JoeyKelly

J Immunother Cancer. 2025 Apr 20;13(4):e011380. doi: 10.1136/jitc-2024-011380.

ABSTRACT

BACKGROUND: Adenoid cystic carcinoma (ACC) is a rare, but lethal cancer with low response rates to systemic therapies, such as cytotoxic chemotherapy and immune-checkpoint inhibitors (ICIs). Despite extensive clinical trials, no effective treatments for patients with recurrent or metastatic ACC are available, and ACC mortality rates remain poor.

METHODS: We employed automated multiplex immunofluorescence (mIF), single-cell RNA sequencing (scRNA-seq) Gene Expression analysis, RNA in-situ hybridization, and spatial transcriptomics analysis to characterize the immune landscape of ACC tumors, ACC metastasis, and normal tissues from regions where ACCs arise. Based on results from these studies, we treated freshly resected ACCs with interferon-γ or a stimulator of the interferon genes (STING) agonist in vitro. Additionally, we included one patient with ACC in a phase 1 clinical study of a novel STING agonist (dazostinag) plus pembrolizumab.

RESULTS: The mIF analysis revealed that ACC tumors are immunologically "cold", with few tumor-infiltrating T-lymphocytes and low programmed death-ligand 1 (PD-L1) expression. The most striking finding was a very low beta-2-microglobulin (B2M) expression in nearly all ACCs, with only focal expression found in some ACC metastases. mIF and RNA sequencing analyses of normal salivary gland and breast tissues revealed a p63+, NFIB+, basal duct cell population, with similarly low B2M/human leukocyte antigen (HLA) class I expression. Spatial transcriptomics analysis of the focally B2M-positive ACC metastases uncovered the genetic pathway driving upregulation of B2M, an interferon-γ program mediating the reintroduction of HLA-I/B2M; the significantly upregulated genes included IRF1, GBP1, and TAP1. On short-term treatment of primary ACC tissues in vitro with interferon-γ or a STING agonist, we observed strongly upregulated HLA class I/B2M expression. Moreover, treatment of a patient with recurrent, metastatic breast ACC with a STING agonist and pembrolizumab led to a partial response with a 70% tumor reduction.

CONCLUSIONS: Low B2M/HLA class I expression may explain why ACCs are immunologically cold and the lack of response to ICIs. Our findings suggest that the normal cell of ACC origin exists in a B2M/HLA-class I low state, and that pharmacologic manipulation with immune activators, such as STING agonists, can restore HLA/B2M in ACCs, as supported by the promising response observed in a patient with metastatic ACC. These findings indicate a potential path to urgently needed immunotherapies.

PMID:40254393 | PMC:PMC12010351 | DOI:10.1136/jitc-2024-011380

Mol Cell Proteomics. 2025 Apr 14:100971. doi: 10.1016/j.mcpro.2025.100971. Online ahead of print.

ABSTRACT

Over the past three decades, the Hunt laboratory has developed advancements in mass spectrometry-based technologies to enable the identification of peptides bound to major histocompatibility complex (MHC) molecules. The MHC class I processing pathway is responsible for presenting these peptides to circulating cytotoxic T cells, allowing them to recognize and eliminate malignant cells, many of which have aberrant signaling. Professor Hunt hypothesized that due to the dysregulation in phosphorylation in cancer, that abnormal phosphopeptides are likely presented by this pathway, and went on to discover the first phosphopeptide presented by the MHC processing pathway. Thereafter, the laboratory continued to sequence MHC-associated phosphopeptides and contributed several improved methods for their enrichment, detection, and sequencing. This manuscript summarizes the most recent advancements in identification of modified MHC-associated peptides and includes the cumulative list of phosphopeptides sequenced by the Hunt lab. Further, many other post-translational modifications (PTMs) were found to modify MHC peptides, including O-GlcNAcylation, methylation, and kynurenine; in total, we present here a list of 2,450 MHC-associated PTM peptides. Many of these were disease specific and found across several patients, thus highlighting their potential as cancer immunotherapy targets. We are sharing this list with the field in hopes that it might be used in investigating this potential. Overall, the Hunt lab's contributions have significantly advanced our understanding of antigen presentation and dysregulation of PTMs, supporting modern immunotherapy and vaccine development efforts.

