Uncovering ectopic GC-like niches for tumor reactive lymphocyte priming in lung adenocarcinoma using Stereo-XCR-seq.

T/B cell receptors (T/BCR), coordinating antigen-targeting immune response, play crucial roles in anti-tumor immune response. Tracking T and B cell clonal evolution in situ at single-cell resolution is essential for understanding the adaptive immune responses. To address the lack of tools for in situ single-cell T/BCR (XCR) sequencing, we develop Stereo-XCR-seq, an efficient method for retrieving and sequencing TCR and BCR from spatial transcriptome cDNA libraries at subcellular resolution. Stereo-XCR-seq enables high-fidelity, unbiased recovery of XCR sequences alongside spatial transcriptomics in extensive tissues, facilitating the identification of heterogeneous lymphoid aggregates with distinct clonal activities in situ. Using Stereo-XCR-seq, we uncover that IgG+ plasma cell aggregates display features of ectopic germinal center-like (GC-like) niches to select tumor-reactive clones from distal immature tertiary lymphoid structures in 11 lung adenocarcinoma (LUAD) tumors. The presence of these IgG+ plasma cell aggregates in LUAD indicates an improved anti-tumor immune surveillance and sensitivity towards immune checkpoint blockade (ICB) therapy. Collectively, Stereo-XCR-seq enables in situ single-cell profiling of T and B cell clonal activities and their interactions with local microenvironment, links tumor reactivity to intratumoral distribution of lymphocytes, and thus offers a versatile tool for dissecting lymphocyte clonal dynamics across human diseases.