Abstract Introduction Tumor neoantigens have emerged as promising candidates for personalized immunotherapies, however, confirming their presentation and immunogenicity remains challenging due to low neoantigen expression and limited detection sensitivity. Moreover, the spatial organization of neoantigen-specific T cell clones relative to their target tumor cells and other cell types within the tumor microenvironment remains incompletely defined due to technological limitations. To bridge this gap, we developed Slide-GoTags. Methods Slide-GoTags integrates single-nucleus spatial transcriptomics with targeted genotyping and TCR sequencing from a single 20 µm tissue section. Slide-tags spatial barcoding and the 10x Genomics 5’ droplet-based platform are followed by targeted RNase H—dependent PCR and long-read sequencing, enabling genotypes and TCR repertoires to be linked to gene expression profiles and spatial coordinates via shared cell barcodes. Results Slide-GoTags was applied to murine and human tumors, revealing colocalization of clonally expanded, neoantigen-specific T cells with tumor cells expressing their cognate neoantigen, validated by known neoantigen-TCR pairs. T cell functional state and clonotype avidity were linked to neoantigen proximity. Moreover, we identified IFN-γ—rich immunogenic niches consistently enriched for neoantigen-specific progenitor-exhausted T cells, marked by enhanced local T cell activation, clonal expansion, and immune-modulatory interactions with antigen-presenting cells within the TME. Spatially resolved receptor—ligand analysis revealed co-stimulatory and inhibitory interactions that shape immune engagement and regulation within these niches. Conclusion In this study, we define the spatial architecture of neoantigen directed T cell immunity shaped by proximity to neoantigen expressing tumor cells, TCR avidity, and receptor-ligand interactions within tumors, providing a framework for spatially informed TCR deorphanization and the development of personalized T cell therapy. Funding Source This work was supported in part by the NIH/NCI (CA276865), the Mark Foundation for Cancer Research Endeavor Award, the Dana-Farber/Harvard Cancer Center Kidney SPORE (P50CA101942), and the Dana-Farber/Harvard Cancer Center Support Grant (P30CA006516). A.N Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Improved methods to identify therapeutically relevant tumor neoantigens and their cognate T cells would aid the development of precision medicines for cancer. Here, we developed Slide-GoTags, a droplet-based single-nucleus spatial transcriptomics approach that characterizes neoantigen-specific immunity by integrating targeted transcript genotyping and T cell receptor (TCR) sequencing with single-nucleus RNA sequencing from the same slice of frozen tissue. Application of Slide-GoTags to mouse and human tumors revealed colocalization of clonally expanded, neoantigen-specific T cells with tumor cells expressing their cognate neoantigen. We also identified distinct spatial immune landscapes shaped by anti-PD1 or anti-CTLA4 blockade in mouse colorectal tumors. Across human tumor types, Slide-GoTags detected TCR–neoantigen interactions through spatial proximity and identified an enrichment of interferon-driven immunogenicity niches in immunologically ‘hot’ tumors compared to ‘cold’ tumors. These niches harbored three T cell clonotypes that colocalized with genotyped neoantigens, highlighting a spatially organized antitumor immune response. Collectively, Slide-GoTags establishes a framework for in situ mapping of T cell–tumor interactions directly from individual tissue. Spatial relationships between tumor neoantigens and cognate T cells are studied.

