ABSTRACT Immune responses correlated with clearance of human papillomavirus type 16 (HPV16) were sought in PVX2 immunotherapy-treated CIN ≤1 patients. PVX2 comprises intramuscular vaccinations with HPV16 E7-targeted pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine (3 mg, weeks 0 and 4) and HPV16 L2E7E6 fusion protein (TA-CIN; 100 µg week 8). Administering PVX2 to 12 women with HPV16+ CIN ≤1 elicited both antibody and CD8+ T-cell responses, including changes in TCR diversity, clonal expansion, and emergence of public clonotypes. HPV16 was not detected in 5/11 evaluable participants at month 6. Post-vaccination E6, E7, and L2-specific antibody titers were higher, but none correlated with clearance. Neither TCR clonal diversity/expansion nor public clonotypes were associated with HPV16 clearance. While the fractions of expanded/new clonotypes were not associated with HPV16 clearance, a higher proportion of vaccine antigen-specific reads was evident post-vaccination in those clearing HPV16. High baseline serum MCP-1 associated with HPV16 persistence, and MIP-1β trended higher post-vaccination in those clearing HPV16. IMPORTANCE HPV16 is a necessary cause of over half of all cervical cancers, and in even higher fractions of other anogenital and oropharyngeal cancers. Treatments for HPV16 infection and associated pre-cancerous lesions are needed given the disease burden and limitations of current treatments. Women with persistent HPV infections endure anxiety and stress due to frequent colposcopies, inconvenience, healthcare costs from active surveillance, and potential to transmit the virus to other sexual partners. Since a majority of patients can spontaneously clear their infections, administration of an immunotherapy may promote the elimination of otherwise persistent infections where disease can progress.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells capable of MR1-dependent immune surveillance, but how intratumoral bacteria modulate MR1 expression in human lung tumors remains unclear. We studied intratumoral MAIT cells from paired single-cell RNA and TCR sequencing datasets of tumor-infiltrating CD3 T cells isolated from non-small cell lung cancer tumors in patients receiving neoadjuvant PD-1 blockade therapy. MAIT cells were subclustered to identify conventional MAIT-associated TCR clonotypes, which were then used to examine how bacterial exposure impacts cell-surface MR1 expression and downstream MAIT TCR activation. We found that select intratumoral Enterococcus species (spp.) did not directly activate MAIT cells but enhanced MR1-dependent MAIT activation in the presence of exogenous 5-OP-RU by increasing cell-surface MR1 expression on antigen-presenting cells including dendritic cells, B cells, and mononuclear phagocytes. This increase in MR1 cell surface expression is modulated through a posttranscriptional mechanism consistent with altered intracellular processing and trafficking of MR1. These findings reveal a role for tumor-associated bacteria in modulating MR1-dependent innate-like T cell activation and provide a basis for future studies examining whether this process influences response to immune checkpoint blockade.

Motivation:The Functional Expansion of Specific T cell (FEST)-based assays combine short-term peptide stimulation with TCR sequencing to identify clonotypes that expand in response to specific antigens. These approaches have proven invaluable for detecting neoantigen-specific T cell responses, guiding vaccine development, and assessing checkpoint blockade efficacy. However, variability introduced by biological and technical replicates poses challenges for reproducibility and interpretation, and existing computational tools do not address replicate-level analysis in these assays. Results:We developed replicateFest, a computational framework implemented as an R package and Shiny web application, to analyze FEST-based TCR-seq data with and without replicates. replicateFest applies Fisher's exact test for non-replicate datasets and negative binomial modeling for replicate experiments, returning adjusted p-values and odds ratios to identify clonotypes significantly expanded in antigen-stimulated conditions. The framework distinguishes FEST-expanded clonotypes (relative to a no-antigen control) and FEST-positive clonotypes (expanded compared to all other conditions). Validation using synthetic datasets confirmed accurate detection of antigen-specific clonotypes. Application to published HIV-1 epitope stimulation data reproduced original findings and demonstrated replicateFest's utility for reproducibility assessment and quality control. Availability and Implementation:replicateFest is freely available under the Apache-2.0 license as an R package at https://github.com/OncologyQS/replicateFest and as an interactive Shiny application at http://www.stat-apps.onc.jhmi.edu/FEST/.

