Adoptive Immunotherapy Research Articles (Page 1)

Regulatory T cells (Tregs) play a crucial role in the immune system, and their dysfunction can lead to the development of autoimmune conditions. In cancer, tumors frequently hijack the immunosuppressive function of Tregs to evade immune responses. Due to their central role in key pathological processes, Tregs have gained increasing attention as promising targets for various clinical applications. However, their relative scarcity (∼5-10% of CD4+ T cells) and instability presents a technical challenge for research and therapeutic development. In congenic animal models used to investigate autologous cell transfer based therapies, this challenge is even greater, as Treg donor animals may only be able to provide cells to a small number of recipient mice. Here we present an optimized protocol for ex vivo editing and expansion of mouse Tregs (mTregs). Since a recent study demonstrated the anti-cancer potential of SRC-3 KO mTregs, we use them here as a case study.

Background Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TILs) is a personalized immunotherapy that has shown promising clinical results in various tumor types. Although TILs are associated with improved survival in patients with ovarian cancer (OC), their therapeutic efficacy remains limited. Therefore, novel strategies to enhance the anti-tumor activity of TILs are needed to improve outcomes in OC treatment. Methods Single cells were isolated from tumor tissues of patients with high-grade serous carcinoma (HGSC) and expanded for 14 days in the presence of IL-2 under four different conditions: (1) control (W), (2) PD-1 antagonist (WI), (3) PD-1 antagonist + IL-15 + IL-21 (WIO), and (4) PD-1 antagonist + IL-15 + IL-21 + GITR-agonist (WIOG). Following validation of TIL purity and activation phenotypes by flow cytometry, RNA sequencing was performed to elucidate the underlying mechanisms. In vitro efficacy was assessed using a 7-AAD/Far-Red cytotoxicity assay against autologous tumor cells, and in vivo efficacy was evaluated in NSG mice bearing subcutaneous patient-derived tumor cell xenografts (PDCX). Results On day 14, the WIOG group showed a 1.3-fold increase in expansion compared to the control group, along with a high CD8 + /Treg ratio (454.6). Furthermore, both CD8 + and CD4 + T cells in the WIOG group exhibited elevated Granzyme B expression. RNA sequencing identified 279 upregulated genes associated with T cell activation ( CSF2, TNFRSF4 ), cytotoxicity ( IFNG, GZMB ), and anti-apoptosis ( BMF, BCL2L1 ). Compared to the controls, the WIOG group demonstrated a 1.9-fold increase in cytolytic activity in vitro and a 56% reduction in tumor growth in the patient-derived tumor cell xenograft (PDCX) model. Conclusions Taken together, we demonstrated that the addition of an agonistic GITR antibody during the early phase of TIL culture increased the CD8 + T cell to Treg cell ratio and enhanced anti-tumor T cell immunity. Enhancing TILs with a GITR agonist may be beneficial for improving the clinical outcomes of TIL-based ACT in OC.

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Neonatal transfer of immature dendritic cell-enriched Flt3L splenocytes significantly reduces the incidence of type 1 diabetes in female NOD mice. Early time points are associated with accumulation of anergic T cells. In adult mice, there is a reduction in CD4 T helper 1 cells and reduced proliferation and perforin of CD8 T cells. Our work demonstrates how targeting the neonatal window of tolerance alters autoimmunity outcome.

Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) can achieve durable responses in patients with metastatic cancers, but the long-term clonal dynamics after multiple administration and synergy with checkpoint blockade remain understudied. We present a longitudinal case study of a patient with treatment-refractory metastatic prostate cancer that achieved complete and durable tumor remission over 5-years after multiple TIL infusions and anti-PD-1 therapy. We performed longitudinal high-throughput T-cell receptor (TCR) sequencing on blood and tumor samples collected over five years to track the persistence and dynamics of TIL-derived and endogenous clonotypes. TIL-derived clonotypes exhibited sustained persistence in blood, with notable clonal expansions correlating with reduced repertoire diversity, increased clonality, and observed clinical response. Multiple TIL administration increased the patient exposure to the therapy, improving its pharmacokinetics profile over time. The third TIL infusion was followed by pembrolizumab administrations, which coincided with the re-expansion of TIL-derived clonotypes and emergence of novel clones. Serial tracking revealed clonotype stability for up to five years post-treatment. Our findings provide insights into the long-term persistence and reactivation of TIL-derived immunity and illustrate the potent synergy between adoptive transfer and PD-1 blockade by enhancing both infused and endogenous tumor-reactive T cell responses, and supporting the integration of longitudinal immunogenomic monitoring in personalized immunotherapy.

