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Background The lack of a definitive cure for Alzheimer's disease (AD) is fueling the search for innovative therapeutic strategies. Having revolutionized cancer immunotherapy, immune cell engineering with chimeric antigen receptors (CAR) is being explored to target AD. Whether CARs can recognize distinct amyloid-β (Aβ) species and tau neurofibrillary tangles (NFTs)—hallmark pathologies of AD—remains unclear. Methods To investigate this, we engineered a series of CARs using single-chain fragment variable (scFv) derived from the variable light and heavy chains of antibodies tested in AD clinical trials. These included E2814 (E2814-CAR), targeting tau; Lecanemab (Lec-CAR) and Aducanumab (Adu-CAR), targeting Aβ; and Donanemab (Don-CAR) and Remternetug (Rem-CAR), targeting the truncated pyroglutamated Aβ species Aβp3–42. To evaluate CAR function, we utilized the murine DO11.10 CD4⁺ T-cell hybridoma line as a scalable and reproducible platform. CAR activation was assessed in response to tau preformed fibrils (PFFs), Aβ 1–42 oligomer-enriched aggregates, and Aβp3–42 aggregates, using flow cytometry for CD69 expression and ELISA for IL-2 secretion. To validate this platform, we tested Adu-CAR in primary mouse CD4⁺ T cells treated with Aβ 1–42 aggregates and assessed activation via flow cytometry for CD69 and CD25 expression. Results DO11.10 cells expressing E2814-CAR—but not Lec-CAR—responded to tau PFFs. In contrast, cells expressing Adu-CAR, and to a lesser extent Lec-CAR—but not E2814-CAR—responded to Aβ 1–42 aggregates. For Aβp3–42 aggregates, Rem-CAR elicited the strongest response, followed by Adu-CAR, while E2814-CAR and Don-CAR showed no activation. The activation of Adu-CAR by Aβ 1–42 aggregates was recapitulated in primary CD4⁺ T cells, as measured by CD69 expression. Conclusions Our findings demonstrate that CARs can detect and discriminate between tau PFFs, Aβ1-42, and Aβp3-42 aggregates. This highlights the potential of repurposing AD antibodies for CAR-based therapies to selectively target tau NFTs and distinct forms of Aβ senile plaques. Graphical Abstract

Although the link between ischemia-reperfusion injury (IRI) and T cell-mediated rejection (TCMR) in kidney transplantation (KT) is well known, the mechanism remains unclear. We investigated essential genes and biological processes involved in interactions between IRI and TCMR. Methods: Renal IRI and TCMR datasets were obtained from the Gene Expression Omnibus database. IRI and TCMR co-expression networks were built using weighted gene co-expression network analysis, and essential modules were identified to acquire shared genes and conduct functional enrichment analysis. Shared genes were used for TCMR consensus clustering, differentially expressed genes (DEGs) were identified, and gene set enrichment analysis (GSEA) was conducted. Three machine learning algorithms screened for hub genes, which underwent miRNA prediction and transcription factor analysis. Hub gene expression was verified, and survival analysis was performed using Kaplan–Meier curves. Results: IRI and TCMR shared 84 genes. Functional enrichment analysis revealed that inflammation played a significant role. Based on shared genes, TCMR was divided into two clusters. GSEA revealed that graft rejection-related pathways varied between the two clusters. TCMR hub genes, guanylate-binding protein 1 (GBP1) and CD69, showed increased expression. Decreased survival rates were found in patients who had undergone KT and had high GBP1 and CD69 levels. Conclusions: The study demonstrates that renal IRI has a potential role in renal TCMR and the pathogenic pathways are potentially inflammation-related.

Acne is a multifactorial disease driven by physiological changes occurring during puberty in the pilosebaceous unit (PSU) that leads to sebum overproduction and a dysbiosis involving notably Cutibacterium acnes . These changes in the PSU microenvironment lead to a shift from a homeostatic to an inflammatory state. Indeed, immunohistochemical analyses have revealed that inflammation and lymphocyte infiltration can be detected even in the infraclinical acneic stages, highlighting the importance of the early stages of the disease. In this study, we utilized a robust multi-pronged approach that included flow cytometry, confocal microscopy, and bioinformatics to comprehensively characterize the evolution of the infiltrating and resident immune cell populations in acneic lesions, beginning in the early stages of their development. Using a discovery cohort of 15 patients, we demonstrated that the composition of immune cell infiltrate is highly dynamic in nature, with the relative abundance of different cell types changing significantly as a function of clinical lesion stage. Within the stages examined, we identified a large population of CD69 + CD4 + T cells, several populations of activated antigen presenting cells, and activated mast cells producing IL-17. IL-17 + mast cells were preferentially located in CD4 + T cell rich areas and we showed that activated CD4 + T cells license mast cells to produce IL-17. Our study reveals that mast cells are the main IL-17 producers in the early stage of acne, underlying the importance of targeting the IL-17 + mast cell/T helper cell axis in therapeutic approaches.