PMID:40239839 | DOI:10.1016/j.mcpro.2025.100971

Cancer Biol Ther. 2025 Dec;26(1):2486141. doi: 10.1080/15384047.2025.2486141. Epub 2025 Apr 8.

ABSTRACT

BACKGROUND: Peptide vaccines offer a direct way to initiate an immunogenic response to a defined antigen epitope. However, peptide vaccines are unstable in vivo, subject to rapid enzymatic proteolysis. Replacement of an α-amino acid residue with a homologous β-amino acid residue (native side chain, but backbone extended by a single CH₂ unit) impairs proteolysis at nearby amide bonds. Therefore, antigen analogues containing α-to-β replacements have been examined for functional mimicry of native all-α antigens. Another group previously took this approach in the ovalbumin (OVA) antigen model by evaluating single α-to-β analogues of the murine major histocompatibility complex (MHC) I-restricted peptide SIINFEKL.

METHODS: We re-examined this set of α/β SIINFEKL antigens. We tested the susceptibility to proteolysis in mouse serum and their ability to activate OVA-antigen-specific CD8 T cells in vitro. Additionally, we tested the α/β antigens in vivo for their ability to induce an antigen-specific immunogenic response in naïve mice and in OVA-expressing tumor-bearing mice.

RESULTS: The α/β antigens were comparable to the native antigen in their susceptibility to proteolysis in serum. Each α/β antigen was capable of activating antigen-specific CD8 T cells in vitro. However, antigen-specific CD8 T cells induced against α/β antigens in vivo were not cross-reactive to the native antigen. Moreover, immunization with α/β analogues did not elicit anti-tumor effects in tumor-bearing mice.

CONCLUSIONS: We conclude that even though α/β analogues of the SIINFEKL antigen can elicit a T cell-based response, this class of backbone-modified peptides is not promising from the perspective of antitumor vaccine development.

PMID:40200635 | DOI:10.1080/15384047.2025.2486141

Front Immunol. 2025 Mar 21;16:1512509. doi: 10.3389/fimmu.2025.1512509. eCollection 2025.

ABSTRACT

Immunotherapy has emerged as a preeminent force in the domain of cancer therapeutics and achieved remarkable breakthroughs. Nevertheless, the high resistance has become the most substantial impediment restricting its clinical efficacy. Beta-2 microglobulin (B2M), the light chain of major histocompatibility complex (MHC) class I, plays an indispensable part by presenting tumor antigens to cytotoxic T lymphocytes (CTLs) for exerting anti-tumor effects. Accumulating evidence indicates that B2M mutation/defect is one of the key mechanisms underlying tumor immunotherapy resistance. Therefore, elucidating the role played by B2M and devising effective strategies to battle against resistance are pressing issues. This review will systematically expound upon them, aiming to provide insight into the potential of B2M as a promising target in anticancer immune response.

PMID:40191187 | PMC:PMC11968357 | DOI:10.3389/fimmu.2025.1512509

Int Immunopharmacol. 2025 Mar 27;153:114531. doi: 10.1016/j.intimp.2025.114531. Online ahead of print.

ABSTRACT

BACKGROUND: Tuberculosis (TB) is an infectious disease transmitted through the respiratory system that affects people worldwide. Bacillus Calmette-Guérin (BCG), the only approved TB vaccine, has been shown to have highly variable protective efficacy in different populations and is ineffective at protecting adults. Therefore, the development of more effective TB vaccines is vital.

