The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) is characterized by restrained function of effector T cells that drives resistance to immunotherapy. While tumor-extrinsic stroma and myeloid cells have been shown to mediate PDAC immune evasion, the role of tumor-intrinsic post-transcriptional gene regulation in driving tumor-immune crosstalk has been relatively unexplored. Here, we report that the RNA-binding protein HuR (ELAVL1) is enriched in human PDAC and negatively correlates with T-cell infiltration. In two immunocompetent, murine models of PDAC, we found that genetic disruption of HuR impaired tumor growth without significantly impacting in vivo proliferation. Comprehensive spatial and flow cytometry profiling of the PDAC TME revealed that genetic disruption of HuR in PDAC enhanced both T-cell number and functional state. Moreover, T-cell depletion abrogated the growth difference caused by HuR loss. Mechanistically, RNA immunoprecipitation sequencing, single-cell RNA sequencing, and orthogonal functional assays in vitro and in vivo showed that HuR stabilized mTOR pathway transcripts critical for metabolic adaptation in PDAC. HuR-driven metabolic reprogramming promoted tumor nutrient dominance and limited nutrient consumption by neighboring tumor-reactive T cells. Accordingly, HuR depletion sensitized PDAC tumors to immune checkpoint blockade and allowed for the expansion of tumor-specific T-cell populations, suggesting that HuR-mediated nutrient dominance and immune evasion have translational relevancy. Overall, we found that the post-transcriptional regulator HuR facilitates immune evasion in PDAC by constraining T-cell function, identifying HuR blockade as a promising therapeutic strategy in combination with immunotherapies.
The therapeutic potential of chimeric antigen receptor (CAR) T cells has long been limited by the static nature of the engineered receptor; once manufactured, the cell is committed to a single antigen, unable to respond to the immune evasion and antigen heterogeneity that define solid tumors. Although solutions such as bispecific constructs and logic-gated circuits have been proposed, each encodes additional complexity into the cell product at the time of manufacture, with no capacity for post-infusion reprogramming. In this issue, Kuo and colleagues report a fundamentally different strategy-the meditope-enabled CAR-in which a small, structurally orthogonal peptide-docking interface is embedded into the extracellular domain of the CAR, converting a fixed cellular product into a programmable platform that can be redirected, selectively expanded, or precisely tracked by administering a modular adapter molecule. See related article by Kuo et al., p. XX.
Abstract Chimeric antigen receptor (CAR) T-cell therapy has become a promising clinical approach against hematologic malignancies, but patients receiving CAR T-cell therapy still present inconsistent clinical outcomes and are complicated by incomplete tumor eradication. The gut microbiome has shown a strong correlation with the therapeutic outcomes of CAR T-cell therapy. However, the underlying mechanism of how gut microbiota affect CAR T-cell therapeutic potency remains undetermined. In this study, we established a syngeneic CD19+ murine lymphoma model that allows for the evaluation of both endogenous immune cells and gut microbiota following CD19–CD28ζ CAR T-cell therapy. Using single-cell transcriptomic analyses, we report that CAR T-cell infusion led to the activation of peripheral and gut-infiltrating endogenous CD8+ T cells toward an effector-like phenotype. In parallel, 16S RNA sequencing revealed substantial alterations in gut microbiota after infusion. The composition of gut bacteria was associated with the activation status of endogenous CD8+ T cells and responsiveness to CAR T-cell therapy. More specifically, we identified gut bacteria strains Turicibacter and Parvibacter as critical determinants of effective CAR T-cell treatment. Supplementation with these species of gut bacteria during CAR T-cell therapy led to superior antitumor efficacy. Furthermore, both strains facilitated CAR T-cell therapy–induced activation of endogenous CD8+ T cells, enhancing their capability to express activation-associated surface markers as well as tumor lysis potency. In summary, our results demonstrate that the gut microbiome plays an essential role in endogenous immune activation after CAR T-cell therapy and provide specific targets for therapeutic interventions.
