Viral transduction of primary T cells enables stable genetic engineering for research and immunotherapy, supporting both transgene overexpression and gene deletion. Although the overall workflow can be similar to transduction in other mammalian cell lines, primary T cell culture imposes distinct requirements such as cell-state-dependent nuances shaped by T cell activation and proliferation which can make it challenging to obtain a sufficient number of genetically engineered T cells. This article provides practical guidance for researchers new to T cells but familiar with basic mammalian cell culture.

Several viral vectors have been developed for gene therapy due to their high transduction efficiency, but some integrate into the host genome, raising safety concerns. Recent studies have identified recombinant adeno-associated virus serotype 6 (rAAV6) as a promising vector for hematopoietic stem and progenitor cell-targeted gene therapy because of its non-pathogenic nature, low integration frequency, and capacity for sustained episomal transgene expression. Nevertheless, its chromosomal integration profile remains incompletely defined, warranting a comprehensive evaluation to assess long-term safety. In this study, human CD34＋ cells were transduced with rAAV6 under varying vector doses and transgene contexts, and integration-site mapping was performed using the integration-site enriched library sequencing approach. Consistent with the largely episomal nature of AAV, high vector sequence alignment rates were observed across all groups. rAAV6 integrations occurred randomly throughout the genome, showing a broad pan-chromosomal distribution without evidence of sequence-specific targeting or clustering. Although integrations were more frequent in CpG islands, commonly located within open chromatin, this pattern likely reflects chromatin accessibility rather than targeting bias. Functional enrichment analysis indicated associations with general cellular and structural processes, without enrichment in oncogenic pathways. Distance-based analysis confirmed that integration sites were mapped at a distance from oncogenes and tumor suppressor genes, even under high-dose conditions. The data support the genomic safety of rAAV6 and its applicability to hematological gene therapy.

Cytokines of the common γ-chain family (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) are pivotal in regulating immune responses and hold significant promise for cancer immunotherapy. However, their clinical efficacy has been hindered by short serum half-life, pleiotropic off-target effects, and dose-limiting systemic toxicities such as cytokine release syndrome. This review provides a comprehensive overview of recent advancements designed to overcome these limitations. Molecular engineering strategies, including PEGylation, Fc-fusion, muteins, and next-generation approaches like pro-cytokines and split-cytokines, which aim to enhance stability and receptor specificity. To minimize systemic toxicity and achieve high, tumor-localized cytokine concentrations, a variety of innovative delivery systems have been developed, ranging from polymer- and lipid-based nanoparticles to biological vehicles such as oncolytic viruses and cell-based therapies. Furthermore, these delivery systems are designed to respond to both intrinsic and extrinsic stimuli, enabling spatial and temporal control over cytokine activity. By synthesizing current progress and remaining challenges, this review outlines the future trajectory of cytokine-based therapeutics in achieving precise and safe anti-tumor immunity.

Colorectal cancer (CRC) remains largely resistant to immune checkpoint inhibitors (ICIs) due to an immunosuppressive tumor microenvironment (TME) shaped by M2-like tumor-associated macrophages (TAMs). Identifying transcriptional regulators of M2-like TAMs in CRC could provide strategies to overcome ICI resistance by reprogramming the TME. In this study, we analyzed single-cell RNA-seq data from CRC patients to identify transcriptional regulators of M2-like TAMs. Notably, MAFB expression was predominantly detected in M2-like TAMs and was significantly higher in mismatch repair-proficient (pMMR) CRC than in mismatch repair-deficient (dMMR) CRC. Moreover, MAFB expression was inversely correlated with relapse-free survival in colon cancer patients. In macrophages, MAFB was induced by the IL-4-STAT6 and IL-10-STAT3 pathways, which drive M2 polarization, and was suppressed by M1-polarizing signals. Myeloid-specific deletion of Mafb, in combination with ICI treatment, reduced colon cancer growth by enhancing anti-tumor immunity through increased activity of M1-like TAMs, which led to increased infiltration of NK cells and activated cytotoxic T cells within the TME. Mechanistically, MAFB acts as a transcriptional activator directly promoting Il4ra, Il10, and Arg1 mRNA expression, supported by the identification of MAF recognition element (MARE) sites within these loci. Consistently, ectopic expression of IL-4 receptor α in Mafb-deficient macrophages restored M2 phenotypes comparable to those of wild-type macrophages. These data highlight the critical cell-intrinsic role of MAFB in regulating M2-like TAMs and provide the first evidence that targeting MAFB enhances ICI efficacy in CRC by reprogramming TAMs toward an anti-tumorigenic M1-like phenotype.

