Tα-1 promotes splenic cDC1 maturation during the phagocytosis of ABs.

Table S1. Candidate miRNAs and their sequencing information Table S2. RNA and miRNA quality metrics of input and IP samples Table S3. Baseline characteristics.

Tα-1 inhibits RNase A-mediated miR146a-5p cleavage to promote DC maturation.

BACKGROUND:Relapsed or refractory (R/R) B-cell non-Hodgkin's lymphoma (B-NHL) remains a major therapeutic challenge despite CD19-directed chimeric antigen receptor (CAR) T-cell therapies, with treatment failure often driven by antigen escape and limited CAR-T persistence. Dual targeting of CD19 and CD20 may mitigate antigen loss. We conducted an expanded phase I/II study of bispecific CD19/20 CAR-T cells and incorporated spatially resolved single-cell transcriptomics to evaluate tumor-intrinsic and microenvironmental factors of clinical response. METHODS:Patients with R/R B-NHL received CD19/20 CAR-T cells following lymphodepletion. Efficacy, safety, CAR-T expansion/persistence, and B-cell reconstitution were assessed. Pretreatment tumor biopsies from five patients with divergent outcomes underwent spatial single-cell transcriptomic profiling. RESULTS:32 patients were treated, including 24 with diffuse large B-cell lymphoma. Among 31 evaluable patients, the best overall response rate was 74%, including 58% achieving complete remission. Median progression-free and overall survival were 6.8 and 22.1 months, respectively. Response rates were higher in patients with normal lactate dehydrogenase. CAR-T expansion peaked on days 7-17, and persistence exceeded 500 days in long-term responders. Cytokine release syndrome occurred in 53% (12% grade ≥3) and immune effector cell-associated neurotoxicity syndrome in 9% (all grade 3), with no lasting deficits. Spatial profiling identified two dominant tumor architectures: (1) a B-cell-dominant phenotype, in which durable remission was associated with heightened apoptotic competence in malignant B cells, and (2) a fibroblast-enriched and monocyte/macrophage-enriched phenotype, in which response correlated with a chemokine-rich, T-cell-permissive microenvironment. CONCLUSIONS:Bispecific CD19/20 CAR-T therapy produced durable clinical activity with manageable toxicity. Spatial single-cell analysis reveals distinct tumor-intrinsic and microenvironmental features associated with CAR-T responsiveness, providing a spatially informed framework for understanding heterogeneous therapeutic outcomes. TRIAL REGISTRATION NUMBER:NCT04723914.

CRISPR‐Cas13d system decreased miR146a-5p expression.

Tα-1 facilitates migration of cutaneous cDC1s during the phagocytosis of ABs.

Diffuse large B-cell lymphoma (DLBCL) exhibits 30–40

Deletion of TLR7 impairs Tα-1-mediated DC maturation when phagocytosis of ABs.

Abstract Chimeric antigen receptor (CAR) T-cell therapy has become a promising clinical approach against hematologic malignancies, but patients receiving CAR T-cell therapy still present inconsistent clinical outcomes and are complicated by incomplete tumor eradication. The gut microbiome has shown a strong correlation with the therapeutic outcomes of CAR T-cell therapy. However, the underlying mechanism of how gut microbiota affect CAR T-cell therapeutic potency remains undetermined. In this study, we established a syngeneic CD19+ murine lymphoma model that allows for the evaluation of both endogenous immune cells and gut microbiota following CD19–CD28ζ CAR T-cell therapy. Using single-cell transcriptomic analyses, we report that CAR T-cell infusion led to the activation of peripheral and gut-infiltrating endogenous CD8+ T cells toward an effector-like phenotype. In parallel, 16S RNA sequencing revealed substantial alterations in gut microbiota after infusion. The composition of gut bacteria was associated with the activation status of endogenous CD8+ T cells and responsiveness to CAR T-cell therapy. More specifically, we identified gut bacteria strains Turicibacter and Parvibacter as critical determinants of effective CAR T-cell treatment. Supplementation with these species of gut bacteria during CAR T-cell therapy led to superior antitumor efficacy. Furthermore, both strains facilitated CAR T-cell therapy–induced activation of endogenous CD8+ T cells, enhancing their capability to express activation-associated surface markers as well as tumor lysis potency. In summary, our results demonstrate that the gut microbiome plays an essential role in endogenous immune activation after CAR T-cell therapy and provide specific targets for therapeutic interventions.

