Abstract 4026: Spatial T-cell receptor profiling uncovers barriers to T-cell engagement and shared tumor-reactive clonotypes in high-risk neuroblastoma

Abstract Background: Survival for patients with high-risk neuroblastoma (NB) is ∼50%. While T-cell infiltration correlates with improved outcomes in some pediatric cancers, T-cell diversity, spatial organization, and antigen specificity remain poorly characterized. We integrated bulk T-cell receptor (TCR) sequencing, spatial transcriptomics, and spatial TCR profiling in NB patients from the Children's Oncology Group phase 3 trial ANBL1531, which evaluated 131I-metaiodobenzylguanidine therapy during frontline induction. Methods: We applied hybrid-capture TCR sequencing to paired biopsy and resection FFPE tumors (10 samples from 5 patients), longitudinal PBMCs (84 samples from 33 patients), and cfDNA (14 samples from 12 patients). Using shared TCRs across sample types and timepoints, we designed a Xenium panel with 377 genes and 100 TCRα/β/γ/δs, enabling spatial mapping across 1.90 million cells (median 138,742, range 17,175-715,510 cells per tumor). We predicted TCR specificity using MixTCRpred, and inferred sequence convergence using GLIPH2 against a database of reference TCRs (4,146 known specificities, 595,689 controls, 16,888 NB from the Precision oncology for young people cohort). Results: PBMC TCR repertoires showed biphasic dynamics, with diversity contracting mid-therapy (Shannon mean 476→151) then recovery post-therapy (413; p=0.0006, linear mixed-effects model), likely corresponding to chemotherapy-driven lymphopenia. In tumors, TCR diversity declined between biopsy and resection while hyperexpanded clonotypes increased (mean 30→59%), indicating potential selective clonal enrichment. Spatial profiling of the 10 tumors revealed immune-tumor compartmentalization, with macrophage-enriched barriers segregating immune cells from clusters of tumor cells. Spatial TCR profiling enabled in situ α/β chain pairing, identifying four TCRα/β clonotypes (all CD8+ effector memory) not resolvable by bulk sequencing. One clonotype with predicted NY-ESO-1 specificity expanded from biopsy to resection (2.22→4.92% of all T cells), suggesting antigen-driven clonal proliferation. Despite this expansion, clonotype spatial positioning remained unchanged, indicating that microenvironmental architecture constrains T-cell localization. GLIPH2 identified 8 NB-restricted TCR sequence motifs shared across 3-7 NB patients. These motifs occurred in individuals with convergent MHC alleles, suggesting recognition of common NB-associated antigens. Conclusions: Integrated TCR and spatial profiling revealed macrophage-mediated compartmentalization and restricted T-cell localization. Clonotypes predicted to be tumor-reactive also expanded during therapy. Shared NB-specific TCR motifs suggest convergent antigen recognition and targets for immunotherapy. Citation Format: Yiyue (January) Jiang, Wenbao Yu, Stephanie Pedersen, Jenna Eagles, Anusha Thadi, Arlene Naranjo, Natalie Bucheimer Collins, Steven DuBois, Rochelle Bagatell, Brian D. Crompton, Kai Tan, Trevor J. Pugh. Spatial T-cell receptor profiling uncovers barriers to T-cell engagement and shared tumor-reactive clonotypes in high-risk neuroblastoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4026.