Germinal center-dependent and -independent immune responses of tumor-infiltrating B cells in human cancers

Abstract

B cells play essential roles in immunity, mainly through the production of high affinity plasma cells (PCs) and memory B (Bmem) cells. The affinity maturation and differentiation of B cells rely on the integration of B-cell receptor (BCR) intrinsic and extrinsic signals provided by antigen binding and the microenvironment, respectively. In recent years, tumor infiltrating B (TIL-B) cells and PCs (TIL-PCs) have been revealed as important players in antitumor responses in human cancers, but their interplay and dynamics remain largely unknown. In lymphoid organs, B-cell responses involve both germinal center (GC)-dependent and GC-independent pathways for Bmem cell and PC production. Affinity maturation of BCR repertoires occurs in GC reactions with specific spatiotemporal dynamics of signal integration by B cells. In general, the reactivation of high-affinity Bmem cells by antigens triggers GC-independent production of large numbers of PC without BCR rediversification. Understanding B-cell dynamics in immune responses requires the integration of multiple tools and readouts such as single-cell phenotyping and RNA-seq, in situ analyses, BCR repertoire analysis, BCR specificity and affinity assays, and functional tests. Here, we review how those tools have recently been applied to study TIL-B cells and TIL-PC in different types of solid tumors. We assessed the published evidence for different models of TIL-B-cell dynamics involving GC-dependent or GC-independent local responses and the resulting production of antigen-specific PCs. Altogether, we highlight the need for more integrative B-cell immunology studies to rationally investigate TIL-B cells as a leverage for antitumor therapies.

Keywords: antibody; cancer; germinal center; plasma cell; tertiary lymphoid structure.

Fig. 1

B-cell responses in secondary lymphoid organs. A Upon primary antigen exposure, naïve B cells may rapidly differentiate into short-lived plasmablasts producing unmutated IgM antibodies or into GC-independent Bmem cells. Naïve B cells differentiating into GC B cells will in turn generate Bmem cells and PC expressing mutated IgG or IgA. B Schematic representation of the GC reaction underlying the cyclic dynamics of affinity maturation. In the DZ, B cells undergo cell division and SHM. In the LZ, B cells undergo affinity-based selection through interactions with FDCs and TFH cells. After clonal selection, GC B cells can differentiate into Bmem cells or PCs or re-enter the DZ. GC-dependent clonal expansion and BCR diversification induce sequential mutations of BCR sequences and intraclonal heterogeneity. C Upon antigen re-exposure, Bmem cells rapidly differentiate into high-affinity plasmablasts producing mutated antibodies or into Bmem cells replenishing the Bmem pool. Bmem cells may also form secondary GCs and undergo SHM to generate a new round of long-lived PC and Bmem cells. D In the case of GC-independent Bmem cell reactivation, clonal expansion without further BCR diversification generates large clonotypes with no intraclonal BCR diversity. Created with BioRender.com

Fig. 2

Integrative methods for studying B-cell responses in tumors. A Depending on the material that is available, different immunology, genomics, and imaging methods may be used to study B-cell responses in cancer patients and their tumors. Blood samples may be used to study antibodies in the serum, B-cell subsets and BCR repertoire among peripheral blood mononuclear cells (PBMCs). Tumor samples may be used fresh for studying the immune cell composition by flow cytometry and single-cell RNA-seq and as a source of live B cells and PCs for single-cell cloning of BCR and production as recombinant monoclonal antibodies. Frozen or formalin-fixed paraffin-embedded (FFPE) tissues may be used for histology, imaging, spatial transcriptomics and BCR sequencing. B A deeper understanding of TIL-B responses and their importance will come from integrating several B-cell-focused analyses on tumor and blood samples from well-annotated clinical sample cohorts. Created with BioRender.com

Fig. 3

GC-TLS displays structural differences from conventional GC-SLO. Multiplex IF staining of a TIL-B infiltrated non-small cell lung cancer (NSCLC) tumor sample compared with a reactional lymph node to assess the presence of B-cell subsets and GC organization. Staining was performed with the Leica Bond Rx. Our multiplex panel was designed for TIL-B identification: mouse anti-CD20 (clone L26, DAKO), rabbit anti-BCL6 (EPR11410-43, Abcam), rabbit anti-IgA (EPR5367-76, Abcam), rabbit anti-CD27 (EPR8569, Abcam), rabbit anti-IgG (EPR4421, Abcam), and anti-pancytokeratin (AE1/AE3/PCK26, Roche). We used the Akoya Bioscience Opal 6-Plex Detection Kit using TSA opal fluorophores (Opal 480, Opal 520, Opal 570, Opal 620 and Opal 690). Slides were counterstained with spectral DAPI (Akoya Bioscience), cover-slipped, and scanned using the PerkinElmer Vectra Polaris System. A–C Representative images of GC-SLO. D–I Representative images of GC-TLS. Low magnification (scale bar = 100 µm) images with all markers are shown in (A, D, G). White squares indicate areas selected for higher magnification views. High magnification (scale bar = 20 µm) images with only the indicated markers are shown in (B, E, H, C, F, I)

Fig. 4

Current knowledge on TIL-B responses in tumors. A Different degrees of TLS maturation and organization: simple immature TLSs are composed of TIL-B and T-cell aggregates (1); GC-TLSs contain TIL-GC structures (2); reactivation of TIL-Bmem in GC-independent (3) and, to a lesser extent, GC-dependent responses (4) differentiates TIL-PCs that migrate to specific stromal areas in tumors (5). B TIL-B may target different types of antigens: foreign antigens present within the tumor may lead to local TIL-PC differentiation (1); self-antigens may be recognized by TIL-PC-derived antibodies (class I or class II) (2); TIL-PC-derived antibodies may recognize tumor-specific antigens or neoantigens released upon cell death (3); and some of the resulting antibodies may coat tumor cells and sensitize them to killing by NK cells or phagocytes (4). Created with BioRender.com