Deciphering the mechanism governing the temporal switch from fetal to adult hemoglobin during erythropoiesis has significant clinical relevance. Here, we identify LDB1 as a pivotal regulator of β-globin switching in erythroid progenitors. The absence of LDB1 in proerythroblasts from mouse fetal liver leads to cell cycle arrest and apoptosis due to the accumulation of reactive oxygen species (ROS), resulting from excessive heme content caused by significant overexpression of embryonic β-globin genes such as Hbb-y and Hbb-bh1. Mechanistically, LDB1 directly enhances the mRNA expression of fetal globin gene repressors, including Bcl11a, Cbfa2t3, and Sox6. Moreover, the LDB1 complex, which includes LMO2 and GATA1, binds directly to enhancer regions of Bcl11a, promoting its transcription. CRISPR/Cas9-mediated LDB1 knockout in human erythroleukemia cells confirmed LDB1 as a key enhancer of BCL11A transcription, reducing its mRNA expression while upregulating transcription of the fetal globin gene HBG. Following chromatin immunoprecipitation (ChIP) assays revealed LDB1 binding to intron 2 enhancers within the BCL11A locus, reinforcing its indispensable role in BCL11A transcription in humans. Consequently, ectopic expression of BCL11A in LDB1-deficient proerythroblasts promotes their proliferation by rescuing them from ROS-mediated apoptosis. These findings highlight the essential role of LDB1 in fetal globin silencing during erythropoiesis.

In this MiniResource, we outlined practical approaches for preparing murine T cells, from isolation to in vitro activation, with emphasis on reproducibility and viability. While specific experimental conditions should be tailored to individual assays, the principles summarized here provide a framework for establishing robust T cell preparation across diverse research settings. By integrating standard methodologies with troubleshooting insights, this resource aims to support both basic immunological studies and future applications in T cell engineering.

Supplementary Data from Epigenetic Repression of STING by MYC Promotes Immune Evasion and Resistance to Immune Checkpoint Inhibitors in Triple-Negative Breast Cancer

Glioblastoma (GBM) stem cells (GSCs) are pivotal in tumor initiation, recurrence, and therapeutic resistance, underscoring their critical role in the complex pathology of GBM. Despite their recognized importance, the mechanisms by which GSCs facilitate immune evasion, especially in emerging immunotherapies, remain incompletely understood. Here, we identify intercellular adhesion molecule 1 (ICAM1) as a key regulator of GSC stemness and tumorigenicity, promoting an immunosuppressive microenvironment via β-catenin/PD-L1 signaling. Mechanistically, ICAM1 interacts with ZNRF3, leading to its autoubiquitination and clearance, stabilizing LRP6, and activating β-catenin signaling, which upregulates PD-L1 expression. Combined treatment with anti-ICAM1 and anti-PD-1 antibodies results in the most effective tumor inhibition and significantly extends survival in ICAM1-overexpressing GBM models. CyTOF and flow cytometry analyses reveal that ICAM1 overexpression reduces cytotoxic CD8+ T cell populations via PD-L1/PD-1 interactions, reversible by PD-1 blockade. Our findings highlight the co-targeting of ICAM1 and PD-1 as a promising strategy against immune evasion in GBM.

Limited T cell infiltration into solid tumors remains one of the major obstacles to successful cancer immunotherapy, particularly for adoptive cell therapy (ACT). Although the chemokine CXCL10 recruits T cells, its direct therapeutic application is hampered by poor pharmacokinetics, systemic leakage, and failure to establish stable concentration gradients required for effective cell migration. To overcome these challenges, we engineered mesenchymal stromal cells (MSCs) to co-express NAD(P)H quinone oxidoreductase 1 (NQO1) for enhanced survival and CXCL10-Fc fusion protein for sustained chemokine delivery (NIP-MSCs). The engineered MSCs exhibited resilience to tumor microenvironment conditions through improved redox homeostasis, resulting in enhanced persistence and sustained IP10-Fc production in vivo. Crucially, tumor-targeted delivery of CXCL10-Fc established potent chemotactic gradients with minimal systemic leakage, dramatically increasing both endogenous and adoptively transferred T cell recruitment to tumor site. In syngeneic mouse models, NIP-MSC treatment significantly suppressed tumor growth through enhanced CD8+ T cell infiltration. When combined with ACT in melanoma models, NIP-MSCs resulted in superior tumor control and significantly prolonged survival compared to conventional approaches. This work validates NIP-MSCs as a promising platform to overcome T cell exclusion and potentiate immunotherapy efficacy in solid tumors.

