Immune evasion remains a major barrier to effective immunotherapy in cancer. Coordinated activation of different immune cell types shapes immune responses within the tumor microenvironment (TME). In this study, we uncovered a spatiotemporal synergy between cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in tumors with low T-cell infiltration. An integrative multiomics approach combining spatial transcriptomics, single-cell RNA sequencing, and high-dimensional flow cytometry demonstrated that vaccination, capable of activating both innate and adaptive immunity, rapidly reshapes the TME. Early infiltration of IRF8+KLRG1+ NK cells established a chemokine-rich niche together with myeloid cells that promoted the recruitment of antigen-primed CXCR3+ CTLs. The accompanying inflammatory monocyte recruitment and dendritic cell activation led to the coordinated structural and chemotactic remodeling of the TME. Functionally, NK-CTL clustering at the tumor margin was associated with enhanced cytotoxic activity and sustained immune engagement. These spatially organized immune interactions involved CCR5-CCL5 and CXCR3-CXCL9 signaling pathways that coordinate communication between innate and adaptive immune compartments. Together, these findings reveal a previously unrecognized NK-CTL cooperative program that promotes the transition of poorly infiltrated tumors toward an inflamed, immune-responsive state. More broadly, this study illustrates how spatially resolved multiomics approaches can uncover immune interactions and provides a conceptual framework for designing next-generation immunotherapies that mobilize coordinated innate and adaptive immunity. SIGNIFICANCE:Early infiltration of NK cells followed by T cell accumulation reshapes the tumor microenvironment to promote antitumor immunity following vaccination, providing a mechanistic rationale for the development of effective vaccine-based immunotherapy strategies.

Tumor-associated macrophage (TAM) heterogeneity significantly influences the tumor microenvironment, positioning TAM inhibition as a promising anticancer strategy. Although several TAM subsets have been described, their functional roles remain unclear. In this study, we identified a distinct subset of CD9hiCD63hiCD206+Class IIlo hypoxic TAMs, located near tumors. These TAMs engage in trogocytosis, acquiring tumor membrane fragments, and cross-dress major histocompatibility complex (MHC)/tumor antigen epitopes. These processes facilitate their recognition by cytotoxic T lymphocytes, enhancing antitumor immune responses. We further found CH25H as a key regulator of TAM cross-dressing, with its inhibition associated with the activity of HIF1-α and VHL. These findings highlight the potential of modulating TAMs as an innovative immunotherapy strategy.

Table S1 The antibodies used in this study Table S2 The specific Primer sequences used for real-time PCR Table S3 TCRbeta deep sequencing of OVA-tetramer+ CD8 T cells in spleen and TIL

Figure S1. Characterization of aAVC-OVA Figure S2. Analysis of T cells in spleen and TIL after immunization of tumor-bearing mice with aAVC-OVA Figure S3. Phagocytosis and maturation of splenic dendritic cells after administration of aAVCs Figure S4. Confirmation of depletion of the XCR1+ subset of DCs in DT-treated XCR1-DTR-venus mice. Figure S5. Induction of an OVA-specific T cell response by aAVC-OVA in a dose-dependent manner

Uncontrolled type 2 immunity by type 2 helper T (Th2) cells causes intractable allergic diseases; however, whether the interaction of CD4 + T cells shapes the pathophysiology of allergic diseases remains unclear. We identified a subset of Th2 cells that produced the serine proteases granzyme A and B early in differentiation. Granzymes cleave protease-activated receptor (Par)-1 and induce phosphorylation of p38 mitogen-activated protein kinase (MAPK), resulting in the enhanced production of IL-5 and IL-13 in both mouse and human Th2 cells. Ubiquitin-specific protease 7 (USP7) regulates IL-4-induced phosphorylation of STAT3, resulting in granzyme production during Th2 cell differentiation. Genetic deletion of Usp7 or Gzma and pharmacological blockade of granzyme B ameliorated allergic airway inflammation. Furthermore, PAR-1 + and granzyme + Th2 cells were colocalized in nasal polyps from patients with eosinophilic chronic rhinosinusitis. Thus, the USP7-STAT3-granzymes-Par-1 pathway is a potential therapeutic target for intractable allergic diseases.