Detection and characterization of antigen‐specific T cells are important for studying immune responses upon infection, vaccination, or autoreactivity. The activation‐induced marker (AIM) assay is a robust technique to identify and characterize antigen‐specific CD4 and CD8 T cells. However, there is variability in the AIM assay, particularly in the type and number of activation markers used. In this study, we set out to define which marker combinations are most suited to optimally detect antigen‐specific CD4 and CD8 T cells and if certain marker combinations preferentially detect specific CD4 T helper subsets. A multiparameter flow cytometry panel, including six common activation markers: CD40L, CD137, CD69, OX40, CD25, and PD‐L1, was used for detecting antigen‐specific T cells following infection (SARS‐CoV‐2 and CMV) or vaccination (mRNA‐1273 SARS‐CoV‐2). We demonstrate that combining multiple activation markers increases the detection frequency of antigen‐specific CD4 T cells compared to commonly used dual marker combinations. In addition, marker combinations including PD‐L1 detected a higher frequency of antigen‐specific CD4 T cells in SARS‐CoV‐2 and CMV infected but not in SARS‐CoV‐2–vaccinated individuals. Certain dual marker combinations preferentially detected specific CD4 T helper subsets. The majority of antigen‐specific CD8 T cells were captured by the dual combination of CD69 plus CD25. In conclusion, combining CD137, CD69, OX40, CD25, and PD‐L1 in an AIM assay results in robust and optimal detection of both specific CD4 T helper subsets and CD8 T cells in different antigenic contexts.

The HIV-1 accessory protein Vpr is known for its profound effect on the host proteome. It degrades many cellular proteins, including transcription factors and DNA-associated proteins. In addition, Vpr activates the nuclear factor of activated T cells (NFAT), a key transcription factor in T cells. However, it has remained unclear to what extent Vpr and consequently NFAT control changes in the transcriptome of HIV-1-infected primary CD4+ T cells. In this study, we show that Vpr significantly alters the transcriptome of CD4+ T cells, with almost half of the deregulated genes being under NFAT control. These changes involve pathways associated with increased immune activation and cell cycle regulation, shedding light on how Vpr contributes to CD4+ T cell depletion and HIV-1 pathogenesis.

Janus kinase inhibitors (JAKis) are widely prescribed for autoimmune diseases, but their use is associated with increased infection risk. The mechanisms underlying this susceptibility remain unclear. CD8 T cells play a central role in antimicrobial defence, yet little is known about how JAKis reprogramme their activation and effector programmes. Here, we investigated naive and memory CD8 T cells from healthy donors stimulated in vitro in the presence of clinically relevant JAK inhibitors targeting JAK1 (JAK1i), JAK1/2 (JAK1/2i), or JAK1/3 (JAK1/3i). Flow cytometry, SCENITH, transmission electron microscopy, and RNA-seq were used to evaluate metabolic and functional programmes. We found that JAKis uncoupled phenotypic activation from metabolic reprogramming. Functionally, JAKi-treated CD8 T cells exhibited reduced activation and produced lower amounts of cytokines and cytotoxic molecules. Notably, even JAKi-treated memory CD8 T cells that upregulated CD69 and CD25 failed to engage glycolysis, showing decreased GLUT1 expression and glucose uptake. SCENITH profiling confirmed diminished glucose dependence and a shift toward mitochondrial reliance, despite reduced mitochondrial potential and structural alterations. Transcriptomic and protein analyses further revealed decreased mTOR activity and increased p53-associated transcripts, consistent with impaired growth and stress signalling. CD8 T cells from rheumatoid arthritis patients under JAKi therapy were analysed ex vivo for translational validation. These cells showed similar metabolic and signalling alterations, underscoring their clinical relevance. Altogether, these findings identify JAKis as disruptors of metabolic and signalling pathways in CD8 T cells, providing a mechanistic link between impaired effector function and the increased infection risk observed in treated patients.

Monoclonal antibodies (mAbs) targeting immune checkpoint pathways such as programmed cell death protein 1 (PD‐1)/PD‐L1 are central to modern immunotherapy, yet scalable methods to assess their functional blockade remain limited. We present a bead‐based flow cytometry assay for quantifying the inhibition of PD‐1/PD‐L1 interaction by antibodies. Recombinant human PD‐1 protein was conjugated to polystyrene beads, and its interaction with recombinant human PD‐L1 protein labeled with a fluorochrome was measured. The inhibitory activity of an anti‐PD‐L1 mAb was quantified based on their ability to disrupt this interaction. The assay was validated for intra‐ and inter‐assay precision, in addition, functionality was confirmed using a T cell coculture assay. The assay demonstrated dose‐dependent inhibition by the αPD‐L1 mAb, with a calculated mean IC 50 of 3.122 µg/mL. The method proved to be reproducible for the determination of antibody blocking activity, with relative standard deviation (RSD) < 20% between three independent runs. At the concentration approximating the IC 50 detected on the bead assay, the antibody significantly restored CD69 expression on the T cell surface ( p = 0.0001) in a coculture in vitro system. In addition, the methodology could successfully distinguish the blocking capacity of two anti‐PD‐L1 antibodies with different affinities. This high‐throughput compatible platform offers a reliable tool for screening PD‐1/PD‐L1 blocking antibodies, supporting immunotherapy discovery and development.