Postweaning multisystemic wasting syndrome (PMWS) is caused by a systemic inflammation after porcine circovirus type 2 (PCV2) infection. It was one of the most economically important pathogens affecting pig production worldwide before PCV2 vaccine was first introduced in 2006. After the development of a vaccine against PCV2a type, pig farms gradually restored enormous economic losses from PMWS. However, vaccine against PCV2a type could not be fully effective against several different PCV2 genotypes (PCV2b - PCV2h). In addition, PCV2a vaccine itself could generate antigenic drift of PCV2 capsid. Therefore, PCV2 infection still threats pig industry worldwide. PCV2 infection was initially found in local tissues including reproductive, respiratory, and digestive tracks. However, PCV2 infection often leads to a systemic inflammation which can cause severe immunosuppression by depleting peripheral lymphocytes in secondary lymphoid tissues. Subsequently, a secondary infection with other microorganisms can cause PMWS. Eleven putative open reading frames (ORFs) have been predicted to encode PCV2 genome. Among them, gene products of six ORFs from ORF1 to ORF6 have been identified and characterized to estimate its functional role during PCV2 infection. Acquiring knowledge about the specific interaction between each PCV2 ORF protein and host protein might be a key to develop preventive or therapeutic tools to control PCV2 infection. In this article, we reviewed current understanding of how each ORF of PCV2 manipulates host cell signaling related to immune suppression caused by PCV2.

Dear Editor, CLDN18.2(CLDN),a member of tight junction protein family,is strictly limited to express on differentiated epithelial cells of the gastric mucosa and abnormal overexpression has been found in many cancers,especially in digestive system malignancies.1 Those features make CLDN a potential therapeutic target.However,monoclonal antibody targeting CLDN induce limited antitumor immune responses in clinical trials and fusion of strong immunomodulators might be needed to enhance its efficacy.High dose IL-2 activates tumor infiltrating lymphocytes(TILs),but the severe toxicity and poor tumor targeting limits its use.

Immune checkpoint blockade (ICB)-based immunotherapy depends on functional tumour-infiltrating lymphocytes (TILs), but essential cytokines are less understood. Here we uncover an essential role of endogenous IL-2 for ICB responsiveness and the correlation between insufficient IL-2 signalling and T-cell exhaustion as tumours progress. To determine if exogenous IL-2 in the tumour microenvironment can overcome ICB resistance, we engineered mesenchymal stem cells (MSCs) to successfully deliver IL-2 mutein dimer (SIL2-EMSC) to TILs. While MSCs have been used to suppress inflammation, SIL2-EMSCs elicit anti-tumour immunity and overcome ICB resistance without toxicity. Mechanistically, SIL2-EMSCs activate and expand pre-existing CD8(+) TILs, sufficient for tumour control and induction of systemic anti-tumour effects. Furthermore, engineered MSCs create synergy of innate and adaptive immunity. The therapeutic benefits of SIL2-EMSCs were also observed in humanized mouse models. Overall, engineered MSCs rejuvenate CD8(+) TILs and thus potentiate ICB and chemotherapy.

Abstract Tumor microenvironment (TME) generates immunosuppressive niche to induce CD8+ tumor infiltrating lymphocytes (TILs) exhaustion. Therapeutic targeting to functionally reinvigorate CD8+ T cells is a promising strategy to enhance antitumor immunity. While interleukin-2 (IL-2) based therapies cause potent T cell activation and proliferation, the clinical application remains challenging due to short half-life and severe toxicity at therapeutic doses. To address this, we engineered mesenchymal stem cells (MSCs) to successfully proliferate and turn on or off CD8 T cell-preferential IL-2 mutein/Fc fusion protein (SIL2-EMSC) to target cytotoxic T cells in the TME. Peritumoral administration of SIL2-EMSCs permits local production of sufficient SIL2 inside the TME and induces complete tumor regression without adverse toxicity. Mechanistically, SIL2-EMSC remodels the TME that activates and expands preexisting CD8+ TILs. Furthermore, local treatment of SIL2-EMSC elicits systemic antitumor responses for the clearance of distal tumor and metastasis. In advanced tumors, SIL2-EMSCs can overcome resistance to immune check blockade (ICB) and β-lapachone (β-lap) chemotherapy. The therapeutic benefits of SIL2-EMSC were also observed in humanized mouse models. Overall, tumor-targeted delivery of cytokines by next generation of MSCs reverses immunosuppressive environment, improves antitumor effects, and synergize with various therapies without adverse toxicity. Citation Format: Joonbeom Bae, Longchao Liu, Casey Timmerman, Eric Hsu, Anli Zhang, Jiankun Zhu, Yang-Xin Fu. Tumor-targeted IL-2 by engineered mesenchymal stem cells reinvigorates CD8+ T cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P058.