Tertiary lymphoid structures (TLS) have emerged as critical immune niches within the tumor microenvironment across various cancers. However, their structural organization and functional roles in non-muscle-invasive bladder cancer (NMIBC) remain poorly characterized. In this study, we comprehensively characterized TLS in NMIBC using en bloc resected primary diagnostic specimens, which enabled high-resolution spatial mapping and quantitative histological assessment across a large cohort of primary tumors (97 patients). Compared with TLS in inflammation-driven cystitis, tumor-associated TLS showed heterogeneous spatial organization, altered immune composition, and disrupted follicular dendritic cell (FDC) networks with reduced high endothelial venules density. TLS density increased with NMIBC grade and stage and correlated with higher recurrence risk and shorter progression-free survival in multivariable analysis. Paired analysis of primary and recurrent tumors showed a reduction in FDC density and a shift from M1 to M2 differentiation in tumor recurrences, suggesting the development of an immunosuppressive microenvironment. Single-cell RNA sequencing of TLS from NMIBC and cystitis samples revealed impaired germinal center activity in tumor-associated TLS, with asynchronous B-cell maturation and reduced B-cell interactions with both T follicular helper cells and FDC. Tumor-associated TLS also showed increased myeloid infiltration and altered dendritic-cell function, including reduced MHC class II antigen presentation and downregulation of co-stimulatory (CD86-CD28) and migration-related (CD99) pathways compared to TLS from cystitis samples. Overall, we found that NMIBC-associated TLS are structurally and functionally impaired, which may limit effective local immune responses and suggests they may be relevant as biomarkers and targets for immunomodulatory therapy in bladder cancer.
The efficacy of immunotherapy in unresectable/recurrent/metastatic head and neck squamous cell carcinoma (U/R/M HNSCC) remains suboptimal compared to primary untreated resectable HNSCC. The immunological and stromal landscape during immunotherapy in U/R/M HNSCC is crucial for understanding drug resistance mechanisms. In this study, single-cell RNA sequencing was performed on 42 samples pre- and post-treatment from patients in our prospective trial (NCT05156970) who received immunochemotherapy. A subset of myofibroblasts expressing CD266 was identified in hampering the response to immunochemotherapy in patients with U/R/M HNSCC. This distinct subtype of cancer-associated fibroblasts (CAFs) enhanced extracellular matrix (ECM) stiffness and cellular tension through a Piezo1-mediated biomechanical mechanism, spatially segregated CD8+ T cell infiltration to establish immune-excluded niches, and ultimately facilitated immune evasion. Mechanistically, PIEZO1 inhibition suppressed YAP activation and reduced FAK phosphorylation, thereby mitigating matrix remodeling effects driven by myCAF CD266. We also observed that Piezo1 inhibition had synergistic effect with PD-1 blockade in HNSCC in vivo. In summary, our findings reveal an immunosuppressive mechanism in which stromal cells regulate ECM stiffness, presenting a potential therapeutic target for U/R/M HNSCC.
Immune checkpoint blockade (ICB) has markedly improved overall survival in various cancers, but is associated with severe and sometimes fatal immune-related adverse events (irAEs). Current management of irAEs involves discontinuation of ICB therapy and administration of immunosuppressive drugs, such as corticosteroids, which have been associated with decreased antitumor efficacy. Although irAE development is associated with ICB response, it is currently unknown whether their underlying mechanisms are shared or distinct. To identify early and targetable drivers of irAEs, we performed proteomic analyses on the serum of patients with cancer treated with anti-PD-1 and anti-CTLA-4 combination ICB. We identified a significantly increased concentration of p40, a subunit of IL-12/IL-23, shortly after the start of ICB but before the onset of clinically apparent irAEs. Importantly, increased p40 levels were not associated with ICB efficacy. Neutralizing p40 mitigated ICB-induced toxicity in various mouse models without impairing ICB-induced antitumor efficacy. In conclusion, we demonstrated that IL-12/IL-23p40 is a key mediator of ICB-induced toxicity while being redundant for ICB antitumor efficacy. This shows that the mechanisms underlying ICB toxicity and efficacy can be uncoupled and provides a rationale for p40 blockade in clinical trials with ICB treatment to prevent irAEs in patients.
