Insights into the heterogeneity of iNKT cells: tissue-resident and circulating subsets shaped by local microenvironmental cues.

Invariant natural killer T (iNKT) cells are a distinct subpopulation of innate-like T lymphocytes. They are characterized by semi-invariant T cell receptors (TCRs) that recognize both self and foreign lipid antigens presented by CD1d, a non-polymorphic MHC class I-like molecule. iNKT cells play a critical role in stimulating innate and adaptive immune responses, providing an effective defense against infections and cancers, while also contributing to chronic inflammation. The functions of iNKT cells are specific to their location, ranging from lymphoid to non-lymphoid tissues, such as the thymus, lung, liver, intestine, and adipose tissue. This review aims to provide insights into the heterogeneity of development and function in iNKT cells. First, we will review the expression of master transcription factors that define subsets of iNKT cells and their production of effector molecules such as cytokines and granzymes. In this article, we describe the gene expression profiles contributing to the kinetics, distribution, and cytotoxicity of iNKT cells across different tissue types. We also review the impact of cytokine production in distinct immune microenvironments on iNKT cell heterogeneity, highlighting a recently identified circulating iNKT cell subset. Additionally, we explore the potential of exploiting iNKT cell heterogeneity to create potent immunotherapies for human cancers in the future.

Control of the Development, Distribution, and Function of Innate-Like Lymphocytes and Innate Lymphoid Cells by the Tissue Microenvironment.

Recently, considerable attention has been directed toward innate-like T cells (ITCs) and innate lymphoid cells (ILCs) owing to their indispensable contributions to immune responses, tissue homeostasis, and inflammation. Innate-like T cells include NKT cells, MAIT cells, and γδ T cells, whereas ILCs include NK cells, type 1 ILCs (ILC1s), type 2 ILCs (ILC2s), and type 3 ILCs (ILC3s). Many of these ITCs and ILCs are distributed to specific tissues and remain tissue-resident, while others, such as NK cells and some γδ T cells, circulate through the bloodstream. Nevertheless, recent research has shed light on novel subsets of innate immune cells that exhibit characteristics intermediate between tissue-resident and circulating states under normal and pathological conditions. The local microenvironment frequently influences the development, distribution, and function of these innate immune cells. This review aims to consolidate the current knowledge on the functional heterogeneity of ITCs and ILCs, shaped by local environmental cues, with particular emphasis on IL-15, which governs the activities of the innate immune cells involved in type 1 immune responses.

Hematopoietic cell-derived IL-15 supports NK cell development in scattered and clustered localization within the bone marrow.

Natural killer (NK) cells are innate immune cells critical for protective immune responses against infection and cancer. Although NK cells differentiate in the bone marrow (BM) in an interleukin-15 (IL-15)-dependent manner, the cellular source of IL-15 remains elusive. Using NK cell reporter mice, we show that NK cells are localized in the BM in scattered and clustered manners. NK cell clusters overlap with monocyte and dendritic cell accumulations, whereas scattered NK cells require CXCR4 signaling. Using cell-specific IL-15-deficient mice, we show that hematopoietic cells, but not stromal cells, support NK cell development in the BM through IL-15. In particular, IL-15 produced by monocytes and dendritic cells appears to contribute to NK cell development. These results demonstrate that hematopoietic cells are the IL-15 niche for NK cell development in the BM and that BM NK cells are present in scattered and clustered compartments by different mechanisms, suggesting their distinct functions in the immune response.

Risk factors associated with overall survival in patients with multiple myeloma following carfilzomib treatment: A retrospective study from a large claims database in Japan.

BACKGROUND: Carfilzomib is a selective proteasome inhibitor approved for treating relapsed or refractory multiple myeloma (RRMM). Carfilzomib improves overall survival (OS) and progression-free survival (PFS); however, treatment with carfilzomib results in a higher incidence of cardiovascular and renal toxicity. More than 70% of patients with RRMM in clinical practice do not meet the eligibility criteria for randomized clinical trials (RCT). OS and PFS are negatively influenced by complications, concomitant medications and prior treatments. Therefore, we assessed the risk factors influencing the OS and time to next treatment (TTNT) in the real world. TTNT has emerged as a relevant alternative clinical endpoint to PFS. METHODS: A retrospective analysis of a large claims database prepared during the post-marketing stages in Japan was performed. The patients treated with carfilzomib for the first time were identified. Multivariable Cox proportional hazards regression analysis was performed to evaluate the risk factors influencing OS and TTNT following carfilzomib treatment. RESULTS: A total of 732 patients with RRMM who received carfilzomib-containing chemotherapy between April 2014 and September 2021 were identified. Multivariable Cox regression analysis for OS and TTNT showed a significantly higher hazard ratio (HR) of 1.48 (95% confidence interval [Cl]: 1.10-2.00; p = 0.010) and 1.38 (95% Cl: 1.15-1.65; p < 0.001), respectively, for patients with renal impairment compared to those without renal impairment. Multivariable Cox regression analysis for OS and TTNT showed a significantly higher HR of 1.80 (95% Cl: 1.27-2.55; p = 0.0010) and 1.38 (95% Cl: 1.14-1.66; p < 0.001), respectively, for patients with prior lenalidomide treatment compared to those without prior lenalidomide treatment. CONCLUSION: Complication of renal impairment and prior lenalidomide treatment could be risk factors influencing OS and TTNT during carfilzomib treatment.

