Tsuneyasu Kaisho

DNA ligase IV (LIGIV or LIG4) is essential for DNA double-strand break (DSB) repair. Hypomorphic LIG4 variants cause LIG4 syndrome, characterized by growth disturbance, increased radiosensitivity, predisposition to malignancies, adaptive immunodeficiency, and inflammatory conditions. Most of these manifestations are recapitulated in hypomorphic Lig4 mutant mice. However, no model mice with defective DSB repair have consistently exhibited inflammation. Here, we generated mutant mice carrying the LIG4 missense variant, p.W447C, found in a patient with LIG4 syndrome. Lig4 ^W447C/W447C mice showed functional defects of LIGIV and manifested growth retardation, increased radiosensitivity, and life-threatening intestinal inflammation under severe adaptive immunodeficiency. The inflammation was dependent on lymphocytes and characterized by marked infiltration of Th1 cells and macrophages, along with elevated expression of IFN-γ–inducible genes. When Ifng was deleted, Th2 and Th17 instead of Th1 cells drove the inflammation. Single-cell RNA-seq analyses with TCR repertoire revealed that T cells from Lig4 ^W447C/W447C mice preferentially used proximal Vα and Jα segments in V regions of TCRα chains and exhibited expansion of several clonotypes, a substantial portion of which were CD4 ⁺ T cells expressing IFN-γ. Thus, our hypomorphic Lig4 mutant mice represent a unique model for studying Th1-skewed intestinal inflammation under severe adaptive immunodeficiency.

Depletion of CD4⁺ cells during an anti-tumor immune response promotes tumor regression and accelerates proliferation of tumor-antigen-specific CD8⁺ T cells in draining lymph nodes (dLNs). However, the effect of the depletion on inter-organ kinetics of antigen presentation and qualitative changes in dendritic cells (DCs) are not yet understood. Here, we established a novel approach for simultaneous detection of cellular movement and cell cycle phase in vivo by KikGR/Fucci mice and used it to examine migratory and LN resident DC (LNDC) dynamics after CD4⁺ cell depletion. We found that CD4⁺ cell depletion enhanced migration of CD11c⁺ MHC class II^high migratory DCs from tumor to dLNs and induced activation-associated phenotypic changes, such as increased MHC class II expression. Despite reduced overall cellularity in tumor dLNs, LNDC numbers were relatively maintained. Within the LNDC compartment, CD4 depletion increased the relative abundance of a CD8α⁺CD11b⁺ subset and promoted influx and cell-cycle activity among newly recruited LNDCs. Publicly available single-cell RNA sequencing data further delineated ligand–receptor expression relationships, including Flt3l–Flt3 and Csf2–Csf2ra/Csf2rb axes, within tumor dLNs. These findings reveal remodeling of DC migration, activation, and LNDC turnover within tumor dLNs in the context of CD4⁺ cell depletion, which enhanced anti-tumor immunity.

Antigen heterogeneity remains a major obstacle to the effective application of chimeric antigen receptor (CAR)-T cell therapy in solid tumors. We investigated the potential of epitope spreading as a strategy to overcome this limitation and examined whether CAR-T cells concomitantly producing IL-7 and CCL19 (7 × 19 CAR-T cells) could act as potent inducers of epitope spreading, as well as the underlying mechanisms. We used a murine model inoculated with a mixture of cancer cell lines-genetically modified to express the CAR target antigen-and their respective parental lines lacking target expression. Following administration of 7 × 19 CAR-T cells, flow cytometry and peptide stimulation assays were performed to evaluate the induction of tumor antigen-specific T cells and dendritic cells within tumor-draining lymph nodes and tumor tissues. We also evaluated the antitumor efficacy of 7 × 19 CAR-T cells derived from an allogeneic donor and their capacity to induce epitope spreading in vivo and ex vivo. In multiple tumor mixture models, 7 × 19 CAR-T cells demonstrated marked antitumor activity and promoted epitope spreading in murine solid tumor models, enabling endogenous T cells to recognize and target tumor-associated antigens. This response was dependent on cross-presentation by defined dendritic cell subsets in both the tumor microenvironment and tumor-draining lymph nodes within 2 weeks of 7 × 19 CAR-T cell administration. Notably, 7 × 19 CAR-T cells derived from allogeneic donors also induced epitope spreading in tumor-bearing hosts, thereby increasing survival. The 7 × 19 CAR system is a promising strategy for overcoming antigen heterogeneity in solid tumors by promoting epitope spreading.

Interleukin-17-producing γδT cells (γδT17 cells) play a dual role in immune regulation, serving as both protectors in various tissues and orchestrators of inflammatory responses in autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis. However, the ontology and repertoires of encephalitogenic γδT17 cells remain unclear. In this study, we demonstrate that the encephalitogenicity of γδT17 cells is conferred through microfold cell (M cell)-dependent uptake of commensal bacteria in Peyer’s patches. Specifically, CXCR6 ^hi Vγ6 ⁺ Vδ1 ⁺ invariant γδT17 cells are activated by specific commensal bacteria such as Lactobacillus spp., which stimulate TCR of CXCR6 ^hi Vγ6 ⁺ Vδ1 ⁺ invariant γδT17 cells. During the early stages of EAE, γδT17 cells infiltrate the central nervous system (CNS), initiating a type 17 inflammatory response. Our findings illustrate that Peyer’s patch M cells serve as a critical bridge, linking the pathological association between commensal bacteria and the onset of CNS inflammation.

Supplementary figures 1-5 Supplementary methods

The ability of conventional type-1 dendritic cells (cDC1) to cross-present tumor antigens to CD8+ T cells is critical for the induction of antitumor CTLs. Mice that are constitutively deficient in cDC1 cells have been reported to fail to respond to immunotherapy strategies based on checkpoint inhibitors. However, further work is needed to clarify the precise time during immunotherapy treatment that cDC1 cells are required for the beneficial effect of treatment. Here, we used a refined XCR1-DTR-Venus transgenic mouse model to acutely deplete cDC1 cells and trace their behavior using intravital microscopy. Diphtheria toxin–mediated cDC1 depletion prior to immunotherapy treatment with anti–PD-1 and/or anti-CD137 immunostimulatory mAbs completely ablated antitumor efficacy. The efficacy of adoptive T-cell therapy was also hampered by prior cDC1 depletion. After the onset of immunotherapy treatment, depletion of cDC1s only moderately reduced the therapeutic efficacy of anti–PD-1 and anti-CD137 mAbs. Intravital microscopy of liver-engrafted tumors revealed changes in the intratumoral behavior of cDC1 cells in mice receiving immunotherapy, and treatment with diphtheria toxin to deplete cDC1s impaired tumor T-cell infiltration and function. These results reveal that the functional integrity of the cDC1 compartment is required at the onset of various immunotherapies to successfully treat established tumors.Significance:These findings reveal the intratumoral behavior of cDC1 dendritic cells in transgenic mouse models and demonstrate that the efficacy of immunotherapy regimens is precluded by elimination of these cells.