METHODS: Three dominant antigens (ESAT6, CFP10, and MPT64) from the region of difference were selected for this study. Their physicochemical properties, spatial structures, and immune responses were evaluated using bioinformatics screening of dominant T cell and B cell epitopes. Three vaccine constructs were developed. After selecting the appropriate linkers, their physicochemical properties, spatial structures, and immune responses of the vaccines were evaluated, and molecular dynamics simulations were performed to test their ability to bind to major histocompatibility complex (MHC) receptors within 100 ns. This process aimed to create highly antigenic vaccine constructs capable of eliciting an immune response. The effects of the vaccine constructs on the host immune response were assessed using enzyme-linked immunosorbent assays, flow cytometry, and hematoxylin and eosin staining.

RESULTS: A novel peptide vaccine, designated ECM-64, was developed by screening six immunodominant peptides from three antigens and constructing independent T-epitope and B-epitope vaccines. Compared weith BCG-immunized mice, ECM-64-immunized mice exhibited a substantial augmentation in Th1/Th2 cytokine secretion and CD3⁺CD4⁺T and CD3⁺CD8^-T lymphocyte counts. ECM-64-specific IgG and IgG1 antibodies were produced after immunization. The immunoinformatics findings were largely consistent with those obtained from the analysis of immunized mice.

CONCLUSION: ECM-64 is a promising multipeptide TB vaccine with the advantage of inducing high levels of Th1/Th2 cytokines, antibodies, and CD3⁺CD4⁺T and CD3⁺CD8^-T lymphocytes in mice. This study also provides preliminary evidence that bioinformatic methods can be used to screen for dominant epitopes. These findings lay the groundwork for the development of peptide-based TB vaccines.

PMID:40154176 | DOI:10.1016/j.intimp.2025.114531

Cancer Cell. 2025 Mar 21:S1535-6108(25)00082-0. doi: 10.1016/j.ccell.2025.03.003. Online ahead of print.

ABSTRACT

Aberrant peptides presented by major histocompatibility complex (MHC) molecules are targets for tumor eradication, as these peptides can be recognized as foreign by T cells. Protein synthesis in malignant cells is dysregulated, which may result in the generation and presentation of aberrant peptides that can be exploited for T cell-based therapies. To investigate the role of translational dysregulation in immunological tumor control, we disrupt translation fidelity by deleting tRNA wybutosine (yW)-synthesizing protein 2 (TYW2) in tumor cells and characterize the downstream impact on translation fidelity and immunogenicity using immunopeptidomics, genomics, and functional assays. These analyses reveal that TYW2 knockout (KO) cells generate immunogenic out-of-frame peptides. Furthermore, Tyw2 loss increases tumor immunogenicity and leads to anti-programmed cell death 1 (PD-1) checkpoint blockade sensitivity in vivo. Importantly, reduced TYW2 expression is associated with increased response to checkpoint blockade in patients. Together, we demonstrate that defects in translation fidelity drive tumor immunogenicity and may be leveraged for cancer immunotherapy.

PMID:40154482 | DOI:10.1016/j.ccell.2025.03.003

J Biol Chem. 2025 May;301(5):108455. doi: 10.1016/j.jbc.2025.108455. Epub 2025 Mar 26.

ABSTRACT

Discovering new bacterial signaling pathways offers unique antibiotic strategies. With current antibiotic classes targeting cell wall synthesis, depolarizing the inner membrane, altering the bacterial metabolome or inhibiting replication or transcription pathways, manipulation of transporters to limit bacterial respiration and thereby pathogenesis has been a decades-long quest. Here we report an inhibitor of multiple bacterial transporters. The inhibitor is the bactericidal N-104 endogenous cleavage fragment of the prosecretory mitogen lacritin. Lacritin is now known to be widely distributed in plasma, cerebral spinal fluid, tears, and saliva. With the bactericidal mechanism determined to be nonlytic by surface plasmon resonance as confirmed by lack of SYTOX Orange entry, we performed an unbiased resistance screen of 3884 Escherichia coli gene knockout strains revealing a complex N-104 polypharmacology. Validation in the virulent Pseudomonas aeruginosa strain PA14-one of three WHO Priority 1: Critical list species-focused on an approach that sequentially couples three transporters and downstream transcription to lethally suppress respiration. By targeting the outer membrane YaiW, cationic N-104 translocates into the periplasm where it ligates inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. With FeoB favoring an anaerobic environment, N-104 promotes the expression of genes regulating anaerobic respiration while largely suppressing those involved in aerobic respiration-a strategy counterproductive under aerobic conditions. This mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with tear thrombin fragment GKY20 as tested on antibiotic-resistant clinical isolates.