Immunotherapies have transformed cancer care; however, tumor intrinsic and extrinsic factors contribute to high variability in therapeutic responses. While tissue injuries can impact cancer recurrence and metastatic spread, little is known about their potential to effect immune checkpoint blockade (ICB) response. In this study, we reveal that distal traumatic muscle injury accelerated progression and impaired adjuvant ICB response of multiple murine tumors. This injury-induced accelerated tumor growth coincided with decreased intra-tumoral density and effector phenotype of tumor-reactive CD8 + T cells and relied on communication through a shared draining lymph node. Enhancing injury repair using a biological scaffold abrogated the injury-induced accelerated tumor growth in an interleukin-4-dependent manner and improved ICB response. In a retrospective cohort analysis of breast cancer patients undergoing ICB treatment, biological scaffold implantation following mastectomy was associated with increased overall survival. This work suggests that injury-driven immune dysfunction may contribute to cancer progression and ICB resistance, but enhancing wound healing with pro-regenerative biomaterials may offer a viable strategy for mitigating adverse cancer outcomes, particularly in the setting of adjuvant and neoadjuvant ICB.

Extracellular matrix (ECM) scaffolds induce type 2 immunity to promote repair. Here, we show that immune cells recruited to ECM-treated murine muscle injuries and clinical soft tissue defects express immune checkpoints. Specifically, T H 2 cells and regulatory T cells (Tregs) increase LAG3 expression, while macrophages express PDL2. TCR analysis and a triple-reporter strain for interleukin (IL)-13 and Treg fate-mapping suggest that Tregs in ECM-treated wounds transition into T H 2-like exTregs that express LAG3. Immune checkpoint inhibition (ICI) significantly stimulated type 2 immunity in ECM-treated wounds, including increased T H 2 cells, Treg transition to T H 2-like exTregs, and pro-regenerative macrophages. Moreover, ICI enhanced muscle repair and reduced fibrosis in ECM-treated wounds. Collectively, these findings show Treg/T H 2 plasticity in wound healing and introduce a novel ICI application to enhance immune-mediated regeneration.

Clonal expansion of HIV-infected CD4+ T cells is a barrier to HIV eradication. We previously described a marked reduction in the frequency of the most clonally expanded, infected CD4+ T cells in an individual with elite control (ES24) after initiating chemoradiation for metastatic lung cancer with a regimen that included paclitaxel and carboplatin. We tested the hypothesis that this phenomenon was due to a higher susceptibility to the chemotherapeutic drugs of CD4+ T cell clones that were sustained by proliferation. We studied a CD4+ T cell clone with replication-competent provirus integrated into the ZNF721 gene, termed ZNF721i. We stimulated the clone with its cognate peptide and then exposed the cells to paclitaxel and/or carboplatin or the antiproliferative drug mycophenolate mofetil. While treatment of cells with the cognate peptide alone led to a marked expansion of the ZNF721i clone, treatment with the cognate peptide followed by culture with either paclitaxel or mycophenolate mofetil abrogated this process. The drugs did not affect the proliferation of other CD4+ T cell clones that were not specific for the cognate peptide. This strategy of antigen-specific stimulation followed by treatment with an antiproliferative agent may lead to the selective elimination of clonally expanded HIV-infected cells.