The ribosomal protein RPS15 is recurrently mutated in chronic lymphocytic leukemia (CLL) and confers adverse prognosis. While translational rewiring is a recognized consequence of RPS15 mutation (RPS15-mut), its impact on genome stability and the precise mechanisms governing RPS15-mut driven B-cell leukemogenesis remain unclear. To delineate the sequence of cellular events that underlie malignant transformation and progression in the context of RPS15 mutation, we functionally interrogated a conditional knock-in mouse model of the RPS15-S138F mutation established using the Cd19-Cre/loxP system (Gutierrez et al, ASH 2020). Despite enrichment for Myc target genes in pre-leukemic Rps15-mutB splenocytes, these cells displayed a hypoproliferative phenotype characterized by increased G1 cell cycle checkpoint activation and reduced ex vivo proliferative capacity accompanied by increased apoptosis, compared to Rps15-WT splenocytes. We hypothesized that decreased proliferation and increased apoptosis could result from a p53-mediated response to cellular stress arising from Rps15 mutation. Informed by the genomic landscape of CLL developing in older Rps15-mut mice that displayed an enrichment of A•T>C•G and C•G>T•A transversions indicative of peroxide-induced DNA damage, we interrogated the transcriptomes of Rps15-mutpre-leukemic B splenocytes, which showed upregulation of reactive oxygen species (ROS) genes (NES 1.5, p<0.05). We experimentally quantified mitochondrial ROS which confirmed increased oxidative stress in Rps15-mut compared to Rps15-WT splenocytes (p<0.001). We reasoned that unresolved oxidative stress could lead to DNA damage accumulation. Accordingly, we demonstrated increased γH2AX accumulation, a marker of DNA damage, in Rps15-mut B splenocytes compared to their Rps15-WT counterparts (p=0.01), as well as hypersensitivity of Rps15-mut splenocytes to oxidative stress overload with parthenolide (p=0.0009). RPS15 is known to stabilize p53 via MDM2 inhibition, hence Rps15-mut cells exhibited reduced p53 expression. Nevertheless, in response to DNA damage, p53 and p21 were significantly induced, the latter a p53-dependent mediator of G1 checkpoint activation, highlighting the functional importance of residual p53 activity in Rps15-mut B cells. Given their heightened level of DNA damage, we further hypothesized that Rps15-mut pre-leukemic B splenocytes are hyper-dependent on DNA damage response (DDR). Conversely, defective DDR could precipitate genomic instability and facilitate acquisition of additional genomic alterations, such as TP53 deletion or loss-of-function mutation, that might promote transition toward a hyperproliferative or malignant state. To determine whether Rps15-mut B splenocytes could effectively respond to further genomic insults, we assessed cellular response to hydroxyurea (HU) and ionizing radiation (IR) that induce replication stress and DNA double-strand breaks (DSBs) respectively. While the ATR-Chk1 response to HU was preserved, the ATM-Chk2 response to IR was defective with diminished phosphorylation of downstream ATM targets and impaired induction of G2/M arrest. Indeed, Rps15-mut B cells subsequently acquired a spectrum of additional mutations and chromosomal alterations (e.g. Myc/Mapk amplification, Trp53 loss) that reflect genomic instability secondary to defective DDR. Finally, we postulated that translational defects directly contribute to oxidative DNA damage accumulation and genomic instability in Rps15-mut B cells. We therefore performed ribosome profiling that showed differential translation efficiency (TE) in 342 genes between Rps15-mutand Rps15-WT B splenocytes, with significant enrichment of DNA replication, DDR and cell cycle genes by GSEA that was recapitulated in RPS15-mut vs WT isogenic HG3 CLL cell lines. In particular, Gpx1, a glutathione peroxidase that functions as a key cellular antioxidant, was confirmed by western blot to be downregulated in Rps15-mut B cells secondary to reduced TE (0.6-fold change vs WT, p=0.018), alongside other proteins important for the amelioration of oxidative DNA damage and DSBs (e.g. Cyb5r4, Otud4, Fance, Tlk1). Our analyses thus link RPS15-induced translational alterations with a cascade of cellular defects that compromise genome stability and highlight the critical nature of these defects in B-cell transformation and CLL progression.

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR015) of the Conference Program/Proceedings. Citation Format: David A. Braun, Derin B. Keskin, Sachet A. Shukla, Bradley A. McGregor, Nicholas R. Schindler, Eryn Blass, Susan Klaeger, Lucas Pomerance, Siranush Sarkizova, Shuqiang Li, Jackson Southard, Giorgia Moranzoni, Christina B. Pedersen, Yiwen Liu, Steven L. Chang, Michelle S. Hirsch, Nicole R. LeBoeuf, Matthew Mossanen, Vipheaviny Chea, Isabel Carulli, Oriol Olive, Ambica Mehndiratta, Haley Greenslade, Giacomo Oliveira, J. Bryan Iorgulescu, Sabina Signoretti, Jon C. Aster, Liudmila Elagina, Ignaty Leshchiner, Gad Getz, Maegan Harden, Stacey Gabriel, Lars R. Olsen, Donna S. Neuberg, Edward F. Fritsch, Nir Hacohen, Kenneth J. Livak, Steven Carr, Patrick A. Ott, Catherine J. Wu, Toni K. Choueiri. Tumor-specific immunity generated by a personalized neoantigen vaccination incorporating locally delivered ipilimumab in renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A013.

