Cutting Edge: Recruitment, Retention, and Migration Underpin Functional Phenotypic Heterogeneity of Regulatory T Cells in Tumors.

Tumor-infiltrating regulatory T cells (Tregs) have been extensively studied as therapeutic targets. However, not all infiltrating T cells exert their functions equally, presumably because of their heterogeneity and substantial turnover in tissues. In this study, we hypothesized that intertissue migration underlies the functional heterogeneity of Tregs. To test this, we applied in vivo photolabeling to examine single-cell diversity of immunosuppressive molecules in mouse Tregs migrating to, remaining in, and emigrating from MC38 tumors. Neuropilin-1 (Nrp1) expression was inversely correlated with that of six other molecules associated with Treg function. Unsupervised clustering analyses revealed that clusters containing Tregs that were retained in tumors expressed high levels of the six functional molecules but not of Nrp1. However, these clusters represented only half of the Tregs migrating to the tumor, suggesting evolving heterogeneity of tumor-infiltrating Tregs. Thus, we propose progressive pathways of Treg activation and migration between tumors and draining lymph nodes.

Epithelial membrane protein 3 (Emp3) downregulates induction and function of cytotoxic T lymphocytes by macrophages via TNF-α production.

Tetraspanin membrane protein, epithelial membrane protein 3 (Emp3), is expressed in lymphoid tissues. Herein, we have examined the Emp3 in antigen presenting cell (APC) function in the CD8+ cytotoxic T lymphocytes (CTLs) induction. Emp3-overexpressing RAW264.7 macrophage cell line derived from BALB/c mice reduced anti-C57BL/6 alloreactive CTL induction, while Emp3-knockdown RAW264.7 enhanced it compared with parent RAW267.4. Emp3-overexpressing RAW264.7 inhibited, but Emp3-knockdown RAW264.7 augmented, CD8+ T cell proliferation, interferon-γ secretion, IL-2 consumption, and IL-2Rα expression on CD8+ T cells. The supernatant from co-culture with Emp3-overexpressing RAW264.7 contained higher amount of TNF-α, and TNF- α neutralization significantly restored all these inhibitions and the alloreactive CTL induction. These results suggest that Emp3 in allogeneic APCs possesses the inhibitory function of alloreactive CTL induction by downregulation of IL-2Rα expression CD8+ T cells via an increase in TNF-α production. This demonstrates a novel mechanism for regulating CTL induction by Emp3 in APCs through TNF-α production.

Sublingual immune cell clusters and dendritic cell distribution in the oral cavity.

The oral mucosa is the first line of defense against pathogenic bacteria and plays a vital role in maintaining tolerance to food antigens and commensal bacteria. We used CD11c reporter mice to visualize dendritic cells (DCs), a key immune cell population, in the oral cavity. We identified differences in DC density in each oral tissue region. Sublingual immune cell clusters (SLICs) extended from the lamina propria to the epithelium, where DCs and T cells resided in close contact with each other and innate lymphoid cells (ILCs). Targeted in situ photolabeling revealed that the SLICs comprised mostly of CD11c+CD11b+ DCs and were enriched for cDC1s and Langerhans cells. Although the frequency of T cell subsets was similar within and outside the SLICs, tissue resident memory T cells were significantly enriched within the clusters and cluster size increased in response to inflammation. Collectively, we found that SLICs form a unique microenvironment that facilitates T cell-DC interactions in the steady state and during inflammation. Since the oral mucosa is an important target for needle-free vaccination and sublingual immunotherapy to induce tolerogenic responses, the novel insight into the localized immunoregulation provided in this study may accelerate the development of these approaches.

Layer-Stacking Sequence Governs Ion-Storage in Layered Double Hydroxides.

In layered materials, the layer-stacking sequence allows the tuning of ion transport and storage properties by modulating the host-ion interactions. However, unlike in the case of cations, the relationship between the stacking sequence and anion transport and storage properties is less clearly understood. Herein, we demonstrate that the stacking sequence governs the nitrate-storage properties of layered double hydroxides (LDHs); the 2H1 polytype enhances the nitrate-storage capacity to 400% of that of the 3R1 polytype. A quartz crystal microbalance with dissipation monitoring combined with multimodal ex situ experiments indicated that the high ion-storage capacity of the 2H1 polytype originates from the soft nature of LDHs lattices, which facilitates nitrate with minimal lattice changes. In contrast, the rigid lattice of the 3R1 sequence requires a notably large lattice expansion, which is detrimental to ion storage. Our findings can aid the rational design of anion-host interaction-derived functionalities.

Critical role of water structure around interlayer ions for ion storage in layered double hydroxides.