CD45 alleviates airway inflammation and lung fibrosis by limiting expansion and activation of ILC2s.

Group 2 innate lymphoid cells (ILC2s) are critical for the immune response against parasite infection and tissue homeostasis and involved in the pathogenesis of allergy and inflammatory diseases. Although multiple molecules positively regulating ILC2 development and activation have been extensively investigated, the factors limiting their population size and response remain poorly studied. Here, we found that CD45, a membrane-bound tyrosine phosphatase essential for T cell development, negatively regulated ILC2s in a cell-intrinsic manner. ILC2s in CD45-deficient mice exhibited enhanced proliferation and maturation in the bone marrow and hyperactivated phenotypes in the lung with high glycolytic capacity. Furthermore, CD45 signaling suppressed the type 2 inflammatory response by lung ILC2s and alleviated airway inflammation and pulmonary fibrosis. Finally, the interaction with galectin-9 influenced CD45 signaling in ILC2s. These results demonstrate that CD45 is a cell-intrinsic negative regulator of ILC2s and prevents lung inflammation and fibrosis via ILC2s.

Lung group 2 innate lymphoid cells differentially depend on local IL-7 for their distribution, activation, and maintenance in innate and adaptive immunity-mediated airway inflammation.

Interleukin-7 (IL-7) is a cytokine critical for the development and maintenance of group 2 innate lymphoid cells (ILC2s). ILC2s are resident in peripheral tissues such as the intestine and lung. However, whether IL-7 produced in the lung plays a role in the maintenance and function of lung ILC2s during airway inflammation remains unknown. IL-7 was expressed in bronchoalveolar epithelial cells and lymphatic endothelial cells (LECs). To investigate the role of local IL-7 in lung ILC2s, we generated two types of IL-7 conditional knockout (IL-7cKO) mice: Sftpc-Cre (SPC-Cre) IL-7cKO mice specific for bronchial epithelial cells and type 2 alveolar epithelial cells and Lyve1-Cre IL-7cKO mice specific for LECs. In steady state, ILC2s were located near airway epithelia, although lung ILC2s were unchanged in the two lines of IL-7cKO mice. In papain-induced airway inflammation dependent on innate immunity, lung ILC2s localized near bronchia via CCR4 expression, and eosinophil infiltration and type 2 cytokine production were reduced in SPC-Cre IL-7cKO mice. In contrast, in house dust mite (HDM)-induced airway inflammation dependent on adaptive immunity, lung ILC2s localized near lymphatic vessels via their CCR2 expression 2 weeks after the last challenge. Furthermore, lung ILC2s were decreased in Lyve1-Cre IL-7cKO mice in the HDM-induced inflammation because of decreased cell survival and proliferation. Finally, administration of anti-IL-7 antibody attenuated papain-induced inflammation by suppressing the activation of ILC2s. Thus, this study demonstrates that IL-7 produced by bronchoalveolar epithelial cells and LECs differentially controls the activation and maintenance of lung ILC2s, where they are localized in airway inflammation.

Liver type 1 innate lymphoid cells lacking IL-7 receptor are a native killer cell subset fostered by parenchymal niches.

Group 1 innate lymphoid cells (G1-ILCs), including circulating natural killer (NK) cells and tissue-resident type 1 ILCs (ILC1s), are innate immune sentinels critical for responses against infection and cancer. In contrast to relatively uniform NK cells through the body, diverse ILC1 subsets have been characterized across and within tissues in mice, but their developmental and functional heterogeneity remain unsolved. Here, using multimodal in vivo approaches including fate-mapping and targeting of the interleukin 15 (IL-15)-producing microenvironment, we demonstrate that liver parenchymal niches support the development of a cytotoxic ILC1 subset lacking IL-7 receptor (7 R- ILC1s). During ontogeny, fetal liver (FL) G1-ILCs arise perivascularly and then differentiate into 7 R- ILC1s within sinusoids. Hepatocyte-derived IL-15 supports parenchymal development of FL G1-ILCs to maintain adult pool of 7 R- ILC1s. IL-7R+ (7R+) ILC1s in the liver, candidate precursors for 7 R- ILC1s, are not essential for 7 R- ILC1 development in physiological conditions. Functionally, 7 R- ILC1s exhibit killing activity at steady state through granzyme B expression, which is underpinned by constitutive mTOR activity, unlike NK cells with exogenous stimulation-dependent cytotoxicity. Our study reveals the unique ontogeny and functions of liver-specific ILC1s, providing a detailed interpretation of ILC1 heterogeneity.