Allogeneic Hematopoietic Stem Cell Transplant (allo-HCT) is an effective treatment for high-risk or relapsed acute leukemia. However, the frequent occurrence of graft-versus-host disease (GVHD) poses significant complications. Modifiable factors such as the gut microbiome and dietary regimen have the potential to influence the frequency and severity of GVHD. Previous studies in mouse models have shown a direct link between obesity and increased severity of GVHD. Analysis of human data has not suggested a causal relationship, however. We hypothesized that dietary fat content prior to transplantation, rather than obesity itself, might affect outcomes, given that increased dietary fat is associated with reduced gut microbiome diversity. In our study, we evaluated the effects of an animal fat-based diet (AFD) and a plant fat-based diet (PFD) in a mouse model of sclerodermatous chronic GVHD (Scl-cGVHD). Mice initially fed normal chow (~10% kcal from fat) were switched to either AFD or PFD (each with ~25% kcal from fat) two weeks before allo-HCT. Mice on the AFD had an elevated serum cytokine response, more severe skin inflammation, and greater intestinal immune dysregulation compared to mice on the PFD. Although both diets reduced gut microbial diversity, the fat source led to distinct microbiota compositions that included two co-abundance guilds (CAGs) that differentiated between the AFD and PFD groups. Our findings demonstrate that even short-term consumption of diets with equivalent macronutrient content, but different fat sources, can modulate the gut microbiome, disrupt intestinal homeostasis, and influence Scl-cGVHD outcomes.

Adoptive cell therapy using ex vivo expanded autologous Tregs could be a novel therapeutic approach for cell-based immunotherapy in dogs. This study aimed to expand dog Treg lymphocytes via the use of tregitopes. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy beagle dogs and stimulated with peptides: human tregitope EEQ; two potential canine tregitopes, EQF and PSV; and whole canine IgG in primary cultures. In addition, lymphocytes were simultaneously stimulated with peptides and canine vaccine antigens. The frequencies of Treg lymphocytes (CD4 + CD25 + Foxp3+, CD4 + Foxp3+) and activated lymphocytes (CD4 + CD25+) were determined by flow cytometry. A statistically significant increase in the frequency of CD4 + Foxp3+ and CD4 + CD25 + Foxp3+ lymphocytes stimulated with PSV and EQF peptides and canine IgG was observed. No increase in the Treg lymphocyte frequency occurred after EEQ Tregitope stimulation or vaccine antigen costimulation. Canine Treg lymphocytes isolated from peripheral blood are sensitive to stimulation with peptides with potential tregitope properties derived from canine proteins.

Xenotransplantation of pig hearts may help address the current human shortage of human donors once rejection is controlled. One innovative approach to combat rejection in humans is the use of regulatory cell (RC) therapy. The term RC refers to all cell populations that share immunosuppressive functions. The use of RC, including mesenchymal stem cells (MSC) and CD4+CD125lowCD25highFoxp3+ T cells (Treg), may potentially reduce or eliminate the need for chronic general immunosuppression (IS). This approach is hypothesized to act by augmenting suppressive immune mechanisms that maintain tolerance by prevailing over the immune effector mechanisms responsible for rejection. Increasing RC numbers through adoptive cell transfer (ACT) and enhancing their functions via chimeric antigen receptor (CAR) technology are two promising strategies for RC therapy applications. During the various steps of rejection, monitoring specific biomarkers can guide the use of the corresponding RC subpopulation, preferably available off-the-shelf, either alone or in combination, administered once or multiple times. In the future, exosomes or RC-derived active molecules (or their antagonists) may supplement or replace whole-cell therapy. With further research, RC therapy, which has not yet been used in clinics to induce functional tolerance to pig heart xenotransplants in humans, has the potential to become a routine, personalized treatment.