Bacillus Calmette-Guérin (BCG) is the standard adjuvant therapy for early-stage bladder cancer (BCa), based on its immunostimulatory activity. However, over half of patients experience BCG failure and subsequent recurrence. The heterogeneity of the tumor microenvironment may influence BCG response, however this relationship is currently not well-established. We found that BCG response did not correlate with the number of tumor-infiltrating T cells but was determined by the pre-treatment immunosuppressive microenvironment. We identified a subset of cancer-associated fibroblasts characterized by microfibrillar-associated protein 2 (MFAP2+ CAF), which impaired BCG-induced antitumoral immunity by interacting with both individual T cells and tertiary lymphoid structures/lymphoid aggregates. This study also revealed a potential stepwise activation of cancer immune evasion factors based on cross-sectional analysis, from increasing MFAP2+ CAF to activation of PD-L1 and then LAG3 on T cells, during early-stage BCa development. These findings have the potential to enable accurate prediction of BCG response. This approach may also be applicable to the investigation of other human cancers.
Chimeric antigen receptor (CAR)-T cell exhaustion constitutes a critical barrier to sustained antitumor efficacy. Through transcriptomic analysis of CAR-T cells from patients with lymphoma, we identified the histone variant macroH2A2 (H2AFY2) as a critical regulator of T cell exhaustion-a finding consistently observed across multiple tumor models. In mice, T cell-specific knockout of H2afy2 promots the expression of inhibitory receptors by activating the nuclear factor kappa-B pathway and increasing chromatin accessibility at the Rela locus, as demonstrated by single-cell RNA-sequencing and assay for transposase-accessible chromatin sequencing. H2AFY2 overexpression in CD8+ T cells induces prominent epigenetic remodeling, characterized by increased H3K27me3 enrichment. Mechanistically, H3K27me3 enrichment at the Rela locus suppresses p65-mediated transcriptional activation, leading to downregulation of the exhaustion-associated transcription factor TOX and consequent amelioration of T cell exhaustion. Furthermore, H2AFY2-overexpressing CAR-T cells sustain lower levels of inhibitory receptors and suppressed tumor recurrence. Collectively, these results define an epigenetic pathway through which H2AFY2 counteracts T cell exhaustion and support the therapeutic potential of H2AFY2-engineered CAR-T cells across tumor types.
In esophageal squamous cell carcinoma (ESCC), chemoradiotherapy potentiates the effects of immune checkpoint inhibitors (ICIs) by activating the tumor-intrinsic innate immune response. However, ESCC cells frequently suppress this activation, which contributes to the high rates of immunotherapy resistance (70-80%) observed clinically. Thus, identifying intracellular suppressors of this innate immune response remains an unmet critical need. Herein, through multi-omic analyses, we identify the chromatin assembly factor CHAF1A as a suppressor of the tumor-intrinsic innate immune response in ESCC. We found that CHAF1A was overexpressed in ESCC and negatively correlated with type I interferon production and CD8+ T-cell infiltration. Mechanistically, CHAF1A maintained heterochromatin silencing mediated by H3K9me3, thereby repressing endogenous retroviruses (ERVs). This suppression prevented the accumulation of double-stranded RNA (dsRNA) and the subsequent activation of the MAVS-IRF3 signaling pathway. Concurrently, CHAF1A preserved genomic stability, limiting the release of double-stranded DNA (dsDNA) and activation of the cGAS-STING pathway. Loss of CHAF1A potentiated the response to immunotherapy through the coordinated activation of these dual pathways. We then performed a small-molecule compound screen and identified a CHAF1A inhibitor, Baimaside, which enhanced the effect of anti-PD-1 therapy to augment antitumor immunity. Collectively, these data indicate that CHAF1A represents a potential therapeutic target for sensitizing ESCC to immunotherapy and provide a potential combination strategy for reversing immunotherapy resistance.
