Colsen

Proc Natl Acad Sci U S A. 2026 Jun 9;123(23):e2602949123. doi: 10.1073/pnas.2602949123. Epub 2026 Jun 1.

ABSTRACT

T cell receptor (TCR) restriction by highly polymorphic major histocompatibility complex (MHC) proteins is a foundation of cellular immunity. Although the effects of MHC polymorphisms on peptide binding and selection are well established, how micropolymorphisms within MHC supertypes impact immune recognition is poorly understood. Here, we identified a mechanism through which the micropolymorphisms in two closely related HLA-A3 superfamily members govern TCR specificity. We previously showed that TCRs specific for a public neoantigen arising from a PIK3CA oncogenic hotspot mutation restricted by HLA-A*03:01 were unable to recognize the same epitope in the context of HLA-A*03:02 despite equivalent processing and presentation by both alleles. We found here that the two micropolymorphisms distinguishing A*03:02 from A*03:01 prevent TCR binding not by altering peptide binding or static structures, but by altering the conformational ensemble of the neoantigen, preventing it from adopting a binding-permissive state. The effect is rooted in how the two polymorphic sites interact with other covarying, evolutionarily coupled polymorphisms, reflecting a cross-groove network of interactions that controls the conformational adaptability of the peptide/HLA complex. We suggest polymorphism-dependent adaptability reflects an evolved feature of class I MHC proteins, further diversifying epitopes and contributing to how TCRs and other immunoreceptors differentiate between antigens. Beyond this mechanistic insight, our findings emphasize the need for high-resolution HLA typing in efforts across immunology, including antigen-specific immunotherapy.

PMID:42224598 | PMC:PMC13227914 | DOI:10.1073/pnas.2602949123

Mol Cell. 2026 May 29:S1097-2765(26)00311-4. doi: 10.1016/j.molcel.2026.05.005. Online ahead of print.

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) evades immune surveillance in part through autophagic capture and lysosomal degradation of major histocompatibility complex class I (MHC-I), though the basis for this vulnerability is unclear. Using synchronized endoplasmic reticulum (ER) exit assays, we show that PDAC cells retain MHC-I in the ER and inefficiently traffic it to the plasma membrane. We identify an autophagic capture complex composed of the ER-phagy receptor TEX264 and the cargo receptor NBR1 that targets MHC-I for degradation. Suppression of either receptor restores total and surface MHC-I levels. Capture is linked to antigen loading, as impaired peptide loading increases MHC-I binding to the TEX264-NBR1 complex, while high-affinity peptides reduce binding and promote increased surface localization. A genome-wide CRISPRi screen identified the ER-localized E3 ligase NFXL1 as a mediator of MHC-I ubiquitylation and capture. Elevated NFXL1 correlates with reduced MHC-I expression and poor prognosis, highlighting a targetable pathway regulating PDAC immunogenicity.

PMID:42214332 | DOI:10.1016/j.molcel.2026.05.005

Angew Chem Int Ed Engl. 2026 May 28:e25040. doi: 10.1002/anie.202525040. Online ahead of print.

ABSTRACT

Immune cell activity is strongly influenced by the nutrients present during activation. The effects of specific fatty acids (FAs) can be particularly complex, with them exerting diverse and sometimes opposing effects on immune cells. These functional differences are thought to stem from structural differences between the FAs, leading to altered cellular handling. However, chemical tools to directly probe these aspects remain limited. Here, we report the design and synthesis of saturated, unsaturated, and polyunsaturated cyclopropenyl fatty acids, each incorporating a minimal one-carbon cyclopropene moiety as a bioorthogonal click handle. This motif enables rapid and live-cell-compatible labeling via the inverse electron-demand Diels-Alder reaction, providing a versatile platform to trace FA behavior in biological systems. Application of these synthetic cyclopropenyl FAs in primary immune cell mixtures reveals distinct uptake patterns between different cell types, with polyunsaturated analogues showing strong uptake across all immune cell types. Complementary metabolic and proteomic analyses suggest that this uptake results in biological differences between high/low uptake immune populations, highlighting the utility of cyclopropenyl FA probes for dissecting lipid uptake in the context of immune cell biology.

PMID:42206416 | DOI:10.1002/anie.202525040

J Struct Biol. 2026 May 25:108329. doi: 10.1016/j.jsb.2026.108329. Online ahead of print.

