Chimeric antigen receptor T (CAR-T) cell therapy has transformed the treatment of B-cell malignancies, but its application to acute myeloid leukemia (AML) remains limited by heterogeneous antigen expression and the scarcity of leukemia-restricted targets. CD38 and CD123 are expressed across overlapping but non-identical AML compartments; combined recognition may therefore broaden target coverage. We generated two reciprocal loop-structured CD38/CD123 CARs, Loop 123 × 38 CAR and Loop 38 × 123 CAR, and compared them with the monovalent CD38 CAR and CD123 CAR. Two public single-cell RNA-sequencing datasets, GSE241989 and SciLifeLab 23715648, were reanalyzed to assess CD38 and IL3RA expression across malignant cell states, individual AML samples, molecular subtypes, and normal hematopoietic progenitor compartments. Antileukemic activity was evaluated against CD38/CD123-expressing AML cell lines and primary AML samples by cytotoxicity, degranulation, and cytokine assays. CAR-T cell phenotype, T cell numbers, apoptosis, and subset-specific CD38 expression were also examined. Reciprocal single-antigen-loss variants were tested in short-term assays and in a mixed-target co-culture assay followed through day 12. Antitumor efficacy was assessed in cell line-derived, patient-derived, and mixed-antigen-loss xenograft models. Hematopoietic toxicity effects were evaluated by colony-forming assays using human umbilical cord blood (UCB) CD34+ progenitors. Across the two single-cell datasets, CD38 and IL3RA showed substantial interpatient heterogeneity and partially complementary transcript coverage. Longitudinal analysis of the CAR-T cell products showed altered expansion and apoptosis in CD38-targeting products, together with selective depletion of CD38-expressing cells within the live GFP+ CAR-T cells. Both loop CAR-T cells recognized both CD38 and CD123 and showed activity against CD38+CD123+ AML cell lines, primary AML samples, and reciprocal single-antigen-loss cells. During the 12-day mixed-target co-culture, the monovalent CAR-T products could not recognize the corresponding single-antigen-loss population, resulting in accumulation of this uncontrolled population, whereas both loop CAR-T products suppressed the outgrowth of both reciprocal antigen-loss populations. Loop CAR-T cell treatment reduced leukemia burden and prolonged survival in xenograft models. In colony formation assay of UCB-derived CD34+ progenitors, there was no significantly reduced colony formation after 24 h co-cultured with either loop CAR-T cells. However, after co-culturing with CAR-T cells for 72 h, the Loop 123 × 38 CAR-T cell treatment group showed a decrease in BFU-E and CFU-GM formation, while Loop 38 × 123 CAR-T cells did not show a significant decrease compared to that of vector-T cells. BFU-E and CFU-GM formation was higher in Loop 38 × 123 CAR-T co-cultured group than that in Loop 123 × 38 CAR-T co-cultured group (P < 0.01 and P < 0.0001, respectively). CD38/CD123 dual recognition retained activity in antigen-heterogeneous AML models, including reciprocal single-antigen-loss populations. Among the two loop configurations, Loop 123 × 38 CAR-T cells showed a stronger overall antileukemic profile, but this was accompanied by greater suppression of BFU-E and CFU-GM formation after prolonged exposure.
Tertiary lymphoid structures (TLSs) are associated with improved anti-tumor immunity and favorable clinical outcomes across multiple cancer types. Therapeutic induction of TLSs is a promising strategy to stimulate potent anti-tumor immunity in cancer patients. However, the impact of tumor heterogeneity on therapeutic TLS induction remains poorly understood. Using four pancreatic cancer models derived from KPC mice, we examined the effect of combined STING and LTβR stimulation on TLS induction. Although treatment elicited variable anti-tumor responses across tumor models, robust TLS formation was observed only in KxPxCx tumors. Comparative transcriptomic analyses demonstrated that TLS-permissive KxPxCx tumors exhibited distinct inflammatory and stromal remodeling programs, including enrichment of fibroblastic, extracellular matrix remodeling, wound-healing, and TGFβ-associated pathways. Despite these differences in transcriptional profiles, the expression of STING- and LTβR-associated proteins and direct tumor cell responsiveness to stimulation did not explain the selective capacity for TLS formation. These findings demonstrate that pancreatic tumor heterogeneity profoundly influences responsiveness to therapeutic TLS induction and identify distinct transcriptional characteristics associated with a TLS-permissive tumor microenvironment.