NK cells are major effector cells involved in the elimination of early tumors and prevent metastasis. They often have an impaired function in patients with cancer. Preclinical studies have demonstrated NK cell activation as the adjunctive effect of invariant NKT (iNKT) cells. Activation of iNKT cells after administration of the glycolipid ligand α-galactosylceramide, loaded with CD1d-expressing human PBMC-derived APCs (APC/Gal), is an attractive cancer therapy to optimize the use of NK cells. However, the subsets of NK cells that are activated following iNKT cell activation as well as the period of NK cell activation remain unclear. In this study, we report that the granzyme B-expressing NK cell response in postoperative lung cancer patients was enhanced 49 d after administration of APC/Gal in a phase II study. We found maximum IFN-γ production on day 49 in 13 out of 27 APC/Gal-treated patients. On day 49, 14 out of 27 patients (51.9%) had higher IFN-γ production by iNKT cells (>6-fold higher than the baseline level). This increment significantly correlated with granzyme B-expressing NK cells. Although IFN-γ production was lower in patients in the nontreated group, we detected maximum IFN-γ production 12 mo after the resection of lung cancer (9 out of 29 patients [31%]). These findings suggest that elimination of cancer cells leads to increased NK cell function, which can be further enhanced by APC/Gal therapy.

Strategies integrating activation of innate and adaptive immu-nity against cancer are desired. We established a novel plat-form, Wilms' tumor antigen 1 (WT1)-expressing artificial adjuvant vector cells (aAVC-WT1), linking invariant natural killer T (iNKT)-mediated dendritic cell activation to T cell im-munity. Here, we report the first-in-human application of aAVC-WT1 in nine patients with relapsed and refractory acute myelogenous leukemia. No dose-limiting toxicities were observed, whereas activation of iNKT and/or NK cells was observed in all patients. Five patients experienced objective leukemic regression, which correlated with WT1-specific T cell responses. Paired single-cell RNA and T cell receptor (TCR) sequencing demonstrated effector CD8+ T cell clones in the bone marrow. Some bone marrow CD8+ T cells under-went transition from pre-existing precursor exhausted T cells to functional T cells or emerged as newly activated T cells, some of which were maintained long term. These demonstrate the feasibility and safety of aAVC-WT1 therapy and the capac-ity of this platform to activate both innate and adaptive immu-nity in humans.

Although PDA1 expression on tumor is related to the prognosis of immune check point blockade (ICB) therapy, a recent study also demonstrated clinical benefits even in patients without PD-LA expression. To understand the relationship between innate resistance and antitumor cytotoxic T lymphocyte (CTL) responses especially against neoantigens, the interaction between PD-L1(+) or genetically PENN-deleted colorectal tumors and CTLs was assessed under an ICC therapy, finding the robust CTL activation in PD-L1-deleted tumor-bearing mice. Using antigen libraries based on immunogenomics, we identified three H2-K-b-restricted, somatic-mutated immunogenic neoantigens by utilizing enhanced CTLs sponses due to PD-L1 deficiency. Furthermore, we identified three T cell receptor (TCR) repertoires relevant to the neoaritigens, confirming the response of TCR-gene-transduced CTLs to parental tumor cells. Notably, neoantige-pulsed dendritic cell (DC) therapy reversed the tumor tolerance. Thus, innate resistance of tumors determines their responsiveness to neoantigens and mixed neoantigen peptides may be useful in DC therapy against innate resistance type tumor.

The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of Pcgf6 in ESCs leads to robust de-repression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells.