Cancer cells comprise a significant proportion of the tumor microenvironment (TME) and often have compromised expression or repression of cyclic GMP-AMP (cGAMP) synthase (cGAS), which prevents effective stimulation of interferon genes (STING) signaling. Here, we leverage the cancer cells and hijack their cellular machinery for increased production of cGAMP, differing from conventional strategies whereby synthetic STING agonists are delivered to immune cells in the TME as a bolus dose, are rapidly cleared and can cause systemic toxicity. Increasing evidence suggests that cGAMP derived from cancer cells can act on proximal immune cells, activating STING, contributing to an antitumor immune response. We used lipid nanoparticles (LNPs) to deliver mRNA coding for cGAS which catalyzes the production of cGAMP. We observed dramatic increases in extracellular and intracellular cGAMP when cancer cells were transfected with cGAS mRNA and genomic DNA, the substrate for cGAS. We confirmed that cGAS and cGAMP are functional due to activation of immune cells, through a combination of extracellular transfer and cell-cell contact mechanisms. Treatment of syngeneic murine melanoma with cGAS LNPs reduced tumor growth significantly and further benefit was observed upon combination with immune checkpoint blockade (anti-PD-1). Moreover, we found increased activation in CD8+ T cells, NK cells, macrophages, and dendritic cells in the TME post treatment with cGAS LNPs. These findings highlight how cancer cells can be used to actively contribute to their own elimination and may be a broadly applicable strategy for delivery of other reprogramming molecules to cancer cells and wider therapeutic combinations.

Local Single-Dose Radiation Improves Adoptive Cell Therapy With Tumor-Infiltrating Lymphocytes.

Adoptive transfer of NK cells engineered with a CD5-based chimeric antigen receptor (SRCD5CAR) to treat invasive fungal infections.

Tumor-targeted delivery of CXCL10 by mesenchymal stromal cells potentiates adoptive T cell therapy to treat solid tumors.

The extracellular accumulation of adenosine is a central mechanism of immune evasion within the tumor microenvironment. Elevated adenosine levels-driven by hypoxia, chronic inflammation, and upregulated ectonucleotidase activity, primarily through ectonucleoside triphophate diphosphoydrolase 1 and ecto-5'-nucleotidase-induce profound immunosuppression and promote tumor progression. In this setting, adenosine acts mainly through 2 G protein-coupled receptors, the adenosine A2A receptor (A2AAR) and the adenosine A2B receptor (A2BAR), which modulate diverse immune and stromal cell populations. A2AAR signaling suppresses the effector activity of cytotoxic T lymphocytes and natural killer cells, whereas A2BAR activation exerts broader effects by amplifying myeloid-derived immunosuppression, driving stromal remodeling, and fostering angiogenesis and metastatic dissemination. This review provides a comprehensive overview of the distinct and converging roles of A2AAR and A2BAR in immune, stromal, and tumor compartments. We critically analyze current strategies for developing selective and dual A2AAR/A2BAR antagonists, with a focus on structure-activity relationships, scaffold optimization, and pharmacokinetic profiling. In addition, we examine ongoing clinical trials and emerging combination therapies involving A2AAR and A2BAR antagonists in conjunction with immune checkpoint inhibitors, adoptive cell therapies, enzymatic axis blockade, radiotherapy, and classical chemotherapy. We also underscore the therapeutic potential of dual A2AAR/A2BAR antagonists as a multitarget approach to counteract overlapping immunosuppressive mechanisms. Overall, targeting the adenosine axis-particularly through dual receptor blockade-represents a promising strategy for reprograming the tumor microenvironment, reinvigorating antitumor immunity, and improving the efficacy of cancer immunotherapy. SIGNIFICANCE STATEMENT: Adenosine signaling via adenosine A2A (A2AAR) and A2B (A2BAR) receptors plays a central role in tumor-induced immunosuppression, limiting the efficacy of cancer immunotherapy. This review provides an integrated analysis of A2AAR and A2BAR functions across immune and stromal compartments, summarizes current selective antagonists (A2AAR and A2BAR) and dual antagonists, and highlights compounds in clinical studies. Moreover, it discusses synergistic combination strategies that integrate adenosine blockade with complementary immunotherapeutic and conventional approaches to enhance antitumor responses.