ABSTRACT

T cell receptor (TCR)-based immunotherapy can drive cancer regression by targeting neoantigens derived from mutations in self-proteins. Most neoantigens result from mutations in solvent-exposed residues creating neoepitopes that allow highly specific TCR recognition. Here, we describe a melanoma neoantigen (Rac1^P29S) caused by a mutation at a primary anchor residue. Unlike typical cases, the immunogenicity of Rac1^P29S stems from this anchor mutation, which permits MHC presentation of the mutant peptide but not the wild-type counterpart. We determined the structures of both the mutant Rac1^P29S-HLA-A2 complex and its complex with the tumor-specific TCR 5934. These structures show how the P29S mutation makes a Rac1 self- peptide visible to T cells. Notably, TCR 5934 primarily engages the C-terminal, non-mutated P8 threonine residue of Rac1^P29S -far from the N-terminal mutated P2 serine. This contrasts with most neoantigen-specific TCRs, which typically focus on the mutated residue to distinguish mutant from wild-type peptides. Together, these findings provide a structural framework to guide the development of TCR-based cancer immunotherapies.

PMID:42190883 | DOI:10.1016/j.jsb.2026.108329

ACS Chem Biol. 2026 May 26. doi: 10.1021/acschembio.6c00291. Online ahead of print.

ABSTRACT

Peptide-based cancer vaccines offer a promising strategy for targeting tumor-specific neoantigens. This approach is increasingly critical as post-translationally modified peptides, driven by altered tumor metabolism, emerge as a unique class of neoantigens. Because these chemically distinct epitopes cannot be genetically encoded by mRNA or viral platforms, synthetic peptide vaccines are poised to be the primary route to targeting these types of neoantigens. Yet, their clinical translation is restricted by poor metabolic stability, limited intracellular permeability, and structural requirements for MHC-I binding and T cell receptor recognition. Although peptidomimetic modifications have been widely explored to improve pharmacokinetics, their impact on antigen presentation and immune recognition remains poorly understood. Here, we undertook a comprehensive evaluation of peptidomimetic modifications within a model MHC-I epitope from ovalbumin (OVA), SIINFEKL, generating a diverse library of systematically modified peptides that incorporate backbone N-methylation, peptoid substitution, and stereochemical inversion. Integrated assays revealed a highly position-dependent tolerance to peptidomimetic modifications, while subsequent combinatorial designs demonstrated nonadditive effects on the balance between immunogenicity and pharmacokinetics. Collectively, these findings provide initial design insights for balancing immune recognition with enhanced stability and permeability in the peptidomimetic antigen design.

PMID:42191216 | DOI:10.1021/acschembio.6c00291

Chembiochem. 2026 May 27;27(10):e70389. doi: 10.1002/cbic.70389.

ABSTRACT

The immunoproteasome (iCP) plays a central role in generating peptides for presentation in major histocompatibility complex-I (MHC-I) complexes, yet chemical tools to selectively exploit this activity for controlled antigen release and MHC-I loading have not been described. Here, we report an iCP-targeted peptide prodrug, mATMW-SIINFEKL, that undergoes selective cleavage within DC2.4 dendritic cells, releasing the model antigen SIINFEKL for efficient MHC-I loading and extracellular display. Flow cytometry and confocal microscopy confirmed dose-dependent intracellular processing and proper cross-presentation of SIINFEKL-H-2K^b complexes. These findings establish a modular platform for designing antigenic prodrugs that can selectively release defined peptides in iCP-expressing cells. Given the conservation of iCP subunits across species and prior functional validation of Ala-Thr-Met-Trp (ATMW)-based probes in human cells, this strategy may be extended to HLA class I loading and display, enabling controlled CD8⁺ T cell activation. The approach also provides opportunities for incorporating bioorthogonal handles for functionalization, imaging, or quantitation of antigen extracellular display. Overall, iCP-targeted peptide prodrugs offer a versatile chemical tool to interrogate the role of iCP activity in shaping the immunopeptidome and to facilitate the development of antigen-specific immunotherapies.

PMID:42178988 | DOI:10.1002/cbic.70389

Front Immunol. 2026 May 7;17:1831303. doi: 10.3389/fimmu.2026.1831303. eCollection 2026.

ABSTRACT

KRas, NRas and HRas mutations are recognized in over 25% of all tumors, with the predominant mutations occurring at amino acids G12 or G13. While small molecule inhibitors of KRas show therapeutic promise, KRas has largely resisted control by immunotherapy in clinical cases, although immune responses may be detected following vaccination. Inflammation is a recognized precursor of most KRas-associated tumors. In inflammation cathepsin B leaks from the lysosome and at the higher pH of the cytoplasm acquires endopeptidase activity, in addition to its exopeptidase role. Cathepsin B is consistently upregulated in tumors and its role in tumorigenesis has been attributed to increased apoptosis and digestion of the extracellular matrix. Here we examine the effect of cathepsin B on neoepitopes in KRas. We predict that cathepsin B cleavage patterns of KRas may lead to the destruction of the G12 and G13 mutant neoepitope peptides that would otherwise bind to MHC I, thereby rendering them immunologically invisible. We review reports of the interaction of cathepsin B with trypsinogen in the pancreas and caspases in inflammasomes and the potential effect of premature activation of trypsin on immune evasion of G12R mutants. We summarize our observations and literature review in a schematic describing the potential role inflammation and the actions of cathepsin B, trypsin, and caspases on the immune evasion of KRas and related Ras family gene products.

