Abstract Objectives Extensive‐stage small cell lung cancer (ES‐SCLC) remains an aggressive malignancy with limited durable responses to immune checkpoint inhibitors (ICIs) and no established predictive biomarkers. This study aimed to evaluate the prognostic and predictive value of tertiary lymphoid structures (TLSs) in patients with ES‐SCLC receiving ICI‐based therapy. Methods In this multicenter retrospective study of 180 treatment‐naïve ES‐SCLC patients subsequently receiving ICI‐based therapy, TLSs were systematically evaluated using H&E staining, multiplex immunofluorescence, and spatial analysis to determine their prognostic and predictive value. Results TLSs were identified in 22.2% of cases and independently associated with improved overall survival (OS) in both univariate (HR = 0.365, 95% CI: 0.217–0.613, P < 0.001) and multivariate (HR = 0.337, 95% CI: 0.199–0.569, P < 0.001) analyses. Crucially, TLS maturation status was associated with clinical outcomes, as patients with mature TLSs containing CD21 + follicular dendritic cells and organized germinal centers experienced significantly longer survival than those with non‐mature TLSs ( P = 0.0049). Beyond mere presence, responders to ICIs exhibited distinct spatial characteristics, including higher TLS densities, denser immune infiltration, and shorter distances between TLS‐resident CD20 + B cells, CD3 + T cells, and tumor cells, suggesting that immune–tumor spatial organization within TLSs reflects functional antitumor engagement. Conclusion TLS maturation and spatial architecture may serve as candidate tissue biomarkers associated with clinical outcomes in ICI‐treated ES‐SCLC, warranting prospective validation alongside SCLC molecular subtyping.
Abstract Large insert gene editing in ex vivo , primary human T cells represents an important approach for engineering next‐generation cellular immunotherapies. While CRISPR‐based editing is well‐established for small genetic modifications, large insert knock‐in remains a technical bottleneck. This review examines current strategies for achieving efficient, precise and safe integration of large genetic elements. We compare delivery methods for CRISPR components, highlighting electroporation of Cas9 ribonucleoproteins as the most efficient but often cytotoxic approach; emerging alternatives such as cell‐penetrating peptides and lipid nanoparticles offer improved cell viability at the expense of editing efficiency. The influence of genomic locus selection, guide RNA design and chromatin context on editing outcomes, as well as the role of homology‐directed repair (HDR) template design is evaluated. Various template formats—including linear double‐ and single‐stranded DNA, plasmids, circular single‐stranded (css) DNA and recombinant adeno‐associated virus (rAAV)—are assessed for their efficiency, toxicity and risk of off‐target integration. Where possible, cross‐study normalisation is used to enable meaningful comparisons. rAAV templates consistently achieve the highest insertion rates but come with concerns regarding cost, size limitations and insertional mutagenesis, whereas non‐viral approaches such as cssDNA templates show promise. Additionally, we review the use of small‐molecule modulators to enhance HDR efficiency, noting trade‐offs between improved insertion rates and potential genomic instability. Overall, successful large insert editing requires balancing of editing efficiency, cell viability and genomic safety considerations. Continued optimisation of delivery systems, template design and repair pathway modulation will be critical for advancing scalable and clinically viable T‐cell therapies.
Abstract Objectives Despite the availability of vaccines, influenza viruses still cause substantial morbidity and mortality. This highlights the need for improved vaccines and novel therapeutics. CD8 + T cells recognise conserved viral epitopes and can provide broad protection, making them attractive targets for next‐generation vaccine design. A key step in this process is to identify T cell epitopes across influenza viruses. Here, we characterised three homologous influenza Polymerase Basic 1 (PB1)‐derived peptides from influenza viruses (PB1‐A 310 , PB1‐B 310 and PB1‐C 312 , respectively) presented by the common, but understudied, HLA‐B*18:01 molecule. Methods We used a thermal melt assay and X‐ray crystallography to assess the stability and structural features of each peptide. CD8 + T‐cell responses were evaluated using intracellular cytokine staining with HLA‐B*18:01 + samples. Results The structures of HLA‐B*18:01 presenting each peptide were solved, including the first structure of HLA‐B*18:01 presenting a Gammainfluenzavirus (formerly influenza C virus) derived peptide. PB1‐C 312 exhibited a higher thermal stability and a different conformation compared to PB1‐A 310 and PB1‐B 310 . CD8 + T cell responses varied among samples, with PB1‐A 310 eliciting the strongest activation. Minimal T cell cross‐reactivity was observed and was mostly between PB1‐A 310 and PB1‐B 310 likely reflecting shared structural features. Conclusion These findings further expand knowledge on peptide presentation by the common HLA‐B*18:01 molecule, and the limitations on CD8 + T cell cross‐recognition of this PB1‐derived epitope across influenza lineages. This highlights the importance of more research on cross‐lineage T cell cross‐reactivity that could inform the design of broadly protective T cell‐based vaccines and therapeutics.