Personalized cancer vaccines can generate circulating immune responses against predicted neoantigen targets, but whether such responses lead to actual immune recognition of a patient’s tumor and consequent clinical activity is largely unknown. To investigate this, we conducted a phase I clinical trial of a personalized neoantigen vaccine as adjuvant therapy in high-risk, completely surgically resected, stage III/IV clear cell renal cell carcinoma (RCC; NCT02950766). This first-in-disease study included several modifications from prior neoantigen vaccine studies, including: (i) the use of an improved antigen prediction tool (HLAthena), a neural network model that was trained on a large HLA class I immunopeptidome dataset; (ii) the subcutaneous and intradermal administration of the vaccine to engage a broader repertoire of antigen-presenting cells in the skin; and (iii) the inclusion of ipilimumab administered subcutaneously next to the vaccination site (for pre-specified cohorts) intended to improve T cell priming and activation at the local draining lymph node. Across all nine patients, a median of 15 vaccinating peptides (range: 8 – 19) were successfully manufactured and administered per study subject, targeting a median of 13 unique mutations (range: 7 – 17). Six of nine patients were vaccinated against a neoantigen formed through a mutation in at least one known RCC driver gene (VHL, PBRM1, BAP1, KDM5C, or PIK3CA). Single-cell transcriptomic and T cell receptor (TCR) sequencing analysis of vaccine site immune populations in the skin revealed a substantial increase in the number of T cells (p = 0.003) and T cell clonotypes (p < 0.001) following vaccination. No patient had a pre-existing ex vivo peripheral T cell response against any of the neoantigen peptides in the vaccine. Following vaccination, all nine patients developed detectable ex vivo peripheral T cell response (by IFN-gamma ELISPOT) against neoantigen peptides in the vaccine. Nearly all detectable ex vivo responses were derived from CD4+ T cells as assessed by flow cytometry analysis, and a substantial proportion of responses (27.4% - 99.8% of total responses for an individual pool of vaccine peptides) were polyfunctional (with T cells producing at least 2 of the following cytokines: IFN-gamma, IL-2, or TNF-alpha). Among peripheral immune responses, there were no observable differences in the quantity or phenotype of T cells responding to vaccine alone versus the vaccine co-administered with ipilimumab. In seven of nine patients, neoantigen vaccine-specific T cells exhibited in vitro anti-tumor reactivity against autologous RCC cells. Clinically, there were no dose limiting toxicities, and no disease recurrences were observed in this high-risk population (median follow-up of 23.9 months at time of data cutoff). Overall, these data suggest that personalized neoantigen vaccines (with or without co-administered subcutaneous CTLA-4 blockade) can induce anti-tumor immune responses in patients with RCC. Citation Format: David A. Braun, Derin B. Keskin, Sachet A. Shukla, Bradley A. McGregor, Nicholas R. Schindler, Eryn Blass, Susan Klaeger, Lucas Pomerance, Siranush Sarkizova, Shuqiang Li, Jackson Southard, Giorgia Moranzoni, Christina B. Pedersen, Yiwen Liu, Steven L. Chang, Michelle S. Hirsch, Nicole R. LeBoeuf, Matthew Mossanen, Vipheaviny Chea, Isabel Carulli, Oriol Olive, Ambica Mehndiratta, Haley Greenslade, Giacomo Oliveira, J. Bryan Iorgulescu, Sabina Signoretti, Jon C. Aster, Liudmila Elagina, Ignaty Leshchiner, Gad Getz, Maegan Harden, Stacey Gabriel, Lars R. Olsen, Donna S. Neuberg, Edward F. Fritsch, Nir Hacohen, Kenneth J. Livak, Steven Carr, Patrick A. Ott, Catherine J. Wu, Toni K. Choueiri. Tumor-specific immunity generated by a personalized neoantigen vaccination incorporating locally delivered ipilimumab in renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR015.

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I–low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

The tumor immune microenvironment plays a critical role in cancer progression and response to immunotherapy in clear cell renal cell carcinoma (ccRCC), yet the composition and phenotypic states of immune cells in this tumor are incompletely characterized. We performed single-cell RNA and T cell receptor sequencing on 164,722 individual cells from tumor and adjacent non-tumor tissue in patients with ccRCC across disease stages: early, locally advanced, and advanced/metastatic. Terminally exhausted CD8+ T cells were enriched in metastatic disease and were restricted in T cell receptor diversity. Within the myeloid compartment, pro-inflammatory macrophages were decreased, and suppressive M2-like macrophages were increased in advanced disease. Terminally exhausted CD8+ T cells and M2-like macrophages co-occurred in advanced disease and expressed ligands and receptors that support T cell dysfunction and M2-like polarization. This immune dysfunction circuit is associated with a worse prognosis in external cohorts and identifies potentially targetable immune inhibitory pathways in ccRCC.