Retinoic acid receptor activity is required for the maintenance of type 1 innate lymphoid cells.

Group 1 innate lymphoid cells (G1-ILCs) are innate immune effectors critical for the response to intracellular pathogens and tumors. G1-ILCs comprise circulating natural killer (NK) cells and tissue-resident type 1 ILCs (ILC1s). ILC1s mainly reside in barrier tissues and provide the initial sources of interferon-γ (IFN-γ) to prime the protecting responses against infections, which are followed by the response of recruited NK cells. Despite such distribution differences, whether local environmental factors influence the behavior of NK cells and ILC1s is unclear. Here, we show that the signaling of retinoic acid (RA), active metabolites of vitamin A, is essential for the maintenance of ILC1s in the periphery. Mice expressing RARα403, a truncated form of retinoic acid receptor α (RARα) that exerts dominant negative activity, in a lymphoid cell- or G1-ILC-specific manner showed remarkable reductions of peripheral ILC1s while NK cells were unaffected. Lymphoid cell-specific inhibition of RAR activity resulted in the reduction of PD-1+ ILC progenitors (ILCPs), but not of common lymphoid progenitors (CLPs), suggesting the impaired commitment and differentiation of ILC1s. Transcriptome analysis revealed that RARα403-expressing ILC1s exhibited impaired proliferative states and declined expression of effector molecules. Thus, our findings demonstrate that cell-intrinsic RA signaling is required for the homeostasis and the functionality of ILC1s, which may present RA as critical environmental cue targeting local type 1 immunity against infection and cancer.

Physiological concentrations of glucocorticoids induce pathological DNA double-strand breaks.

Steroid hormones induce the transcription of target genes by activating nuclear receptors. Early transcriptional response to various stimuli, including hormones, involves the active catalysis of topoisomerase II (TOP2) at transcription regulatory sequences. TOP2 untangles DNAs by transiently generating double-strand breaks (DSBs), where TOP2 covalently binds to DSB ends. When TOP2 fails to rejoin, called "abortive" catalysis, the resulting DSBs are repaired by tyrosyl-DNA phosphodiesterase 2 (TDP2) and non-homologous end-joining (NHEJ). A steroid, cortisol, is the most important glucocorticoid, and dexamethasone (Dex), a synthetic glucocorticoid, is widely used for suppressing inflammation in clinics. We here revealed that clinically relevant concentrations of Dex and physiological concentrations of cortisol efficiently induce DSBs in G1 phase cells deficient in TDP2 and NHEJ. The DSB induction depends on glucocorticoid receptor (GR) and TOP2. Considering the specific role of TDP2 in removing TOP2 adducts from DSB ends, induced DSBs most likely represent stalled TOP2-DSB complexes. Inhibition of RNA polymerase II suppressed the DSBs formation only modestly in the G1 phase. We propose that cortisol and Dex frequently generate DSBs through the abortive catalysis of TOP2 at transcriptional regulatory sequences, including promoters or enhancers, where active TOP2 catalysis occurs during early transcriptional response.

A circulating subset of iNKT cells mediates antitumor and antiviral immunity.

Invariant natural killer T (iNKT) cells are a group of innate-like T lymphocytes that recognize lipid antigens. They are supposed to be tissue resident and important for systemic and local immune regulation. To investigate the heterogeneity of iNKT cells, we recharacterized iNKT cells in the thymus and peripheral tissues. iNKT cells in the thymus were divided into three subpopulations by the expression of the natural killer cell receptor CD244 and the chemokine receptor CXCR6 and designated as C0 (CD244-CXCR6-), C1 (CD244-CXCR6+), or C2 (CD244+CXCR6+) iNKT cells. The development and maturation of C2 iNKT cells from C0 iNKT cells strictly depended on IL-15 produced by thymic epithelial cells. C2 iNKT cells expressed high levels of IFN-γ and granzymes and exhibited more NK cell-like features, whereas C1 iNKT cells showed more T cell-like characteristics. C2 iNKT cells were influenced by the microbiome and aging and suppressed the expression of the autoimmune regulator AIRE in the thymus. In peripheral tissues, C2 iNKT cells were circulating that were distinct from conventional tissue-resident C1 iNKT cells. Functionally, C2 iNKT cells protected mice from the tumor metastasis of melanoma cells by enhancing antitumor immunity and promoted antiviral immune responses against influenza virus infection. Furthermore, we identified human CD244+CXCR6+ iNKT cells with high cytotoxic properties as a counterpart of mouse C2 iNKT cells. Thus, this study reveals a circulating subset of iNKT cells with NK cell-like properties distinct from conventional tissue-resident iNKT cells.