Colorectal cancer liver metastasis (CRLM) is shaped by interactions between malignant epithelial plasticity and the immune microenvironment, yet the epithelial programs involved remain poorly defined. Single-cell RNA sequencing data from primary colorectal tumors and liver metastases were integrated to characterize epithelial and immune heterogeneity. Epithelial subclustering, pseudotime analysis, functional enrichment, CellChat-based communication inference, transcription factor screening and scTenifoldKnk virtual knockout analysis were performed to identify regulatory circuits. Spatial transcriptomics data from GSE225857 were used to assess the tissue-level organization of the JUN-MIF-CD74 axis. TCGA-COAD and GSE39582 cohorts, in vitro JUN knockdown and overexpression experiments, ChIP-qPCR, dual-luciferase reporter assays, ELISA, functional assays, immunohistochemistry of clinical CRLM specimens and in vivo MC38-based syngeneic subcutaneous tumor and intrasplenic liver metastasis models were used for validation. We identified a secretory epithelial state, Epi_4, characterized by SPINK4, TFF3, FCGBP and REG4 expression and enriched in oxygen-response and mitochondrial programs. JUN emerged as an Epi_4-associated regulator, with MIF identified as a major downstream candidate. Cell–cell communication analysis identified predicted MIF–CD74-related interactions between Epi_4 and myeloid populations, while exploratory spatial transcriptomic analysis suggested more prominent JUN–MIF–CD74 co-enrichment in the analyzed primary colorectal tumor sections than in liver metastatic sections. JUN bound the MIF promoter and increased MIF expression and secretion. JUN overexpression enhanced wound closure, cell viability and clonogenicity, whereas JUN knockdown produced opposite effects. In vivo, Jun knockdown suppressed syngeneic tumor growth and experimental liver metastasis and reduced Mif expression. Clinical analyses further supported a positive association between JUN and MIF. These findings support an Epi_4-associated JUN-dependent MIF transcriptional regulatory program and predicted MIF–CD74-related epithelial–myeloid interactions in colorectal cancer. Although JUN perturbation affected tumor-associated phenotypes and MIF expression, whether MIF functionally mediates these phenotypes remains unresolved, and the predicted MIF–CD74-related epithelial–myeloid interactions require further functional validation. The associated spatial signals were more prominent in primary colorectal tumors than in established liver metastases, suggesting a potential association with metastatic competence rather than liver-metastasis-specific activation.
Melanoma remains a major global health burden, with more than 510,000 new cases and 96,000 deaths projected worldwide by 2040. Over the past decade, neoantigen-based cancer vaccines have emerged as a promising immunotherapeutic strategy for the treatment of melanoma. Nevertheless, beyond the challenges associated with antigen delivery, an important unanswered question is whether combining multiple neoantigens within a single vaccine construct maintains neoantigen-specific T-cell recognition of individual neoepitopes. To address this issue, we developed a nanovaccine platform based on polyethyleneimine–poly(lactic-co-glycolic acid) (PEI-PLGA) nanoparticles (NPs) encapsulating patient-specific multiepitope neoantigen peptides and coated with poly(I:C), enabling the delivery of multiepitope neoantigen peptides and adjuvant within a single formulation. Following physicochemical characterization, their immunogenic potential was assessed ex vivo using peripheral blood mononuclear cells (PBMCs) isolated from patients with melanoma. Activation-induced marker (AIM) assays and IL-2 cytokine secretion were used to assess the effects of delivering multiepitope neoantigens and poly(I:C), which significantly enhanced T-cell activation in two of the three tested patients. Importantly, nanovaccine stimulation elicited immune responses against the 15mer-epitope melanoma-associated antigen using ELISPOT. Analysis using the individual three individual peptide epitopes identified pre-existing immune recognition of specific regions contained preferential immune recognition of specific regions within the multiepitope. These findings demonstrate immune recognition of the multiepitope antigen delivered by the nanovaccine and identify the peptide region(s) most frequently recognized within this sequence, but do not establish induction or maintenance of peptide-specific responses. Collectively, these findings demonstrate that multiepitope PEI-PLGA nanovaccines allow neoantigen-specific T-cell responses while evaluating baseline T-cell reactivity against individual neoantigens, supporting their development as a personalized immunotherapeutic strategy for melanoma.
Immune checkpoint inhibitors (ICIs) targeting CTLA-4 and PD-1 improve cancer outcomes but can disrupt peripheral tolerance, triggering immune-related adverse events (irAEs). While T-cell-mediated mechanisms have been extensively studied, the contribution of B cells remains incompletely understood. We performed phenotypic, functional, and signaling analyses of peripheral B cells in melanoma (PREDIMEL) and pan-cancer (AMICI) cohorts. PREDIMEL included 37 patients in PRED_1 (22 with irAEs; 23