It is well established that CD4⁺ T cells play a critical role in facilitating immune checkpoint therapy (ICT). Although CD4+ T-cell function in lymph nodes during CD8⁺ T-cell priming has been well investigated, their requirement at the effector phase in the tumor is only now beginning to be appreciated. Herein, we used our major histocompatibility complex class II-negative (MHC-II-) sarcoma models to confirm that CD4⁺ T cells are essential not only during T-cell priming, but also to maintain T-cell effector function within the tumor. Depleting CD4⁺ T cells at the effector phase, after CD8+ T-cell priming had occurred, abolished ICT-induced tumor rejection despite the detection of tumor-specific CD8⁺ T cells and their intratumoral accumulation. CD4⁺ T cells were required for functional reinvigoration of CD8⁺ tumor-infiltrating lymphocytes (TIL) by ICT, leading to enhanced cytokine production, expression of cytotoxicity, and reduced exhaustion-without affecting CD8+ T-cell proliferation. Mechanistically, CD4⁺ T-cell function at the effector phase did not require CD40/CD40L signaling, which is necessary for efficient priming, but rather depended on IL-2 and IFNγ. Using a TCR-mimic monoclonal antibody (1G10) specific for the dominant neoantigen:I-Aᵇ complex on antigen-presenting cells formed during T3 sarcoma challenge, we further showed that ongoing MHC-II neoantigen presentation was necessary to sustain CD4⁺ T-cell help after priming. These findings reveal temporally distinct requirements for CD4⁺ T-cell help and establish a need for continuous CD4⁺/CD8⁺ T-cell cooperation as a prerequisite for anti-PD-1/anti-CTLA-4 ICT efficacy against MHC-II- tumors.
Regulatory T cells (Treg) act as a powerful barrier to effective antitumor immunity. Although manipulating Treg is a promising anticancer strategy, doing so while sparing general immune tolerance has been a challenge. Identifying factors specifically expressed in tumor-infiltrating Treg is therefore important for better understanding cancer pathogenesis and identifying novel therapeutic targets that enhance antitumor immunity. We show that T cell Immunoglobulin and Mucin 3 (Tim-3) expression on tumor Treg is required for the function and survival of these cells, in part through Akt and FOXO1 signaling. Deleting Tim-3 in Treg leads to delayed tumor-specific T-cell exhaustion and lower tumor burden, without altering peripheral homeostasis. Similar effects were noted when Tim-3 was only deleted from half of the Treg or when deletion was delayed until after tumor inoculation. Moreover, Treg-specific deletion of Tim-3 cooperated with PD-1 checkpoint blockade to sensitize an immunotherapy-resistant tumor model. In addition, a decrease in Tim-3+ tumor Treg correlated with responsiveness to PD-1/LAG-3 combination checkpoint blockade in a human clinical trial. Overall, our data provide evidence that Tim3-expressing Treg are a promising target to modulate tumor-specific immune responses.
Glioblastoma multiforme (GBM) is a lethal brain tumor with limited treatment options. Tumor-associated macrophages and microglia (TAMs) drive immune suppression and tumor progression, making them a key therapeutic target for GBM. Enhancing TAM phagocytosis in GBM has shown promise, particularly with innate checkpoint inhibitors, such as CD47-blocking antibodies. However, small molecule approaches, which offer tunable and potentially synergistic mechanisms, remain underexplored in this context. In this study, we conducted a large-scale small molecule screen on primary TAMs isolated directly from GBM patient tumors, testing 1,365 compounds to identify drugs that enhance TAM phagocytosis. This screen revealed enrichment for histone deacetylase (HDAC)-targeting drugs among the top hits. HDAC inhibitors enhanced phagocytosis of cancer cells across multiple primary human TAM-GBM combinations, and synergized with CD47 blockade ex vivo. In a xenograft GBM model, Pracinostat suppressed tumor growth and extended survival, with additive benefit when combined with CD47 antibodies. RNA-sequencing and H3K27Ac CUT&Tag profiling of Pracinostat-treated TAMs in vivo revealed a two-tier mechanism: transcriptional reprogramming toward a pro-inflammatory state via NF-κB activation, and epigenetic priming of FcγR-mediated phagocytic machinery, providing a mechanistic basis for the observed synergy with CD47 blockade. Our findings establish a patient-first functional screening platform for identifying TAM-reprogramming therapeutics in GBM, validate HDAC inhibitors as a lead class that potentiates innate checkpoint immunotherapy, and provide additional candidate compounds for clinical investigation.