PMID:40154612 | PMC:PMC12147183 | DOI:10.1016/j.jbc.2025.108455

J Immunother Cancer. 2025 Mar 18;13(3):e010179. doi: 10.1136/jitc-2024-010179.

ABSTRACT

BACKGROUND: Breast cancer (BC) continues to be a major health concern with 250,000 new cases diagnosed annually in the USA, 75% of which are hormone receptor positive (HR+), expressing estrogen receptor alpha (ER) and/or the progesterone receptor (PR). Although ER-targeted therapies are available, 30% of patients will develop resistance, underscoring the need for new non-ER/estrogen-based treatments. Notably, HR+BCs exhibit poor lymphocyte infiltration and contain an immunosuppressive microenvironment, which contributes to the limited efficacy of immunotherapies in HR+BC. In this study, we demonstrate that PR/progesterone signaling reduces major histocompatibility complex (MHC) Class I expression, facilitating immune evasion and escape from immune-based clearance of PR+tumors.

METHODS: To determine the effect of PR/progesterone on MHC Class I expression, we treated human and mouse mammary tumor cell lines with progesterone and/or interferon (IFN) and measured expression of genes involved in antigen processing and presentation (APP), as well as surface MHC Class I expression. We used the OT-I/SIINFEKL model antigen system to measure the impact of progesterone on immune cell-mediated killing of modified tumor cells. We also analyzed two large BC clinical cohorts to determine how PR expression correlates with APP gene expression and MHC Class I expression in ER-positive tumors.

RESULTS: In vitro, we show that PR/progesterone signaling reduces APP gene expression and MHC class I expression in human and breast mammary tumor cell lines. PR-mediated attenuation of APP/MHC Class I expression is more pronounced in the presence of IFN. In immune cell killing assays, PR-expressing mammary tumor cells treated with progesterone are protected from immune-mediated cytotoxicity. We demonstrate that PR expression in vivo prevents immune-mediated rejection of xenoantigen-modified mammary tumor cell lines through mechanisms involving MHC Class I expression and CD8 T cells. Data analysis of two large BC cohorts reveals lower APP gene expression and MHC Class I expression in ER/PR-positive tumors compared with ER-positive/PR-negative tumors. These findings show that HR+BCs, specifically PR+tumors, downregulate APP/MHC class I machinery through PR/progesterone signaling. Use of pharmacological PR/progesterone inhibitors may reverse these effects in patients with BC, thereby improving immunosurveillance and response to immunotherapies.

PMID:40102028 | PMC:PMC11927445 | DOI:10.1136/jitc-2024-010179

Cancer Res. 2025 Mar 18. doi: 10.1158/0008-5472.CAN-24-2553. Online ahead of print.

ABSTRACT

Neoantigens represent a class of antigens within tumor microenvironments that arise from diverse somatic mutations and aberrations specific to tumorigenesis, holding substantial promise for advancing tumor immunotherapy. However, only a subset of neoantigens effectively elicits anti-tumor immune responses, and the specific neoantigens recognized by individual T cell receptors (TCRs) remain incompletely characterized. Therefore, substantial research has focused on screening immunogenic neoantigens, mainly through their major histocompatibility complex (MHC) presentation and TCR recognition specificity. Given the resource-intensiveness and inefficiency of experimental validation, predictive models based on artificial intelligence (AI) have gradually become mainstream methods to discover immunogenic neoantigens. Here, we provided a comprehensive summary of current AI methodologies for predicting neoantigens, with a particular focus on their capability to model peptide-MHC (pMHC) and pMHC-TCR binding. Furthermore, a thorough benchmarking analysis was conducted to assess the performance of antigen presentation predictors for scoring the immunogenicity of neoantigens. AI models have potential applications in the treatment of clinical diseases, although several limitations must first be overcome to realize their full potential. Anticipated advancements in data accessibility, algorithmic refinement, platform enhancement, and comprehensive validation of immune processes are poised to enhance the precision and utility of neoantigen prediction methodologies.