Novel clinical cancer biomarkers are needed to better adjudicate the use of emerging therapies like immunotherapy in multimodal care. We evaluated plasma neutrophil extracellular traps (NETs) levels as one such biomarker due to the multifaceted and targetable roles of NETs in immune checkpoint inhibition resistance. Recently, histone H3 cleavage (H3Clip) was described as a sensitive and specific marker of NET release (NETosis) in humans. We therefore sought to develop H3Clip as a cancer biomarker for patients with resectable non-small cell lung cancer (NSCLC). We analyzed pre-treatment plasma samples from 47 patients with NSCLC who were assigned to up-front surgery, and 163 patients treated with neoadjuvant nivolumab monotherapy or nivolumab plus chemotherapy regimens as part of the phase II NADIM (NCT03081689), NADIM II (NCT03838159), and J1414 (NCT02259621) clinical trials. Circulating H3Clip levels were measured and correlated with the clinical outcomes of pathological complete response (pCR), major pathological response (MPR), recurrence free survival (RFS), progression-free survival (PFS) and overall survival (OS). We found that baseline high H3Clip level was associated with significantly reduced OS and RFS in patients with NSCLC undergoing up-front surgery. Additionally, high H3Clip at baseline evaluation was associated with significantly shorter OS and PFS among patients receiving neoadjuvant chemo-immunotherapy. Among those who achieved a major pathological response to chemo-immunotherapy, high H3Clip was similarly associated with shorter OS and PFS. Among patients achieving a complete or partial clinical response to chemoimmunotherapy, high H3Clip was strongly associated with reduced OS and PFS. In this subgroup, the absolute difference in 30-month OS and PFS between the high and low H3Clip groups was 31% and 52%, respectively. High H3Clip patients were significantly less likely to achieve an MPR (p=0.0233; low vs high H3Clip: 76.92% vs. 52.50%) and pCR (p=0.0095; low vs. high H3Clip: 58.97% vs. 30.00%) to neoadjuvant chemo-immunotherapy. Finally, high H3Clip level was associated with shorter OS and PFS, and lower rates of pCR in the presence of high-risk clinical demographic features and low tumor PD-L1 expression. We demonstrate that H3Clip is a novel biomarker of prognosis and response to neoadjuvant chemo-immunotherapy in NSCLC through analysis of several phase II neoadjuvant chemo-immunotherapy trials. H3Clip is strongly associated with OS, PFS, RFS, MPR, and pCR and provides added clinical utility on top of routine demographic variables and biomarkers for risk stratification. NETs could thus represent both an important biomarker of response to chemo-immunotherapy and therapeutic target given their established effects on the tumor immune microenvironment. Muhammad H. Shahzad, Alberto Cruz-Bermudez, Roni F. Rayes, Ernest Nadal, Meghan L. De Meo, Mark Sorin, Simon Milette, Lyndon C. Walsh, Daniela F. Quail, Logan Walsh, Alex Martinez-Marti, Reyes Bernabe-Caro, Amelia Insa, Bartomeu Massuti, Belen Sierra-Rodero, Atocha Romero, Betty Giannias, Sara Najmeh, Pierre-Olivier Fiset, David S. Mulder, Lorenzo E. Ferri, Valsamo Anagnostou, Gavin Pereira, Kellie N. Smith, Drew Pardoll, Patrick Forde, Jonathan Cools, Mariano Provencio, Jonathan D. Spicer. NETosis-specific clipped histone H3 is a novel biomarker of response to neoadjuvant chemo-immunotherapy in resectable lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Mechanisms of Cancer Immunity and Cancer-related Autoimmunity; 2025 Sep 24-27; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(9 Suppl):Abstract nr B020.

KM curvces for recurrence based on stage and RVT

The role of mucosal invariant T cells (MAITs), in the lung tumor microenvironment re-mains poorly understood, especially in the setting of immune checkpoint inhibitors. We identified intratumoral MAIT cells from paired single cell RNA and TCR sequencing datasets of tumor infil-trating CD3 T cells isolated from non-small cell lung cancer tumors in patients receiving neoadju-vant PD-1 blockade therapy. MAIT cells were subclustered to identify conventional MAIT-associ-ated TCR clonotypes predicted to recognize intratumoral bacteria, which we then tested for func-tional recognition using a MAIT TCR capture functional assay. Strikingly, although not directly recognized by MAIT cells, previously identified probiotic Enterococcus spp and detected in the intratumoral microbiome of lung cancer patients, selectively synergized with exogenous riboflavin biosynthesis-derived metabolites to induce expression of MR1 by antigen presenting cells, includ-ing dendritic cells, B cells and mononuclear phagocytes. Boosting of MR1 cell surface expression resulted from perturbation of endo-lysosomal vacuolar pathway by Enterococcus and recycling of early endosomal MR1 to the cytoplasmic membrane. Riboflavin auxotrophic Enterococcus spp may therefore exercise their beneficial immunomodulatory functions upon immune checkpoint blockade treatment, at least in part, by promoting intratumoral MR1 expression and innate like T cell activation. Our results indicate that composition of the intratumoral microbiome during im-mune checkpoint inhibitor treatment has the potential to impact the function of human intratumoral MAIT cells. ### Competing Interest Statement The authors have declared no competing interest. Commonwealth foundation Willowcroft foundation

