Recent studies suggest that CD4+ T cells can exert potent anti-tumor effects and improve immunotherapy efficacy by aiding CD8+ T cells. However, characterizing the mechanism of CD4+ T cells' anti-tumor activity has been challenging due to inaccurate major histocompatibility complex class II (MHC-II) peptide prediction algorithms and the lack of high-quality reagents for immune monitoring. To address this, we developed MHC2-substitution of CLIP and analytical LCMS evaluation (MHC2-SCALE), a streamlined approach combining affinity optimized class II-associated invariant chain peptide (CLIP) exchange technology, high throughput 2D-LCMS analysis, and rapid generation of peptide-bound MHC-II monomers for subsequent multimer assembly. We validated MHC-II peptide candidates predicted by the immune epitope database (IEDB) algorithm, as well as uncovered many true and immunogenic MHC-II binders that were not predicted by IEDB. Thus, MHC2-SCALE expands the opportunities for discovering, tracking, and phenotyping antigen-specific CD4+ T cells in preclinical and clinical settings, thereby improving therapies for cancer, autoimmunity, or infectious diseases.
© 2025 The Author(s).

Cytotoxic lymphocytes are crucial to our immune system, primarily eliminating virus-infected or cancerous cells via perforin/granzyme killing. Perforin forms transmembrane pores in the plasma membrane, allowing granzymes to enter the target cell cytosol and trigger apoptosis. The prowess of cytotoxic lymphocytes to efficiently eradicate target cells has been widely harnessed in immunotherapies against haematological cancers. Despite efforts to achieve a similar outcome against solid tumours, the immunosuppressive and acidic tumour microenvironment poses a persistent obstacle. Using different types of effector cells, including therapeutically relevant anti-CD19 CAR T cells, we demonstrate that the acidic pH typically found in solid tumours hinders the efficacy of immune therapies by impeding perforin pore formation within the immunological synapse. A nanometre-scale study of purified recombinant perforin undergoing oligomerization reveals that pore formation is inhibited specifically by preventing the formation of a transmembrane β-barrel. The absence of perforin pore formation directly prevents target cell death. This finding uncovers a novel layer of immune effector inhibition that must be considered in the development of effective immunotherapies for solid tumours.
© 2025. The Author(s).

Public health alarm concerning the emerging fungus Candida auris is fueled by its antifungal drug resistance and propensity to cause deadly outbreaks. Persistent skin colonization drives transmission and lethal sepsis although its basis remains mysterious. We compared the skin colonization dynamics of C. auris with its relative C. albicans , quantifying skin fungal persistence and distribution and immune composition and positioning. C. auris displayed a higher propensity to colonize hair follicles and avidly bound to human hair. While C. albicans triggered an effective sterilizing type 3/17 antifungal immune response driven by IL-17A/F-producing lymphocytes, C. auris triggered a type 1, IFNγ-driven immune response targeting hair follicles. Rather than promoting fungal clearance, IFNγ enhanced C. auris skin colonization by acting directly on keratinocytes impairing epithelial barrier integrity and repressing antifungal defense programs. C. auris exploits focal skin immune responses to create a niche for persistence in hair follicles.

Radiotherapy induces a type I interferon-mediated (T1IFN-mediated) antitumoral immune response that we hypothesized could be potentiated by a first-in-class ataxia telangiectasia mutated (ATM) inhibitor, leading to enhanced innate immune signaling, T1IFN expression, and sensitization to immunotherapy in pancreatic cancer. We evaluated the effects of AZD1390 or a structurally related compound, AZD0156, on innate immune signaling and found that both inhibitors enhanced radiation-induced T1IFN expression via the POLIII/RIG-I/MAVS pathway. In immunocompetent syngeneic mouse models of pancreatic cancer, ATM inhibitor enhanced radiation-induced antitumoral immune responses and sensitized tumors to anti-PD-L1, producing immunogenic memory and durable tumor control. Therapeutic responses were associated with increased intratumoral CD8+ T cell frequency and effector function. Tumor control was dependent on CD8+ T cells, as therapeutic efficacy was blunted in CD8+ T cell-depleted mice. Adaptive immune responses to combination therapy provided systemic control of contralateral tumors outside of the radiation field. Taken together, we show that a clinical candidate ATM inhibitor enhances radiation-induced T1IFN, leading to both innate and subsequent adaptive antitumoral immune responses and sensitization of otherwise resistant pancreatic cancer to immunotherapy.

Immune checkpoint blockade (ICB) can produce durable responses against cancer. We and others have found that a subset of patients experiences paradoxical rapid cancer progression during immunotherapy. It is poorly understood how tumors can accelerate their progression during ICB. In some preclinical models, ICB causes hyperprogressive disease (HPD). While immune exclusion drives resistance to ICB, counterintuitively, patients with HPD and complete response (CR) following ICB manifest comparable levels of tumor-infiltrating CD8+ T cells and interferon γ (IFNγ) gene signature. Interestingly, patients with HPD but not CR exhibit elevated tumoral fibroblast growth factor 2 (FGF2) and β-catenin signaling. In animal models, T cell-derived IFNγ promotes tumor FGF2 signaling, thereby suppressing PKM2 activity and decreasing NAD+, resulting in reduction of SIRT1-mediated β-catenin deacetylation and enhanced β-catenin acetylation, consequently reprograming tumor stemness. Targeting the IFNγ-PKM2-β-catenin axis prevents HPD in preclinical models. Thus, the crosstalk of core immunogenic, metabolic, and oncogenic pathways via the IFNγ-PKM2-β-catenin cascade underlies ICB-associated HPD.
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