PMID:40101113 | DOI:10.1158/0008-5472.CAN-24-2553

Mol Cell Proteomics. 2025 Apr;24(4):100943. doi: 10.1016/j.mcpro.2025.100943. Epub 2025 Mar 11.

ABSTRACT

Protein posttranslational modifications have traditionally been challenging to identify due to their dynamic regulation and typically low stoichiometry. Methods for phosphopeptide enrichment from complex proteomes developed in the Hunt lab in the late 1990's and early 2000's launched the field of phosphoproteomics, the large-scale analysis of protein phosphorylation sites. To improve phosphopeptide tandem mass spectra and address the further challenge of identifying other labile posttranslational modifications such as glycosylation or tyrosine sulfation, the Hunt lab invented and disseminated electron transfer dissociation, a novel method for peptide and protein fragmentation. Here we provide a brief historical accounting of these discoveries and their ensuing applications.

PMID:40081537 | PMC:PMC12018110 | DOI:10.1016/j.mcpro.2025.100943

Int J Cancer. 2025 Mar 12. doi: 10.1002/ijc.35409. Online ahead of print.

ABSTRACT

A previously reported clinical trial in familial adenomatous polyposis (FAP) patients treated with erlotinib plus sulindac (ERL + SUL) highlighted immune response/interferon-γ signaling as a key pathway. In this study, we combine intermittent low-dose ERL ± SUL treatment in the polyposis in rat colon (Pirc) model with mechanistic studies on tumor-associated immune modulation. At clinically relevant doses, short-term (16 weeks) and long-term (46 weeks) ERL ± SUL administration results in near-complete tumor suppression in Pirc colon and duodenum (p < 0.0001). We identify a low-dose threshold for significant antitumor activity in Pirc rats given SUL at 125 ppm in the diet plus ERL at 5 mg/kg body weight via twice-weekly oral gavage (SUL125 + ERL5 × 2). Longitudinal analyses show diminished expression of MHC class I and II genes in polyps larger than Grade 5, a novel finding in the Pirc model. Treatment with ERL ± SUL upregulates the corresponding MHC and immune-associated factors in a subset of Pirc colon polyps, Pirc tumor cell lines, murine colon carcinoma cells, and FAP patient-derived organoids, with Nlrc5 playing a critical role in this effect. Imaging mass cytometry reveals that SUL125 + ERL5 × 2 increases tumor-associated Cd4⁺ T cells by ~2.6-fold (p < 0.05), with no apparent effect on Cd8⁺ T cells. The treatment also increases tumor-associated Cd68⁺ cells (p < 0.05) and decreases Foxp3⁺ (p < 0.01) and Arg1⁺ (p < 0.05) cells. Thus, intermittent low-dose ERL + SUL treatment enhances tumor-associated MHC expression and remodels the immune cell niche toward a more permissive "helper" immune microenvironment. We conclude that early immune-interception strategies targeting interferon-γ signaling may benefit FAP patients at drug doses below the clinical standard of care.

PMID:40072251 | DOI:10.1002/ijc.35409

bioRxiv [Preprint]. 2025 Feb 24:2023.06.20.545103. doi: 10.1101/2023.06.20.545103.