PMID:42183221 | PMC:PMC13189885 | DOI:10.3389/fimmu.2026.1831303

Mol Cell Proteomics. 2026 May 18:101590. doi: 10.1016/j.mcpro.2026.101590. Online ahead of print.

ABSTRACT

Mass spectrometry (MS)-based immunopeptidomics is a powerful approach for untargeted discovery of peptides presented on major histocompatibility complex (MHC) molecules, which can guide the selection of vaccine antigens and immunotherapy targets. First-generation immunopeptidomics workflows require processing of hundreds of millions of cells using lengthy, manual procedures. More recent approaches focus on increasing either sensitivity or throughput, but rarely combine both aspects. Here, we describe a semi-automated immunopeptidomics platform that combines high sensitivity with high throughput by implementing highly optimized conditions for isolation of MHC class I and II peptides in a 96-well positive-pressure device. Lysis in a small volume of 100 μl allows efficient MHC capture in a 96-well filter plate with optimal pore size for automated washing, elution and C18 purification steps. Upon analysis of 25% of the eluate from 16 million cells, our workflow identified over 13,500 MHC I and 6,000 MHC II peptides on a timsTOF SCP mass spectrometer, operating in DDA-PASEF mode. Exploring the sensitivity limits of our platform, we identified up to 1,000 MHC I peptides, including hundreds of predicted binders, from as few as 20,000 JY cells. Validating the platform's performance for quantitative biological discovery, we report the identification of known and novel bacterial immunopeptides from U937 macrophages infected with Listeria monocytogenes or Bacillus Calmette-Guérin (BCG). Together, our optimized immunopeptidomics platform enables robust immunopeptide detection from lower-input samples in a high-throughput fashion, enabling its use for biological applications where sample amounts are limiting.

PMID:42155729 | DOI:10.1016/j.mcpro.2026.101590

bioRxiv [Preprint]. 2026 May 9:2026.05.08.723607. doi: 10.64898/2026.05.08.723607.

ABSTRACT

Genetic mutations that drive cancer often occur in tumor suppressor proteins, including the p53 transcription factor which is altered in ∼40-50% of cases ^1,2 . However, current therapies fail to target most such mutations because the mutant proteins typically lack defined drug-binding pockets, and restoring the endogenous function has proven challenging. Here, we programmed CRISPR-Cas12a2, an RNA-guided nuclease with trans -nucleolytic cleavage activities ^3,4 , to selectively kill cancer cells by targeting cancer-specific transcripts. This approach eliminates cells by inducing trans chromatin cleavage, triggering DNA damage and cell death. Unlike existing methods, RNA-guided Cas12a2 senses cellular RNA signatures to shred chromatin, enabling precise targeting of undruggable mutations. Transcript-activated chromatin shredding provides an innovative paradigm to develop precision disease treatments for undruggable targets.

PMID:42146678 | PMC:PMC13174608 | DOI:10.64898/2026.05.08.723607

Mol Cell Proteomics. 2026 May 7:101581. doi: 10.1016/j.mcpro.2026.101581. Online ahead of print.

ABSTRACT

Treatment with immune checkpoint inhibitors in colorectal cancer (CRC) has largely benefited patients with microsatellite instability-high (MSI-H) and not the larger proportion of patient with microsatellite-stable (MSS) tumors. This clinical dichotomy has fueled the view that high mutational burden is the dominant driver of tumor immunogenicity and that MSS CRC fails to respond because it is "antigen poor". To directly test this premise and define the origins of presented tumor antigens, we integrated HLA class I immunopeptidomics and matched RNA-seq from 26 primary CRC tumors spanning MSI-H and MSS subtypes. Using patient-specific canonical and cancer-specific proteogenomic databases, we identified 115,292 unique MHC-associated peptides (MAPs) across 61 HLA alleles, with a mean of 9,292 MAPs per tumor and no significant difference in MAP counts between MSI-H and MSS tumors. In toto, we identified 266 tumor antigens, all coded by unmutated genomic sequences, comprising 70 aberrantly expressed tumor-specific antigens (aeTSAs) and 196 tumor-associated antigens (TAAs). In our cohort, MSS tumors presented more TAAs and a comparable number of aeTSAs per tumor relative to MSI-H tumors. In TCGA-COAD stratified analyses (483 tumors), MSS tumors yielded more presentable aeTSAs and TAAs per patient than MSI-H tumors. Across both subtypes, aeTSAs arose predominantly from intronic translation, UTR usage, retroelement activation, and germline-like transcription, including recurrent aeTSAs from PIWIL1, L1TD1, and endogenous retroviral loci. Together, these data demonstrate that MSS CRC is not antigen poor and highlight non-canonical translation as a major, previously underappreciated contributor to the CRC immunopeptidome.

PMID:42106149 | DOI:10.1016/j.mcpro.2026.101581