Recent efforts in vaccine development have targeted spike proteins from evolving SARS-CoV-2 variants. In this study, we analyzed T cell responses to the XBB.1.5 and BA.2.86 subvariants in individuals who previously received bivalent vaccines containing mRNA for ancestral and BA.5 spike proteins. T cell-mediated cytokine responses to spike proteins from both variants were largely preserved. To determine the mechanism of this preserved recognition, we utilized the functional expansion of specific T cells (FEST) assay to distinguish between the presence of T cells that cross-recognized ancestral and variant epitopes versus distinct populations of T cells that were mono-reactive for ancestral or variant epitopes. We found the majority of spike-specific T cells cross-recognized the ancestral spike and the XBB.1.5 and BA.2.86 subvariants, with less than 10% of T cells being mono-reactive for either variant. Interestingly, immunization with the XBB.1.5 monovalent booster vaccine did not significantly increase the percentage of XBB.1.5 mono-reactive T cells. Our results suggest a potential limitation in the induction of mono-reactive T cell responses by variant-specific booster vaccines.

Immune checkpoint blockade (ICB) is standard of care in advanced diffuse pleural mesothelioma (DPM), but its role in the perioperative management of DPM is unclear. In tandem, circulating tumor DNA (ctDNA) ultra-sensitive residual disease detection has shown promise in providing a molecular readout of ICB efficacy across resectable cancers. This phase 2 trial investigated neoadjuvant nivolumab and nivolumab/ipilimumab in resectable DPM along with tumor-informed liquid biopsy residual disease assessments. Patients with resectable epithelioid/biphasic DPM enrolled sequentially to nivolumab 240 mg every 2 weeks (q2w) for three cycles (Arm A, n = 16) or nivolumab 3 mg kg-1 q2w for three cycles plus ipilimumab 1 mg kg-1 on cycle 1 (Arm B, n = 14), followed by surgery, optional chemotherapy and/or radiotherapy, and nivolumab 480 mg q4w for 1 year. Co-primary endpoints included safety and feasibility; key exploratory endpoints included progression-free survival (PFS), overall survival (OS) and ctDNA analyses. The trial met its primary endpoints, and, in Arms A and B, 81.3% and 85.7% of patients proceeded to surgery, respectively. Treatment was safe, with a single dose-limiting toxicity in each arm. In Arm A, median PFS and OS were 9.6 months (95% confidence interval (CI): 2.5-27.7) and 19.3 months (95% CI: 14.9-34.7), respectively. In Arm B, median PFS and OS were 19.8 months (7.1-not reached) and 28.6 months (20.4-not reached), respectively. Persistent ctDNA was detected during neoadjuvant therapy in patients who did not undergo complete surgical resection due to disease progression (Fisher's exact test, P = 0.00013). Patients with detectable ctDNA on cycle 3 and pre-surgery had shorter PFS (log-rank test, P = 0.027 and P = 0.0059, respectively); this association was more pronounced when quantitative ctDNA changes were considered (log-rank test, P = 1.8 × 10-6). Our findings support the feasibility of neoadjuvant ICB and the clinical utility of ctDNA analyses to capture residual disease in resectable DPM. ClinicalTrials.gov identifier: NCT03918252 .