Abstract Objectives Despite their anti‐cancer properties, natural killer (NK) cells are discarded during CAR T cell manufacturing. NK cells display promising responses as a cell therapy, exhibiting remarkable safety at high doses. Moreover, NK cells have complementary cytokine profiles and distinct antigen‐recognition pathways to supplement CAR T cell therapies. We demonstrate parallel expansion of NK cells from the normally discarded PBMC of CAR T cell production for downstream combination therapies against solid and blood‐borne malignancies. Methods CAR T cells were manufactured from buffy coats with initial isolation of T cells from PBMC. NK cells were rapidly expanded in parallel by stimulating the remaining PBMC with membrane‐bound (mb)IL‐21 + mbIL‐15 feeder cells. Expanded NK cells were assessed through spectral flow cytometry and applied as a pretreatment prior to CAR Tcell infusion against xenograft tumor models in NOD‐scid IL2Rgammanull (NSG) mice. Results Applying feeder cells to residual PBMC stimulated 5000‐fold expansions of pure NK cells. Expanded NK cells expressed various activation receptors and displayed strong in vitro cytotoxicity, which was further improved in CAR T‐conditioned medium. A pretreatment of NK cells enhanced existing CAR T cell therapies, improving in vivo tumor clearance and survival against breast cancer, acute lymphoblastic leukaemia and non‐Hodgkin's lymphoma. While NK cells eliminated CD19−/− variants in vitro, they were unable to suppress antigen‐escape relapse in vivo. Conclusion This study encourages the optimal use of leukapheresis product. By recycling discarded PBMC into NK cells, CAR T cell therapies can be effectively enhanced, improving tumor clearance and survival.
Abstract Objectives To evaluate the prognostic value of peripheral immune markers and their static and dynamic changes during different treatment stages in patients with hepatitis B virus (HBV)‐related unresectable hepatocellula carcinoma (uHCC) receiving transarterial chemoembolisation (TACE) combined with immune checkpoint inhibitors (ICIs) and anti‐vascular endothelial growth factor (anti‐VEGF) antibodies or tyrosine kinase inhibitors (TKIs). Methods This single‐centre retrospective study included patients with HBV‐related uHCC treated with TACE combined with ICls and anti‐VEGF antibodies or TKIs between July 2019 and July 2024. Peripheral blood immune indicators were collected at baseline (Cycle 0), the second treatment cycle (Cycle 2) and the fourth treatment cycle (Cycle 4). The primary outcome was overall survival (OS), while secondary outcomes included progression‐free survival (PFS), objective response rate (ORR) and disease control rate (DCR), with tumour response assessed at the first treatment evaluation (at the end of the fourth treatment cycle) based on mRECIST criteria. Longitudinal changes between adjacent time points were calculated (Δ: Cycle 2 ‐ Cycle 0; Δ2: Cycle 4 ‐ Cycle 2). Both static values and dynamic changes in immune markers were subjected to LASSO‐Cox and stepwise multivariate Cox regression analyses to identify independent predictors of OS. Based on the significant variables, a prognostic nomogram and an immune‐based risk score model were developed. To further assess prognostic heterogeneity, patients were stratified by K‐means clustering according to the temporal patterns of the most predictive immune marker, and survival outcomes were compared across the resulting subgroups. Results A total of 67 HBV‐related uHCC patients treated with TACE plus ICIs and anti‐VEGF antibodies/TKIs were included. The cohort had a median follow‐up of 18.2 months, with a median overall survival (mOS) of 30.4 months and a median progression‐free survival (mPFS) of 11.0 months. At the first evaluation, the objective response rate (ORR) was 53.7%, and the disease control rate (DCR) was 86.6%. LASSO‐Cox regression identified several static and dynamic immune markers associated with OS. Ultimately, CD4⁺/CD8⁺ ratio at Cycle 4 (HR = 0.58, p = 0.035), Δ2 NLR (HR = 1.66, p = 0.014), and Δ2 CD3⁺CD4⁺ (HR = 0.04, p = 0.007) were confirmed by stepwise multivariate Cox regression. A prognostic nomogram incorporating these variables demonstrated favourable predictive performance (C‐index = 0.72; AUCs = 0.750, 0.722 and 0.854 for 1‐, 2‐ and 3‐year OS, respectively). The derived immune‐based risk score effectively stratified patients into high‐ and low‐risk groups with significantly different OS (11.5 vs. 34.0 months, p < 0.001) and PFS (7.5 vs. 13.8 months, p = 0.006). The risk score showed a positive correlation with Δ2 NLR (r = 0.50) and negative correlations with CD4⁺/CD8⁺ ratio (r = −0.64) and Δ2 CD3⁺CD4⁺ (r = −0.45). Based on CD3⁺CD4⁺ trajectory clustering, three distinct immune dynamic subtypes were identified, among which the continuous decline group exhibited the poorest prognosis (p = 0.042). Conclusion This study demonstrates the prognostic relevance of peripheral immune markers in patients with HBV‐related uHCC receiving combination therapy. Immune dynamics observed during later treatment stages, rather than baseline measurements, were more strongly associated with overall survival. A risk model based on the CD4⁺/CD8⁺ ratio at Cycle 4, Δ2 NLR and Δ2 CD3⁺CD4⁺ enabled effective prognostic stratification and may provide a useful framework for risk assessment in this patient population.