AbstractStrategies to reprogram the tumor microenvironment are being explored to improve cancer immunotherapy. In one approach, we have targeted dendritic cells (DC) to improve their function with adjuvant vector cells (aAVC) that are engineered from NKT ligand-loaded CD1d+ allogeneic cells transfected with tumor antigen mRNAs. Here, we report the finding that this approach also programs local immune responses by establishing tertiary lymphoid structures (TLS), which include expanded antigen-specific CD8+ T-cell clones, mobilized DCs, and normalized tumor vasculature. aAVC therapy also expanded specific Vβ-expressing antitumor T-cell clones, leading to the formation of long-term memory T cells. When combined with PD-1 blockade, aAVC infusion triggered regression of poorly immunogenic tumor cells that did not respond to PD-1 blockade alone, as well as expansion of antigen-specific CD8+ T-cell clones in the tumor. The findings of this study help to inform a next-generation platform for the generation of efficacious cancer vaccines. Cancer Res; 76(13); 3756–66. ©2016 AACR.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population with the ability to suppress immune responses and are currently classified into three distinct MDSC subsets: monocytic, granulocytic and non-monocytic, and non-granulocytic MDSCs. Although NK cells provide an important first-line defense against newly transformed cancer cells, it is unknown whether NK cells can regulate MDSC populations in the context of cancer. In this study, we initially found that the frequency of MDSCs in non-Hodgkin lymphoma (NHL) patients was increased and inversely correlated with that of NK cells, but not that of T cells. To investigate the regulation of MDSC subsets by NK cells, we used an EL4 murine lymphoma model and found the non-monocytic and non-granulocytic MDSC subset, i.e., Gr1+CD11b+Ly6GmedLy6Cmed MDSC, is increased after NK cell depletion. The MDSC population that expresses MHC class II, CD80, CD124, and CCR2 is regulated mainly by CD27+CD11b+NK cells. In addition, this MDSC subset produces some immunosuppressive cytokines, including IL-10 but not nitric oxide (NO) or arginase. We also examined two subsets of MDSCs (CD14+HLA-DR− and CD14− HLA-DR− MDSC) in NHL patients and found that higher IL-10-producing CD14+HLA-DR−MDSC subset can be seen in lymphoma patients with reduced NK cell frequency in peripheral blood. Our analyses of MDSCs in this study may enable a better understanding of how MDSCs manipulate the tumor microenvironment and are regulated by NK cells in patients with lymphoma.

Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and β chain mRNA (the Vα24 and Vβ11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide (α-GalCer), they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and β chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer.

Significance Both natural killer (NK) cells and γδT cells, classified as innate immune cells, recently have been shown to have features of memory cells. However, after activation, a memory fate of invariant NK T cells (iNKT cells) has not been identified. Here we show the presence of effector memory-like KLRG1 + (Killer cell lectin-like receptor subfamily G, member 1–positive) iNKT cells in the lung. The KLRG1 + iNKT cells are able to recognize and respond to an antigen in the context of CD1d and can persist for a long time and then mount a potent secondary response upon encountering with the same antigen months later. In addition, we suggest that the KLRG1 + iNKT cells could contribute extensively to immune surveillance, especially in preparation for a possible encounter with tumor diseases.

In the developing neocortex, neural precursor cells (NPCs) sequentially generate various neuronal subtypes in a defined order. Although the precise timing of the NPC fate switches is essential for determining the number of neurons of each subtype and for precisely generating the cortical layer structure, the molecular mechanisms underlying these switches are largely unknown. Here, we show that epigenetic regulation through Ring1B, an essential component of polycomb group (PcG) complex proteins, plays a key role in terminating NPC-mediated production of subcerebral projection neurons (SCPNs). The level of histone H3 residue K27 trimethylation at and Ring1B binding to the promoter of Fezf2, a fate determinant of SCPNs, increased in NPCs as Fezf2 expression decreased. Moreover, deletion of Ring1B in NPCs, but not in postmitotic neurons, prolonged the expression of Fezf2 and the generation of SCPNs that were positive for CTIP2. These results indicate that Ring1B mediates the timed termination of Fezf2 expression and thereby regulates the number of SCPNs.

In the developing neocortex, neural precursor cells (NPCs) sequentially generate various neuronal subtypes in a defined order. Although the precise timing of the NPC fate switches is essential for determining the number of neurons of each subtype and for precisely generating the cortical layer structure, the molecular mechanisms underlying these switches are largely unknown. Here, we show that epigenetic regulation through Ring1B, an essential component of polycomb group (PcG) complex proteins, plays a key role in terminating NPC-mediated production of subcerebral projection neurons (SCPNs). The level of histone H3 residue K27 trimethylation at and Ring1B binding to the promoter of Fezf2, a fate determinant of SCPNs, increased in NPCs as Fezf2 expression decreased. Moreover, deletion of Ring1B in NPCs, but not in postmitotic neurons, prolonged the expression of Fezf2 and the generation of SCPNs that were positive for CTIP2. These results indicate that Ring1B mediates the timed termination of Fezf2 expression and thereby regulates the number of SCPNs.