Multiplex B Cell Characterization in Blood, Lymph Nodes, and Tumors from Patients with Malignancies

Abstract

B lymphocytes contribute to immune surveillance, by tumor-specific Abs and Ag presentation to T lymphocytes, but are insufficiently studied in humans. In this article, we report a flow cytometric investigation of B lymphocyte subpopulations in blood, lymph nodes (LNs), and malignant tissues from 20 patients operated on because of advanced solid tumors. The CD19+ compartment in peripheral blood was essentially unaltered in patients, as compared with healthy control subjects. In metastatic LNs, signs of B lymphocyte activation were observed, as evidenced by increased proportions of plasmablasts and CD86-expressing cells. In tumor-infiltrating B lymphocytes (TIL-B), both switched memory cells and plasmablasts were expanded, as compared with nonmalignant epithelium. Moreover, pronounced skewing of Igλ/Igκ ratio was evident among TIL-Bs. By spectratype analysis on IgH, we confirmed a monoclonal expansion of the Vh7 family in TIL-B, also present in a tumor-associated LN. Sequencing the clonally expanded Vh7 revealed signs of somatic hypermutation. In conclusion, B lymphocytes in cancer patients exhibit signs of activation in tumor-associated tissues, likely induced by recognition of tumor Ags. Increased numbers of switched memory cells and plasmablasts in combination with clonal expansion and signs of somatic hypermutation suggest a CD4+ T lymphocyte–dependent antitumoral response, which may be exploited for immunotherapy.

A specific immune and lymphatic profile characterizes the pre-metastatic state of the sentinel lymph node in patients with early cervical cancer

“…In addition, experimental studies revealed that B cells can both positively and negatively regulate T cell mediated antitumor immune response, by acting as antigen presenting cells or via chemokine secretion. [36][37][38][39] Altogether our data shed light on a subcapsular B cell rich microenvironment whose function in generating a specific pre-metastatic niche for metastase hosting remains to be determined. Of utmost interest, we establish for the first time a link between lymphatic vessels, CD20…”

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

“…These effects may at least be partly target specific. 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].…”

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

“…Our studies, initially focused on CD4 + T cells infiltrating breast tumors, were quickly presented with a major technical problem due to cleavage of surface CD4 receptors using standard enzymatic digestion protocols [4][5][6][7][8] . We tested a variety of collagenase enzymes with or without DNase and hyaluronidase inhibitors, finding that collagenase I and II completely removed CD4 from the cell surface, despite high lymphocyte viability ( Figure 3A).…”

“…Dissociating tumor tissue while maintaining cellular integrity is technically challenging and has traditionally been performed using a combination of mechanical and enzymatic steps to prepare single cell suspensions [4][5][6][7][8] . This approach involves lengthy incubation periods and is associated with a significant reduction in cell viability as well as the loss of cell surface receptors by enzymatic cleavage.…”

“…Currently, the majority of studies designed to characterize TIL have employed enzymatic digestion (hours to O/N) frequently coupled with some form of mechanical dissection [4][5][6][7][8] . Expansion of TIL for further analysis or therapy generally involves the subsequent ex vivo cultivation of TIL or tumor tissue fragments with stimulatory agents (days to weeks) [13][14][15] .…”