Abstract Although mutant-specific KRAS inhibitors are approved to treat cancer, a deeper understanding of intratumoral changes driven specifically by KRAS inhibition is needed to maximize therapeutic responses. In this study, we used single-cell RNA sequencing, flow cytometry, and spatial transcriptomics to distinguish mechanisms of tumor control after KRASG12C inhibition [KRAS(G12C)i] or MEK inhibition (MEKi). Despite both inhibiting the MAPK pathway, KRAS(G12C)i and MEKi drive the adaptation of distinct neoplastic cell fates affecting metabolism and cell-cycle regulation, and additive tumor suppression is observed after co-administration. KRAS(G12C)i results in the emergence of a specific, cDC1-driven mature conventional dendritic cell (cDC) state. Coculture of treated neoplastic cells with cDC1s is sufficient to upregulate maturation markers such as CCR7, and intercellular communication analyses suggest that activation is augmented through nonimmune mediators. Both KRAS(G12C)i and MEKi increase infiltration of cytotoxic T cells, but MEKi, which also targets nonmalignant cells, is associated with a reduced capacity for T-cell proliferation and degranulation, consistent with distinct adaptive immune activation mechanisms. We observe that combination treatment of KRAS(G12C)i with anti–PD-1 immunotherapy further expands effector T-cell states, increases clonal persistence, and induces proinflammatory macrophages associated with higher overall survival that were largely absent after KRAS(G12C)i alone. Furthermore, combination treatment enhances intercellular communication networks among non–PD-1+–expressing cells that can perpetuate cDC activation. Our findings delineate distinct tumor and immune responses to KRAS and MEK inhibition and identify molecular features of the responding tumor microenvironment that may be leveraged to improve therapeutic efficacy.
Abstract Tumor masses often exhibit heterogeneity, including escape variant clones that lack antigen-presenting machinery and/or tumor antigens, which poses a major challenge to immunotherapy. Ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, has been shown to effectively induce cell death in various tumor cells. Recent studies have reported that IFNγ suppresses the expression of system Xc−, thereby enhancing the induction of ferroptosis. Based on this, we hypothesized that combining immunotherapy with ferroptosis inducers could enhance antitumor effects against both antigen-positive and antigen-negative tumor cells. We found that combining RSL3, a ferroptosis inducer, with MART-1–specific T-cell receptor–engineered T cells eradicates a heterogeneous tumor model consisting of human melanoma cells and their β2-microglobulin knockout counterparts. In NOD.Cg-PrkdcscidIl2rgtm1Sug/ShiJic mice, this combination therapy demonstrates a significant antitumor effect against tumors with heterogeneity. These findings suggest that integrating ferroptosis inducers with immunotherapy could overcome the limitations imposed by escape-variant tumor clones, offering a promising strategy for cancer treatment.
