Novel Ganglioside Antigen Identified by B Cells in Human Medullary Breast Carcinomas: The Proof of Principle Concerning the Tumor-Infiltrating B Lymphocytes

Abstract

The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region VH-JH, Vκ-Jκ, and Vλ-Jλ genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected VH and Vκ clones and tested for binding activity. Our data analysis revealed that some of the VH-JH, Vκ-Jκ, and Vλ-Jλ region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment κ minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the VH of which proved to be the overexpressed VH3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.

The Microenvironment of Germ Cell Tumors Harbors a Prominent Antigen-Driven Humoral Response

“…Characterization of TIL B cells of infiltrating ductal carcinoma revealed that these cells have undergone clonal expansion and isotype switching and have accumulated somatic mutations (15,16). Recombinant Igs derived from medullary breast carcinoma B cell infiltrates were shown to bind a ganglioside present in several breast cancer cell lines (25). This strongly suggests that, at least some, of the infiltrating B cells are driven by a specific tumor Ag.…”

Interrogation of individual intratumoral B lymphocytes from lung cancer patients for molecular target discovery

“…To date, very few targets of antibodies produced by B cells located within a tumor have been directly identified. Kotlan et al [23] constructed an scFv library from IgG variable chain genes derived from human medullary breast carcinomainfiltrating B lymphocytes and isolated an scFv that binds to gangliosides, which had been previously identified as TAAs. Also, Yasuda et al [42] engrafted human lung tumor tissue in SCID mice and identified antibodies to mutated p53 in the mouse serum.…”

“…Analysis of B cells from tumors and metastatic lymph nodes found hypermutated antibody genes and expansion of class-switched memory cells and plasmablasts in patients with bladder cancer [17]. Intratumoral germinal centers (GCs) have been reported to be present in NSCLC [18][19][20], breast cancer [21][22][23][24], colon cancer [25], and germ cell cancers [26]. In NSCLC, GCs are associated with early-stage disease [19], suggesting that a humoral immune response may hold these tumors in check.…”

The Immune Microenvironment of Human Tumors: General Significance and Clinical Impact

“…These dual effects may be due to the tumor cells and/or the inflammatory context. The case of B lymphocytes is also unclear: they correlate with tumor growth and invasion in murine models [44][45][46][47] but scarce observations associate a B cells signature to good prognosis in several human cancers [48][49][50]. Intratumoral NK cells have been reported to down regulate their activating receptors and to be associated to larger breast [51] and lung [28] tumors, but without clear impact on patients survival.…”