This survey reports on the current practice and management of hemorrhagic cystitis (HC) among the EBMT centers. The survey response rate was 27%. The diagnostic search of BK polyomavirus (BKPyV) was available in 93% of centers and performed in urine (100%), blood/plasma (51%), and serum (12%). Patient screening for BKPyV infection was performed in 14% of centers before hematopoietic cell transplantation (HCT). In comparison, surveillance for BKPyV infection was performed after HCT in 39% of centers, mainly with a frequency of 1-2 times/week. Preventative measures are based mainly on hyperhydration and sodium 2-mercaptoethanesulphonate (Mesna), while the use of bladder catheter is limited; moreover, 23% of centers used quinolones prophylaxis.The primary therapeutic interventions are hyperhydration, Mesna, analgesics, and sustained platelet transfusions; bladder irrigation by urinary catheter is adopted by 47-50% of centers. In the case of BKPyV infection, 85% of centers use intravenous or intrabladder administration of cidofovir at a dosage ranging from 1 to 5 mg/kg. Centers report a miscellaneous range of alternative therapeutic measures (adoptive immunotherapy, hyperbaric oxygen therapy, fibrin glue/platelet-rich plasma, leflunomide, sodium hyaluronate) and the need for invasive procedures. This survey confirms that the management of HC needs further education, clinical research, and innovation.

Oncolytic viruses (OVs), a kind of emerging therapeutics for treating tumors, are characterized by high replication efficiency, superior killing effects, and few adverse reactions, which have shown great application prospects in preclinical tumor treatment trials. To overcome the limitations of OV monotherapy, recent studies have found that combination therapy with other anti-tumor therapeutics, especially with immunotherapy, yields promising outcomes in tumor eradication. Due to the advancements in genetic engineering, the combination of OVs with novel immunotherapy, including cellular immunotherapy, adoptive cellular immunotherapy, immune checkpoint inhibitors, cancer vaccines, cytokines, and bi- or tri-specific T cell engagers, has greatly improved clinical outcomes and quality of life of tumor patients. In this review, we systematically summarize the latest progress of OVs combined with immunotherapy in tumor treatment and highlight the future directions of the combination strategies, which will promote the clinical application of OVs in tumor therapy.

Tumour-infiltrating lymphocyte therapy comes of age in the era of genetic engineering.

Microbiota-derived butyrate potentiates MSLN CAR-T cell therapy by metabolic reprogramming and extracellular matrix remodeling.