Abstract Tumors remodel the extracellular matrix (ECM) and glycosylation, yielding epitopes with restricted or limited detectability in normal adult tissues. In this study, we evaluated the O-glycosylated type III connecting segment domain of fibronectin (Tn-FN) as a chimeric antigen receptor (CAR) T-cell target. FDC6-BBζ CAR T cells recognizing Tn-FN were benchmarked against extra domain B-FN–targeted L19-BBζ and Tn-MUC1–targeted 5E5-BBζ. FDC6-BBζ mediated robust, antigen-dependent activation and cytotoxicity, outperforming L19-BBζ and matching 5E5-BBζ in vitro and in NOD/SCID gamma xenografts of prostate cancer. FDC6-BBζ and 5E5-BBζ CAR T cells achieved durable tumor control with increased intratumoral CD3+ infiltration and reduced tumor–collagen overlap. Cytotoxicity required an intact tumor IFNγ receptor 1 (IFNγR1); L19-BBζ further depended on Fas, whereas FDC6-BBζ and 5E5-BBζ were less Fas-dependent. Tumoral Toll-like receptor (TLR) 2/6 or TLR4 agonism restored FDC6-BBζ killing of IFNγR1-deficient targets and induced broad inflammatory and stress–response programs. Pharmacologic perturbation implicated caspase-dependent mechanisms and a contribution from inflammasome-linked signaling, whereas ferroptosis blockade did not abrogate restored killing. These findings establish Tn-FN as a glycoform-restricted, ECM-derived CAR target and show that innate agonists can reprogram tumor state to overcome resistance from impaired IFNγ signaling.
Metastatic disease is the leading cause of breast cancer-related deaths, with more than 42,000 patients predicted to die from breast cancer this year. Immunotherapies, which reengage the immune system to fight cancer cells, have shown only modest success in metastatic triple negative breast cancer (TNBC), and a deeper understanding of the anti-tumor immune response is essential to expand the scope of immunotherapy in this disease context. Though T cells have maintained a spotlight in immunotherapy, NK cells are the first responders to metastatic sites, and my data demonstrate a critical role for cytotoxic NK cells in controlling metastatic outgrowth. However, tumor cells develop various mechanisms to evade NK cells, and a comprehensive understanding of NK-cancer cell crosstalk is lacking. To address this gap in knowledge, we conducted a novel, in vivo CRISPR/Cas9 screen in NK cell-competent and -depleted models using a single guide RNA library designed identify druggable genes that have known inhibitors and promote breast cancer metastasis in an NK cell-dependent manner. Top hits from this screen included the protein Phosphatidyl-inositol Transfer Protein Alpha (PITPα; gene name Pitpna), which facilitates the transfer of phosphatidylinositol lipids between membrane. To better understand the role of PITPα in TNBC, we knocked down Pitpna in the murine TNBC cell line E0771, which is syngeneic to C57BL/6 mice, and tested the consequences of Pitpna knockdown in vivo. We observed that Pitpna knockdown reduces both early metastasis and primary tumor growth, suggesting that targeting PITPα could impact both metastatic and primary disease. Intriguingly, Pitpna knockdown had no effects on cancer cell growth in vitro, suggesting that the effects of Pitpna knockdown are dependent on effects from the tumor microenvironment. To interrogate this further, we performed flow cytometry analysis on metastatic lungs and primary tumors with and without Pitpna knockdown and evaluated the presence and phenotypes of immune cell subsets. We observed that Pitpna knockdown is associated with a shift in NK cell maturation state from regulatory to cytotoxic, and increased levels of degranulating NK cells. Altogether, this study identifies PITPα as a novel druggable target that promotes resistance to cytotoxic NK cells and contributes to TNBC metastasis. Sheera Rosenbaum, Erin Citarella, Vadym Zaberezhnyy, Molishree Joshi, James Costello, Michael Verneris, Jill Slansky, Heide Ford. PITPα-mediated tumor signaling shapes NK cell phenotypes and promotes breast cancer metastasis [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A048.