Background:Ulcerative Colitis (UC) is characterized by chronic, relapsing and remitting inflammation in the colon and rectum. Pathogenic T cell activity is thought to play a major role in this process. T cell effector function is determined by the T cell receptor (TCR) and the antigen it recognizes. Examining the TCR repertoire can provide key insights into the adaptive immune response. Objective:To characterize the longitudinal TCR repertoire of patients with UC across disease activity to determine if recurrent antigen(s) are responsible for active inflammation. Design:Bulk TCR Vβ sequencing was done on colon tissue of 20 patients with UC across multiple time points of disease. Corresponding clinical metadata was also obtained over the same time period for each patient to map their clinical disease course. The top ten most highly abundant clones from each time point were longitudinally tracked and correlated with disease phenotype. Results:Seventy-five percent of patients did not have overlapping abundant TCR clones across multiple time points of disease. The remaining 25% of patients had one to five TCR clones present in high abundance in their tissue during every time point analyzed. Conclusion:These results demonstrate that most patients with UC do not share a similar TCR repertoire over time, indicating that times of inflammation are associated with unique antigen exposures. A smaller group of patients have persistent, private TCR clones with high abundance, 60% of whom had more unremitting, active disease.

Purpose: Co-mutations of the Kirsten rat sarcoma virus (KRAS) and serine/threonine kinase 11 (STK11) genes in advanced non-small cell lung cancer (NSCLC) are associated with immune checkpoint blockade (ICB) resistance. Although neoadjuvant chemoimmunotherapy is now a standard-of-care treatment for resectable NSCLC, the clinical and immunologic impacts of KRAS and STK11 co-mutations in this setting are unknown.Experimental Design: We evaluated and compared recurrence-free survival of resectable KRAS-mutated NSCLC tumors, with or without co-occurring STK11 mutations, treated with neoadjuvant ICB. Single-cell transcriptomics was performed on tumor-infiltrating T cells from seven KRASmut/STK11wt tumors and six KRAS and STK11 co-mutated (KRASmut/STK11mut) tumors.Results: Relative to KRASmut/STK11wt tumors, KRASmut/STK11mut exhibited significantly higher recurrence risk. Single-cell transcriptomics showed enhanced oxidative phosphorylation with evidence of decreased prostaglandin E2 signaling and increased IL-2 signaling in CD8+ tumor-infiltrating lymphocytes (TIL) from KRASmut/STK11mut tumors, a finding that was mirrored in KRASwt tumors that relapsed. TILs from KRASmut/STK11mut tumors expressed high levels of molecules associated with tumor residence, including CD39 and ZNF683 (HOBIT).Conclusions: These divergent T-cell transcriptional fates suggest that T-cell maintenance and residence may be detrimental to antitumor immunity in the context of neoadjuvant ICB for resectable NSCLC, regardless of KRAS mutation status. Our work provides a basis for future investigations into the mechanisms underpinning prostaglandin E2 signaling and IL-2 signaling as they relate to T-cell immunity to cancer and to divergent clinical outcomes in KRASmut/STK11mut NSCLC treated with neoadjuvant ICB.

BackgroundAnti-3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR)-positive immune-mediated necrotising myopathy (IMNM) is characterised by the presence of IgG autoantibodies against HMGCR and a strong association with specific HLA-DR alleles. Although these findings implicate HMGCR-specific CD4+T-cells in the disease’s pathogenesis, no such cells have been described. In this study, we aimed to identify and characterise HMGCR-reactive CD4+T-cells and assess their presence in affected muscle tissue from patients with anti-HMGCR+IMNM.MethodsPeripheral blood mononuclear cells from patients with anti-HMGCR+IMNM (n=10) and dermatomyositis (DM; n=10) were stimulated with HMGCR protein and peptides identified using a natural antigen processing assay (NAPA; n=6). CD4+T-cell activation was assessed by CD154 upregulation via flow cytometry. T-cell receptor β(TCR) sequencing was performed on paired HMGCR-reactive T-cells and muscle biopsy tissue (n=5).ResultsCD4+T-cell responses to HMGCR protein were higher in patients with anti-HMGCR+IMNM compared with DM (median 0.06 vs 0.00, p=0.0059). These responses were enriched in Th1-Th17 cells, and when present, they positively correlated with anti-HMGCR antibody levels (r2=0.89, p=0.0012). NAPA revealed convergent presentation of seven HMGCR core peptides, with substantial overlap in the peptide repertoires between patients. These HMGCR peptides elicited robust CD4+T-cell responses, with 9/10 anti-HMGCR+IMNM patients responding to at least one peptide, compared with 1/10 DM (p=0.0003). Analysis of HMGCR-reactive TCRs β yielded antigen-reactive motifs that were enriched in muscle biopsies (projection score 0.03 vs 0.63, p=0.007).ConclusionHMGCR-antigen-reactive CD4+T-cells are present in the circulation and target tissue of patients with anti-HMGCR+IMNM, suggesting an active role for these cells in the pathogenesis of anti-HMGCR+IMNM.