Water-containing layered materials have found various applications such as water purification and energy storage. The highly structured water molecules around ions under the confinement between the layers determine the ion storage ability. Yet, the relationship between the configuration of interlayer ions and water structure in high ion storage layered materials is elusive. Herein, using layered double hydroxides, we demonstrate that the water structure is sensitive to the filling density of ions in the interlayer space and governs the ion storage. For ion storage of dilute nitrate ions, a 24% decrease in the filling density increases the nitrate storage capacity by 300%. Quartz crystal microbalance with dissipation monitoring studies, combined with multimodal ex situ experiments and theoretical calculations, reveal that the decreasing filling density effectively facilitates the 2D hydrogen-bond networking structure in water around interlayer nitrate ions along with minimal change in the layered structure, leading to the high storage capacity.

Immunogenic tumor cell death promotes dendritic cell migration and inhibits tumor growth via enhanced T cell immunity.

Immunogenic tumor cell death enhances anti-tumor immunity. However, the mechanisms underlying this effect are incompletely understood. We established a system to induce tumor cell death in situ and investigated its effect on dendritic cell (DC) migration and T cell responses using intravital photolabeling in mice expressing KikGR photoconvertible protein. We demonstrate that tumor cell death induces phagocytosis of tumor cells by tumor-infiltrating (Ti)-DCs, and HMGB1-TLR4 and ATP-P2X7 receptor signaling-dependent Ti-DC emigration to draining lymph nodes (dLNs). This led to an increase in anti-tumor CD8+ T cells of memory precursor effector phenotype and secondary tumor growth inhibition in a CD103+ DC-dependent manner. However, combining tumor cell death induction with lipopolysaccharide treatment stimulated Ti-DC maturation and emigration to dLNs but did not improve tumor immunity. Thus, immunogenic tumor cell death enhances tumor immunity by increasing Ti-DC migration to dLNs where they promote anti-tumor T cell responses and tumor growth inhibition.

Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disorder with a high prevalence, especially in industrialized countries. Dysbiosis of the intestinal microbiota has been observed in RA patients. For instance, new-onset untreated RA (NORA) is associated with the underrepresentation of the Clostridium cluster XIVa, including Lachnospiraceae, which are major butyrate producers, although the pathological relevance has remained obscure. Follicular regulatory T (TFR) cells play critical regulatory roles in the pathogenesis of autoimmune diseases, including RA. Reduced number of circulating TFR cells has been associated with the elevation of autoantibodies and disease severity in RA. However, the contribution of commensal microbe-derived butyrate in controlling TFR cell differentiation remains unknown. METHODS: We examined the contribution of microbe-derived butyrate in controlling autoimmune arthritis using collagen-induced arthritis (CIA) and SKG arthritis models. We phenotyped autoimmune responses in the gut-associated lymphoid tissues (GALT) in the colon and joint-draining lymph nodes in the CIA model. We developed an in vitro CXCR5+Bcl-6+Foxp3+ TFR (iTFR) cell culture system and examined whether butyrate promotes the differentiation of iTFR cells. FINDINGS: Microbe-derived butyrate suppressed the development of autoimmune arthritis. The immunization of type II collagen (CII) caused hypertrophy of the GALT in the colon by amplifying the GC reaction prior to the onset of the CIA. Butyrate mitigated these pathological events by promoting TFR cell differentiation. Butyrate directly induced the differentiation of functional TFR cells in vitro by enhancing histone acetylation in TFR cell marker genes. This effect was attributed to histone deacetylase (HDAC) inhibition by butyrate, leading to histone hyperacetylation in the promoter region of the TFR-cell marker genes. The adoptive transfer of the butyrate-treated iTFR cells reduced CII-specific autoantibody production and thus ameliorated the symptoms of arthritis. INTERPRETATION: Accordingly, microbiota-derived butyrate serves as an environmental cue to enhance TFR cells, which suppress autoantibody production in the systemic lymphoid tissue, eventually ameliorating RA. Our findings provide mechanistic insights into the link between the gut environment and RA risk. FUNDING: This work was supported by AMED-Crest (16gm1010004h0101, 17gm1010004h0102, 18gm1010004h0103, and 19gm1010004s0104 to KH), the Japan Society for the Promotion of Science (JP17KT0055, JP16H01369, and JP18H04680 to KH; JP17K15734 to DT), Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects (KH), Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research (DT), the SECOM Science and Technology Foundation (KH), the Cell Science Research Foundation (KH), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (DT), the Suzuken Memorial Foundation (KH and DT), the Takeda Science Foundation (KH and DT), The Science Research Promotion Fund, and The Promotion and Mutual Aid Corporation for Private Schools of Japan (KH).