vivo IgA but not IgG autoantibodies reactive to BC-associated antigens were linked with germinal center and early memory B cell maturation and the presence of tertiary lymphoid structures suggesting that these TIL-B are activated in the tumor microenvironment. Overall, our results extend the current understanding of the antigen specificity, the biological and the clinical significance of IgG and IgA autoantibodies produced by BC TIL-B in situ.

Keywords: IgA; IgG; autoantibodies; breast cancer; tertiary lymphoid structures; tumor-infiltrating B cells.

Figure 1

Characteristics of the autoantibody prevalence in breast tissue supernatants and plasma. (A) IgG-TAA and IgA-TAA scores in tissue supernatants and (B) plasma samples. Box plots show mean and 0.1–0.9 quantiles, whiskers show maximal values, dots represent individual samples. The Mann-Whitney U test was used to compare the scores between PB-BC and PB-HD, and Kruskal-Wallis test was used to compare the scores among SN-BC, SN-AT and SN-MR samples. (C) Venn diagrams showing number of patients with concordant or mutually exclusive IgG and IgA responses to CTAG1B and ANKRD30BL in SN-BC samples. (D) Venn diagrams showing the prevalence of anti-CTAG1B and anti-ANKRD30BL IgG and IgA antibodies in SN-BC, SN-AT, and PB-BC samples from BC patients.

Figure 2

Association of the IgG-TAA and IgA-TAA scores with clinicopathological features. (A) IgG-TAA scores and (B) IgA-TAA scores in SN-BC samples from patients stratified according to hormone receptor status, Ki67 level, grade and stage. Box plots show mean and 0.1–0.9 quantiles, whiskers show maximal values, dots represent individual samples. M-WU, Mann-Whitney U test, KW, Kruskal-Wallis test.

Figure 3

Association of IgA class autoantibodies with the immunological makeup of BC. (A) Representative images of TLS-negative (left) and TLS-positive (right) tumors determined by dual IHC using CD3 plus CD20. (B) Anti-COPS4 IgA levels in TLS-positive and TLS-negative tumors. (C) Representative FACS scatter-plots of B cell maturation stages in IgA-negative (upper) and IgA-positive (lower) tumors. (D) Percentage of B cells at various maturation stages in IgA-positive and IgA-negative SN-BC samples. Bm1, resting naïve; Bm2, activated naïve; Bm2′, pre-germinal center; Bm3-4, germinal center; eBm5, early memory; Bm5, late memory; *t-test, P < 0.05.

Figure 4

Prognostic significance of IgG class autoantibodies and association with tumor infiltrating CD8⁺ T cell counts. (A) Kaplan Meier plot showing the association between IgG-TAA score and RFS. (B) Kaplan Meier plot showing the association between intratumoral CD19⁺ B cell count and RFS. (C) Kaplan Meier plot showing the association between CD19⁺ B cell count in the adjacent tissues and RFS. (D) Tumor infiltrating CD8⁺ T cell counts in IgG-positive and IgG-negative SN-BC samples. *t-test, P < 0.05.