Identifying tumor-specific T cell clones that mediate immunotherapy responses remains challenging. Mutation-associated neoantigen (MANA) -specific CD8+ tumor-infiltrating lymphocytes (TIL) have been shown to express high levels of CXCL13 and CD39 (ENTPD1), and low IL-7 receptor (IL7R) levels in many cancer types, but their collective relevance to T cell functionality has not been established. Here we present an integrative tool to identify MANA-specific TIL using weighted expression levels of these three genes in lung cancer and melanoma single-cell RNAseq datasets. Our three-gene “MANAscore” algorithm outperforms other RNAseq-based algorithms in identifying validated neoantigen-specific CD8+ clones, and accurately identifies TILs that recognize other classes of tumor antigens, including cancer testis antigens, endogenous retroviruses and viral oncogenes. Most of these TIL are characterized by a tissue resident memory gene expression program. Putative tumor-reactive cells (pTRC) identified via MANAscore in anti-PD-1-treated lung tumors had higher expression of checkpoint and cytotoxicity-related genes relative to putative non-tumor-reactive cells. pTRC in pathologically responding tumors showed distinguished gene expression patterns and trajectories. Collectively, we show that MANAscore is a robust tool that can greatly enrich candidate tumor-specific T cells and be used to understand the functional programming of tumor-reactive TIL. Although individual genes that distinguish tumor-reactive CD8+ T cells from bystander T cells in tumors have been described, a functionally meaningful integrative signature has not been established. Here authors show that mutation-associated neoantigen-specific CD8+ tumor-infiltrating lymphocytes can be recognized by MANAscore, an algorithm that uses weighted expression levels of CXCL13, ENTPD1 and IL7R in single-cell RNAseq datasets of lung cancer and melanoma patients as input.

OBJECTIVE:Since anti-tumor immunity is enhanced by vaccination of mice adjacent to human papillomavirus type 16 (HPV16+) tumors, we examined whether HPV16 L2E7E6 fusion protein (TA-CIN) vaccination in the thigh of HPV16+ cervical cancer patients would be more immunogenic than their arm. METHODS:HPV16+ cervical cancer (stage IB1-IVA) patients, who had completed standard-of-care treatment within the past year and absent evidence of disease (NED), were enrolled in a pilot study (NCT02405221). Participants were randomized 1:1 to receive three 100 μg TA-CIN monthly intramuscular immunizations either in the arm or thigh and followed for two years for safety (CTCAEv4.0), immune response, and recurrence. RESULTS:Fifteen patients were enrolled (median age 44, range 35-83 years); one patient experienced a non-vaccine-related adverse event after one vaccination and withdrew. Treatment-related adverse events (n = 8) were grade 1, primarily at the injection site, and self-resolved. No recurrence was observed. TA-CIN-specific antibody titers tended to be higher in thigh-vaccinated patients. Bulk TCRseq revealed significant increases in expanded and de novo T cell clones following thigh-vaccination compared with the arm. No correlation with prior treatment modality was observed. E6-, E7-, and L2-specific TCR clones expanded, although L2-specific T cell responses were predominant. One month post-vaccination, scRNAseq revealed significant expansion of MAIT and cytotoxic CD8+ T cells, and both expanded and novel TCR clonotypes were identified in the latter. CONCLUSIONS:Thigh or arm vaccination with TA-CIN was well tolerated, but the former elicited higher CD8 T cell and antibody responses in HPV16+ cervical cancer patients with NED after primary therapy.