Adoptive cell transfer (ACT) is a promising approach to cancer immunotherapy, but its efficiency fundamentally depends on the extent of tumor-specific T cell enrichment within the graft. This can be estimated via activation with identifiable neoantigens, tumor-associated antigens (TAAs), or living or lysed tumor cells, but these approaches remain laborious, time-consuming, and functionally limited, hampering clinical development of ACT. Here, we demonstrate that homology cluster analysis of T cell receptor (TCR) repertoires efficiently identifies tumor-reactive TCRs allowing to: (1) detect their presence within the pool of tumor-infiltrating lymphocytes (TILs); (2) optimize TIL culturing conditions, with IL-2_low/IL-21/anti-PD-1 combination showing increased efficiency; (3) investigate surface marker-based enrichment for tumor-targeting T cells in freshly isolated TILs (enrichment confirmed for CD4⁺ and CD8⁺ PD-1⁺/CD39⁺ subsets), or re-stimulated TILs (informs on enrichment in 4-1BB-sorted cells). We believe that this approach to the rapid assessment of tumor-specific TCR enrichment should accelerate T cell therapy development.

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

The current understanding of humoral immune response in cancer patients suggests that tumors may be infiltrated with diffuse B cells of extra-tumoral origin or may develop organized lymphoid structures, where somatic hypermutation and antigen-driven selection occur locally. These processes are believed to be significantly influenced by the tumor microenvironment through secretory factors and biased cell-cell interactions. To explore the manifestation of this influence, we used deep unbiased immunoglobulin profiling and systematically characterized the relationships between B cells in circulation, draining lymph nodes (draining LNs), and tumors in 14 patients with three human cancers. We demonstrated that draining LNs are differentially involved in the interaction with the tumor site, and that significant heterogeneity exists even between different parts of a single lymph node (LN). Next, we confirmed and elaborated upon previous observations regarding intratumoral immunoglobulin heterogeneity. We identified B cell receptor (BCR) clonotypes that were expanded in tumors relative to draining LNs and blood and observed that these tumor-expanded clonotypes were less hypermutated than non-expanded (ubiquitous) clonotypes. Furthermore, we observed a shift in the properties of complementarity-determining region 3 of the BCR heavy chain (CDR-H3) towards less mature and less specific BCR repertoire in tumor-infiltrating B-cells compared to circulating B-cells, which may indicate less stringent control for antibody-producing B cell development in tumor microenvironment (TME). In addition, we found repertoire-level evidence that B-cells may be selected according to their CDR-H3 physicochemical properties before they activate somatic hypermutation (SHM). Altogether, our work outlines a broad picture of the differences in the tumor BCR repertoire relative to non-tumor tissues and points to the unexpected features of the SHM process.

Advances in high-dimensional flow cytometry and single-cell RNA sequencing (scRNA-seq) have enhanced our understanding of the heterogeneity of tumor-infiltrating B cells (TIBs). Subpopulations of TIBs exhibit diverse, sometimes opposing roles in tumor control, influenced by surface molecule, cytokine, and transcription factor expression. IgA and IgG expression in tumors have shown predictive value in melanoma and KRAS-mutated, but not KRAS wild-type, lung adenocarcinoma (LUAD). To investigate the functional differences between B cells producing these isotypes, we performed bulk transcriptome analysis of tumor-infiltrating surface-IgA+ (sIgA+) and sIgG+ memory B cells in LUAD. In LUAD, sIgA+ B cells overexpressed FCRL4, PDCD1, and RUNX2, suggesting an atypical chronically antigen-stimulated phenotype with features of exhaustion. Public scRNA-seq data revealed FCRL4-expressing TIBs as a distinct cluster with upregulation of genes involved in IFNγ and IFNα responses. sIgG+ B cells from LUAD overexpressed IL5RA, indicating a role for IL-5 in class-switch recombination to IgG. TCGA LUAD cohort analysis showed that the FCRL4/CD20 expression ratio correlates with lower survival, reinforcing FCRL4 as a marker of dysfunctional TIBs. Additionally, in renal cancer, high IGHA1/IGHG1 ratios were linked to worse survival. These findings suggest that the IgA/IgG ratio in tumors reflects not only the TME cytokine environment, but also functional differences in B cell populations, providing insights into their diverse roles in tumor progression.