Cellular immunotherapy has revolutionized the treatment of hematologic malignancies yet has had limited success in the solid tumor microenvironment (TME). While insufficient nutrients can lead to T cell metabolic stress in the TME, the glutamine antagonist DON can paradoxically enhance antitumor immunity. Because DON inhibits both essential and nonessential enzymes whose impairment may contribute to dose-limiting toxicities, mechanisms underlying DON-induced antitumor activity have remained unclear. Here, we aimed to identify specific DON targets that increase T cell antitumor activity and test if more selective inhibition of glutamine metabolism could replicate the effects of DON with reduced toxicity. CRISPR screening in the TME of DON-relevant glutamine metabolizing enzymes identified some targets that were essential in tumor-infiltrating CD8 T cells, but that tumor-infiltrating CD8 T cells lacking the DON target glutamine synthetase (GS) were enriched. Upon adoptive T cell transfers, GS-deficient CD8+ T cells displayed improved survival, a higher proportion TCF-1+ Tox- stem-like cells, and greater antitumor and memory function. GS converts glutamate to glutamine and GS-deficient cells exhibited increased intracellular glutamate and reduced glutathione levels, which correlated with enhanced mitochondrial respiration and resistance to reactive oxygen species. Pharmacological inhibition of GS reduced tumor burden in multiple orthotopic murine tumor models in a manner dependent on adaptive immunity. Our findings establish GS as a key metabolic regulator of CD8+ T cells stress resilience in the TME. By preserving intracellular glutamate, GS inhibition reprograms T cells for improved survival and function, offering a promising therapeutic strategy to enhance immune-based cancer treatments.

Natural killer (NK) cells mediate anti-tumor immunity through integrated signaling of inhibitory and activating receptors. The efficacy of NK cell adoptive transfer therapy varies among patients due to heterogeneous receptor-ligand expression. This study aimed to develop a predictive model based on receptor-ligand interactions to determine NK cells' therapeutic effects. Through analyses of receptor-ligand expression profiles of NK and tumor cells and assessment of NK cell cytotoxicity, we developed a machine learning-based random forest model using 11 key receptor-ligand pairs selected through database mining and experimental screening. Flow cytometry was used to obtain receptor-ligand profiles, and combined predictors were calculated for each pair. The model was validated using independent datasets and evaluated for generalizability across different tumor types. The model showed significant predictive performance, achieving an accuracy of 84.2% and an area under the curve (AUC) of 0.908 in ovarian cancer cohorts. This predictive capability was validated in both in vitro experiments and clinical samples, revealing complex non-linear interactions between receptor-ligand expression and NK cell killing efficacy. Cancer-specific ligand expression patterns were identified. While showing optimal performance in studied cancer types, it exhibited moderate applicability to other cancers and demonstrated potential compatibility with transcriptomic data for prediction. This model provides tools and foundations for the precise treatment of tumors using NK immune cells and may be applied in clinical practice.

Melanin-rich cytologic specimens, particularly those from melanocytic lesions, present diagnostic challenges due to pigment-induced obscuration of cellular details and interference with immunocytochemistry (ICC) interpretation. These limitations are especially pronounced in cell transfer preparations, which differ significantly from tissue sections in cellular distribution and density. Existing bleaching protocols are inconsistent, often incomplete in pigment removal and can compromise cellular morphology. This study aimed to develop and evaluate an automated platform incorporating optimised melanin bleaching, ICC and cytomorphologic staining to enhance diagnostic accuracy in heavily pigmented cytologic samples. Ten melanoma cell transfer smears were processed using an optimised protocol. Slides underwent melanin bleaching with 10% hydrogen peroxide at 60°C for 25 min, followed by automated ICC for Melan-A and SOX-10. Chromogenic detection was performed using either 3,3'-diaminobenzidine (DAB) or alkaline phosphatase (AP). In a parallel workflow, Papanicolaou (Pap) staining was performed after bleaching to assess cytomorphologic preservation. The integrated protocol completed bleaching and staining within 2 h, with bleaching effectively removing melanin pigment while enhancing nuclear and cytoplasmic visibility without compromising morphological detail. Post-bleaching Pap staining preserved cytologic features, enabling accurate morphological interpretation. Both markers exhibited strong, specific immunoreactivity with either chromogen; however, AP yielded superior contrast and clearer antigen localisation. In contrast, residual melanin occasionally masked DAB signals, limiting interpretability. This automated protocol, combining melanin bleaching, Pap staining and ICC, improves visualisation and diagnostic interpretation of melanin-rich cytologic specimens. The bleaching step preserves cellular and antigenic integrity, while AP chromogen provides enhanced clarity in the presence of residual pigment. This reproducible and practical workflow facilitates more accurate cytopathologic evaluation of melanocytic lesions.