ABSTRACT

Gram-negative bacterial pathogens pose a significant challenge in drug development due to their outer membranes, which impede the permeation of small molecules. The lack of widely adoptable methods to measure the cytosolic accumulation of compounds in bacterial cells has hindered drug discovery efforts. To address this challenge, we developed the CHloroalkane Azide Membrane Permeability (CHAMP) assay, specifically designed to assess molecule accumulation in the cytosol of Gram-negative bacteria. The CHAMP analysis utilizes biorthogonal epitopes anchored within HaloTag-expressing bacteria and measures the cytosolic arrival of azide-bearing test molecules through strain-promoted azide-alkyne cycloaddition. This workflow allows for robust and rapid accumulation measurements of thousands of azide-tagged small molecules. Our approach consistently yields a large number of accumulation profiles, significantly exceeding the scale of previous measurements in Escherichia coli ( E. coli ). We have validated the CHAMP assay across various chemical and biological contexts, including hyperporinated cells, membrane-permeabilized cells, and E. coli strains with impaired TolC function, a key component of the efflux pump. The CHAMP platform provides a simple, high-throughput, and accessible method that enables the analysis of over 1,000 molecules within hours. This technique addresses a critical gap in antimicrobial research, potentially accelerating the development of effective agents against Gram-negative pathogens.

PMID:40027664 | PMC:PMC11870406 | DOI:10.1101/2023.06.20.545103

Cell. 2025 Feb 27:S0092-8674(25)00145-X. doi: 10.1016/j.cell.2025.01.043. Online ahead of print.

ABSTRACT

Vitamin C (vitC) is essential for health and shows promise in treating diseases like cancer, yet its mechanisms remain elusive. Here, we report that vitC directly modifies lysine residues to form "vitcyl-lysine"-a process termed vitcylation. Vitcylation occurs in a dose-, pH-, and sequence-dependent manner in both cell-free systems and living cells. Mechanistically, vitC vitcylates signal transducer and activator of transcription-1 (STAT1)- lysine-298 (K298), impairing its interaction with T cell protein-tyrosine phosphatase (TCPTP) and preventing STAT1-Y701 dephosphorylation. This leads to enhanced STAT1-mediated interferon (IFN) signaling in tumor cells, increased major histocompatibility complex (MHC)/human leukocyte antigen (HLA) class I expression, and activation of anti-tumor immunity in vitro and in vivo. The discovery of vitcylation as a distinctive post-translational modification provides significant insights into vitC's cellular function and therapeutic potential, opening avenues for understanding its biological effects and applications in disease treatment.

PMID:40023152 | DOI:10.1016/j.cell.2025.01.043

Int J Mol Sci. 2025 Feb 14;26(4):1613. doi: 10.3390/ijms26041613.

ABSTRACT

The combination of chemotherapeutic agents with immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment. However, its success is often limited by insufficient immune priming in certain tumors, including pediatric malignancies. In this report, we explore clinical trials currently investigating the use of immunogenic cell death (ICD)-inducing chemotherapies in combination with ICIs for both adult and pediatric cancers. Given the limited clinical data available for pediatric tumors, we focused on recent preclinical studies evaluating the efficacy of these combinations in neuroblastoma (NB). Finally, to address this gap, we propose an innovative strategy to assess the impact of ICD-inducing chemotherapies on antitumor immune responses in NB. Using tumor spheroids derived from a transgenic NB mouse model, we validated our previous in vivo findings concerning how anthracyclines, specifically mitoxantrone and doxorubicin, significantly enhance MHC class I surface expression, stimulate IFNγ and granzyme B production by CD8⁺ T cells and NK cells, and promote immune cell recruitment. Importantly, these anthracyclines also upregulated PD-L1 expression on NB spheroids. This screening platform yielded results similar to in vivo findings, demonstrating that mitoxantrone and doxorubicin are the most potent immunomodulatory agents for NB. These data suggest that the creation of libraries of ICD inducers to be tested on tumor spheroids could reduce the number of combinations to be tested in vivo, in line with the principles of the 3Rs. Furthermore, these results highlight the potential of chemo-immunotherapy regimens to counteract the immunosuppressive tumor microenvironment in NB, paving the way for improved therapeutic strategies in pediatric cancers. They provide compelling evidence to support further clinical investigations of these combinations to enhance outcomes for children with malignancies.

PMID:40004078 | PMC:PMC11855819 | DOI:10.3390/ijms26041613