The immune-excluded tumor immune microenvironment (TIME) limits responses to ICIs. Cancer-associated fibroblasts are the most abundant stromal population and key regulators of immune suppression; however, the upstream cues that program pathogenic CAF states and the mechanisms of the immune-excluded TIME remain poorly defined. Here, by combining single-cell RNA sequencing and functional validation, we report that tumor cell-released autophagosome (TRAP) programs inflammatory CAFs (iCAFs) and triggers cathepsin L-dependent intracellular cleavage of C3 into C3a via the HSP70-TLR4-MyD88-ERK/p38 pathway. iCAF-derived C3a affects C3a on TAMs, promotes TAM accumulation in the iCAF-rich stroma, limits TIL trafficking into tumor nests, and reinforces an immune-excluded TIME. Disrupting the TRAP-iCAF-C3a/C3aR axis remodels the immune-excluded TIME and sensitizes tumors to anti-PD-L1 therapy. In clinical cohorts, plasma TRAP and C3a levels increased with disease stage, and their combination improved the discrimination of patients with breast cancer from controls (AUC = 0.96). These data define a TRAP-driven stromal-immune circuit that promotes immune exclusion and suggest that the C3a-C3aR axis is a potential target for enhancing ICI efficacy.

Tumor appearance and weight measurements in mouse transplantation model.

AB-borne ssRNAs are required for Tα-1 to promote DC maturation.

Tα-1 does not exacerbate DDP-induced liver or kidney toxicity.

Tα-1 facilities DCs maturation by AB-borne miR146a-5p.

Tα-1 augments DCs-mediated T-cell activation through a ssRNA-TLR7 axis.

Tα-1 promotes AB-pulsed DC maturation during the uptake of ABs.

Abstract Chemotherapy induces cancer cell apoptosis and the release of apoptotic bodies (AB) that are poorly immunogenic or immunosuppressive, creating a major barrier to the success of coadministered or second-line immunotherapies. In this study, we found reduced circulating levels of thymosin alpha-1 (Tα-1), a key endogenous peptide hormone with immunomodulatory activity, after chemotherapy treatment in patients with multiple types of cancer and in mice bearing established tumors. Tα-1 bound to tumor ABs and interacted with AB-borne microRNAs, including miR-146a-5p, following the phagocytosis of ABs into the endolysosomal compartment of dendritic cells (DC). The interaction with Tα-1 protected miR-146a-5p from lysosomal RNase A–mediated degradation, allowing miR-146a-5p–mediated activation of Toll-like receptor 7 (TLR7) that licenses DC maturation, migration to tumor-draining lymph nodes, and presentation of tumor antigens to activate tumor-specific CD8+ T cells. Therapeutic Tα-1 supplementation produced strong synergy with chemotherapy to control established tumors in mice with high miR-146a-5p expression in a TLR7-dependent manner. These findings establish Tα-1 as a pivotal endogenous microRNA chaperone that unlocks a critical limiting step of DC licensing, empowering robust antitumor immunity after chemotherapy. Significance: Tα-1 promotes miR146a-5p-mediated TLR7 signaling in dendritic cells to enhance chemotherapy-induced immunity, offering a foundation for developing Tα-1-based approaches to elicit or amplify immune responses against diverse tumors.

Tα-1-promoted DC maturation in vivo is associated with AB-borne ssRNAs.

Tα-1 combined with DDP enhances anti-tumor immunity in melanoma mice.

Although chemotherapy-free regimens have improved initial remission rates in patients with newly diagnosed Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph + B-ALL), outcomes for relapsed or refractory (R/R) Ph + B-ALL remain poor. This study reports the efficacy and safety of CD19 chimeric antigen receptor (CAR) T-cell therapy in R/R Ph + B-ALL. This study retrospectively analyzed 93 patients with R/R Ph + B-ALL who received CD19 CAR T-cell therapy across China between August 2015 and March 2024. We evaluated the overall response rates, long-term efficacy, safety, and prognostic factors associated with CD19 CAR T-cell therapy. Complete remission (CR) or CR with incomplete hematologic recovery rate was 87.1