The MYC oncogene is frequently amplified in triple-negative breast cancer (TNBC). Here, we show that MYC suppression induces immune-related hallmark gene set expression and tumor-infiltrating T cells in MYC-hyperactivated TNBCs. Mechanistically, MYC repressed stimulator of interferon genes (STING) expression via direct binding to the STING1 enhancer region, resulting in downregulation of the T-cell chemokines CCL5, CXCL10, and CXCL11. In primary and metastatic TNBC cohorts, tumors with high MYC expression or activity exhibited low STING expression. Using a CRISPR-mediated enhancer perturbation approach, we demonstrated that MYC-driven immune evasion is mediated by STING repression. STING repression induced resistance to PD-L1 blockade in mouse models of TNBC. Finally, a small-molecule inhibitor of MYC combined with PD-L1 blockade elicited a durable response in immune-cold TNBC with high MYC expression, suggesting a strategy to restore PD-L1 inhibitor sensitivity in MYC-overexpressing TNBC.

It is known that tumor-reactive T cells are initially activated in the draining lymph node, but it is not well known whether and how tumor-infiltrating lymphocytes (TILs) are reactivated in the tumor microenvironment (TME). We hypothesize that defective T cell receptor (TCR) signaling and cosignals in the TME limit T cell reactivation. To address this, we designed a mesenchymal stromal cell-based delivery of local membrane-bound anti-CD3 and/or cosignals to explore their contribution to reactivate T cells inside the TME. Combined anti-CD3 and CD40L rather than CD80 led to superior antitumor efficacy compared with either alone. Mechanistically, TCR activation of preexisting CD8+ T cells synergized with CD40L activation of DCs inside the TME for optimum tumor control. Exogenous TCR signals could better reactivate TILs that then exited to attack distal tumors. This study supplies further evidence that TCR signaling for T cell reactivation in the TME is defective but can be rescued by proper exogenous signals.

As a potent lymphocyte activator, interleukin-2 (IL-2) is an FDA-approved treatment for multiple metastatic cancers. However, its clinical use is limited by short half-life, low potency, and severe in vivo toxicity. Current IL-2 engineering strategies exhibit evidence of peripheral cytotoxicity. Here, we address these issues by engineering an IL-2 prodrug (ProIL2). We mask the activity of a CD8 T cell-preferential IL-2 mutein/Fc fusion protein with IL2 receptor beta linked to a tumor-associated protease substrate. ProIL2 restores activity after cleavage by tumor-associated enzymes, and preferentially activates inside tumors, where it expands antigen-specific CD8 T cells. This significantly reduces IL-2 toxicity and mortality without compromising antitumor efficacy. ProIL2 also overcomes resistance of cancers to immune checkpoint blockade. Lastly, neoadjuvant ProIL2 treatment can eliminate metastatic cancer through an abscopal effect. Taken together, our approach presents an effective tumor targeting therapy with reduced toxicity.

Bispecific T-cell engagers (BiTEs) preferentially targeting tumour-associated antigens and stimulating CD3-mediated signalling are being used in patients to treat acute B-cell lymphoblastic leukemia. However, the potency of BiTEs in solid tumours is limited by their short half-life and their severe toxicity at relevant therapeutic doses. Here we report the design and in vivo performance of a bispecific antibody that simultaneously targets the murine T-cell co-receptor CD3ε and the murine immune checkpoint programmed-death ligand 1 (PD-L1). In multiple syngeneic tumour models, the bispecific antibody generated higher antitumour immune responses than conventional BiTEs targeting tumour-associated antigens and CD3ε. We found that the durable antigen-specific T-cell responses resulted from the rejuvenation of CD8 T cells, owing to the blockade of PD-L1 on dendritic cells (but not on tumour cells) and co-stimulation by B7-1&2 (a peripheral membrane protein on dendritic cells). Bispecific T-cell engagers targeting dendritic cells rather than tumour cells may represent a general means of T-cell rejuvenation for durable cancer immunotherapy.

The single-cell RNA seq data of day 10 and day 20.

Bispecific T-cell engagers (BiTEs) that preferentially target tumor-associated antigens (TAA) to reengage CD3 signaling have been approved to treat acute B-cell lymphoblastic leukemia. However, their applications in solid tumors have been hampered due to short half-life, weak anti-tumor activity, and severe toxicity at therapeutic doses. To explore new targets, we designed a bispecific antibody (BsAb) which simultaneously targets CD3 and immune checkpoint PD-L1. Compared with conventional TAA based targeting, PDL1xCD3 generates far superior anti-tumor immune responses in vivo. Mechanistically, blockade of PD-L1 on dendritic cells instead of tumor cells can potently rejuvenate preexisting tumor reactive CD8 T cells in a B7-1/2 dependent manner for a durable anti-tumor responses. This study argues that targeting DC-T cell instead of current tumor-T cell can achieve much better T cell rejuvenation in BsAb therapy.