Abstract Elevated neutrophil-to-lymphocyte ratio (NLR) has been associated with platinum resistance and poor outcomes in high-grade serous ovarian cancer (HGSOC), but the biological basis of this association remains unclear. We retrospectively analyzed 434 patients with HGSOC treated with platinum-based chemotherapy and found that elevated NLR was an independent predictor of platinum resistance. To further investigate the cellular basis of this association, we performed immunohistochemistry (IHC), proteomic profiling, and single-cell RNA sequencing on patient samples. These analyses identified a distinct subpopulation of V-domain immunoglobulin suppressor of T-cell activation–positive neutrophils (VISTA+Neus) enriched in resistant tumors. High VISTA+Neus density was linked to shorter progression-free survival and showed greater predictive value for resistance than total neutrophils. Spatial and multicolor IHC analyses further showed that VISTA+Neus were associated with reduced CD8+ T-cell infiltration and cytotoxic features. Functional validation in an immunocompetent mouse model showed that anti-VISTA plus cisplatin reduced tumor growth, decreased neutrophil abundance, and increased CD8+ T-cell infiltration and granzyme B expression. CD8+ T-cell depletion markedly attenuated the therapeutic benefit of the combination. These findings support VISTA+Neus as a potential immunosuppressive neutrophil subset associated with platinum resistance and CD8+ T-cell suppression in HGSO, and support further investigation of VISTA-targeted strategies and the potential biomarker value of VISTA+Neus.
Recurrent respiratory papillomatosis (RRP) is a chronic, debilitating disease of the airway primarily caused by infection with human papillomavirus (HPV) types 6 and/or 11 and characterized by recurrent, benign tumor growth with potential for malignant transformation. Current standard of care consists of repeated surgical removal of papillomas, which can lead to lasting airway damage and impaired vocal function. Thus, a non-surgical approach to treat RRP is paramount. Previously, we described CD8+ T cell responses associated with clinical response to INO-3107, a DNA immunotherapy designed to generate T cells capable of targeting HPV-infected cells, in adult RRP patients during a Phase 1/2 trial (NCT04398433). Here, we describe CD4+ T cell responses in these patients. INO-3107 was administered during study weeks 0, 3, 6 and 9. Peripheral blood mononuclear cells (PBMCs) obtained at screening and post-treatment were assessed by multiparametric flow cytometry and RNA sequencing. Formalin-fixed, paraffin-embedded papilloma tissue obtained prior to INO-3107 treatment and at the end of the 52-week study (EOS) was subjected to RNA sequencing. RNA sequencing data additionally underwent single sample gene set enrichment analysis (ssGSEA). Clinical response was defined as any reduction in frequency of RRP surgical interventions in the 52 weeks following dose 1 of INO-3107 compared to the 52 weeks prior. Following INO-3107 treatment, activated HPV-specific CD4+ T cells were found to be elevated in PBMCs of responders compared to non-responders. ssGSEA assessment of airway tissue revealed enrichment in CD4+ T cell signatures, inclusive of Th1 and effector memory populations, when comparing screening to EOS. Enrichment of CD4+ T cell signatures was significantly higher in responders but not in non-responders. Enrichment of a Th2 signature was observed in post-treatment PBMCs from non-responders as compared to screening. Expression of HLA class II genes in airway tissue increased significantly from screening to EOS in responders but decreased in most non-responders. Conversely, expression of these genes in post-treatment PBMCs increased in non-responders but decreased in responders. Mechanisms of immune escape may play a role in mediating clinical responses in recurrent respiratory papillomatosis (RRP) as evidenced by enrichment of Th2 cells in PBMCs and decreased expression of HLA class II genes in airway tissue of non-responders. However, DNA immunotherapy INO-3107 induced activation and enrichment of pro-inflammatory CD4+ T cell responses, inclusive of Th1 and effector memory signatures, in blood and airways of RRP patients. These immune responses were associated with clinical responses during the 52-week trial. Additionally, responder airways displayed increased expression of HLA class II genes responsible for proper binding, processing, and presentation of HPV antigens to CD4+ T cells, which may, in part, activate CD8+ T cell responses described previously (doi: 10.1038/s41467-025-56729-6). Emma L. Reuschel, Albert J. Sylvester, Sarah A. Marcus, Sadie Wisotsky, Alex Dolgoter, Katherine S. Reed, Grace S. Tan, Jeffrey M. Skolnik, Elisabeth B. Gillespie. Treatment of Recurrent Respiratory Papillomatosis with DNA immunotherapy INO-3107 induces activation and enrichment of pro-inflammatory CD4+ T cell responses in blood and airways of patients with clinical response [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A060.