Identification and isolation of antigen-specific T cells for downstream transcriptomic analysis is key for various immunological studies. Traditional methods using major histocompatibility complex (MHC) multimers are limited by the number of predefined immunodominant epitopes and MHC matching of the study subjects. Activation-induced markers (AIM) enable highly sensitive detection of rare antigen-specific T cells irrespective of the availability of MHC multimers. Herein, we have developed an AIM assay for the detection, sorting and subsequent single-cell RNA sequencing (scRNA-seq) analysis of hepatitis C virus (HCV)-specific T cells. We examined different combinations of the activation markers CD69, CD40L, OX40, and 4-1BB at 6, 9, 18 and 24 h post stimulation with HCV peptide pools. AIM+ CD4 T cells exhibited upregulation of CD69 and CD40L as early as 6 h post-stimulation, while OX40 and 4-1BB expression was delayed until 18 h. AIM+ CD8 T cells were characterized by the coexpression of CD69 and 4-1BB at 18 h, while the expression of CD40L and OX40 remained low throughout the stimulation period. AIM+ CD4 and CD8 T cells were successfully sorted and processed for scRNA-seq analysis examining gene expression and T cell receptor (TCR) usage. scRNA-seq analysis from this one subject revealed that AIM+ CD4 T (CD69+ CD40L+) cells predominantly represented Tfh, Th1, and Th17 profiles, whereas AIM+ CD8 T (CD69+ 4-1BB+) cells primarily exhibited effector and effector memory profiles. TCR analysis identified 1023 and 160 unique clonotypes within AIM+ CD4 and CD8 T cells, respectively. In conclusion, this approach offers highly sensitive detection of HCV-specific T cells that can be applied for cohort studies, thus facilitating the identification of specific gene signatures associated with infection outcome and vaccination.

In the rheumatoid arthritis (RA) synovium, resident fibroblast-like synoviocytes (FLS) express MHC class II molecules (HLA-D) but lack the costimulatory signals typically required for T cell activation. Here, we demonstrate that antigen presentation by FLS induces a distinct T cell activation state characterized by high CD69 yet reduced CD25 and HLA-DR expression, suppressed proliferation, and decreased effector cytokine production compared with professional antigen-presenting cells (APCs), such as macrophages. FLS were also capable of suppressing macrophage-induced T cell activation, underscoring their dominant immunomodulatory role in the synovial microenvironment. Mechanistically, we identify indoleamine 2,3-dioxygenase–mediated (IDO1-mediated) tryptophan depletion as the primary driver of FLS-induced T cell hyporesponsiveness. Spatial transcriptomics revealed colocalization of IDO1 and CD69 within ectopic lymphoid structures in RA synovium, further supporting the in vivo relevance of this pathway. These findings provide the groundwork for positioning FLS as critical T cell regulators in RA and highlight the importance of preserving their immunosuppressive properties when therapeutically targeting pathogenic FLS functions.

The ectocervix is part of the lower female reproductive tract (FRT), which is susceptible to sexually transmitted infections (STIs). Comprehensive knowledge of the phenotypes and T cell receptor (TCR) repertoire of tissue-resident memory T cells (TRMs) in the human FRT is lacking. We took single-cell RNA-Seq approaches to simultaneously define gene expression and TCR clonotypes of the human ectocervix. There were significantly more CD8 + than CD4 + T cells. Unsupervised clustering and trajectory analysis identified distinct populations of CD8 + T cells with IFNG hi GZMB lo CD69 hi CD103 lo or IFNG lo GZMB hi CD69 med CD103 hi phenotypes. Little overlap was seen between their TCR repertoires. Immunofluorescence staining showed that CD103 + CD8 + TRMs were preferentially localized in the epithelium, whereas CD69 + CD8 + TRMs were distributed evenly in the epithelium and stroma. Ex vivo assays indicated that up to 14% of cervical CD8 + TRM clonotypes were HSV-2 reactive in HSV-2–seropositive persons, reflecting physiologically relevant localization. Our studies identified subgroups of CD8 + TRMs in the human ectocervix that exhibited distinct expression of antiviral defense and tissue residency markers, anatomic locations, and TCR repertoires that target anatomically relevant viral antigens. Optimization of the location, number, and function of FRT TRMs is an important approach for improving host defenses to STIs.