Interleukin-7 receptor signaling is crucial for enhancer-dependent TCRδ germline transcription mediated through STAT5 recruitment.

γδ T cells play important roles in immune responses by rapidly producing large quantities of cytokines. Recently, γδ T cells have been found to be involved in tissue homeostatic regulation, playing roles in thermogenesis, bone regeneration and synaptic plasticity. Nonetheless, the mechanisms involved in γδ T-cell development, especially the regulation of TCRδ gene transcription, have not yet been clarified. Previous studies have established that NOTCH1 signaling plays an important role in the Tcrg and Tcrd germline transcriptional regulation induced by enhancer activation, which is mediated through the recruitment of RUNX1 and MYB. In addition, interleukin-7 signaling has been shown to be required for Tcrg germline transcription, VγJγ rearrangement and γδ T-lymphocyte generation as well as for promoting T-cell survival. In this study, we discovered that interleukin-7 is required for the activation of enhancer-dependent Tcrd germline transcription during thymocyte development. These results indicate that the activation of both Tcrg and Tcrd enhancers during γδ T-cell development in the thymus depends on the same NOTCH1- and interleukin-7-mediated signaling pathways. Understanding the regulation of the Tcrd enhancer during thymocyte development might lead to a better understanding of the enhancer-dependent mechanisms involved in the genomic instability and chromosomal translocations that cause leukemia.

Androgens Alleviate Allergic Airway Inflammation by Suppressing Cytokine Production in Th2 Cells.

Asthma is more common in females than males after adolescence. However, the mechanism of the sex bias in the prevalence of asthma remains unknown. To test whether sex steroid hormones have some roles in T cells during development of asthma, we analyzed airway inflammation in T cell-specific androgen receptor (AR)- and estrogen receptor (ER)-deficient mice. T cell-specific AR-deficient male mice developed severer house dust mite-induced allergic airway inflammation than did control male mice, whereas T cell-specific ERα- and ERß-deficient female mice exhibited a similar degree of inflammation as for control female mice. Furthermore, administration of dihydrotestosterone reduced cytokine production of Th2 cells from control, but not AR-deficient, naive T cells. Transfer of OT-II transgenic AR-deficient Th2 cells into wild-type mice induced severer allergic airway inflammation by OVA than transfer of control Th2 cells. Gene expression profiling suggested that the expression of genes related with cell cycle and Th2 differentiation was elevated in AR-deficient Th2 cells, whereas expression of dual specificity phosphatase (DUSP)-2, a negative regulator of p38, was downregulated. In addition, a chromatin immunoprecipitation assay suggested that AR bound to an AR motif in the 5' untranslated region of the Dusp2 gene in Th2 cells. Furthermore, the Dusp2 promoter with a wild-type AR motif, but not a mutated motif, was transactivated by dihydrotestosterone in a reporter assay. Finally, forced expression of DUSP-2 by retrovirus vector reduced IL-4 expression in Th2 cells. Thus, these results suggest that androgen signaling suppresses cytokine production of Th2 cells by inducing DUSP-2, explaining, in part, the sex bias of asthma after adolescence.

Control of immunity and allergy by steroid hormones.

Steroid hormones, especially glucocorticoids, androgens, and estrogens, have profound influence on immunity. Recent studies using cell-type specific steroid hormone receptor-deficient mice have revealed the precise roles of some of these hormones in the immune system. Glucocorticoids are known to have strong anti-inflammatory and immunosuppressive effects and pleiotropic effects on innate and adaptive immune responses. They suppress the production of inflammatory cytokines by macrophages and DCs and the production of IFN-γ by NK cells, thus inhibiting innate immunity. By contrast, glucocorticoids enhance the immune response by inducing the expression of IL-7R and CXCR4 in T cells and the accumulation of T cells in lymphoid organs in accordance with the diurnal change of the glucocorticoid concentration. Thus, glucocorticoids suppress innate immunity but enhance adaptive immunity. Androgens suppress the homeostasis and activation of ILC2s and the differentiation of Th2 and Th17 cells and enhance the suppressive function of Tregs, thereby alleviating allergic airway inflammation. Thus, these steroid hormones have pleiotropic functions in the immune system. Further investigations are awaited on the regulation of immunity and allergy by estrogens using cell-specific steroid hormone receptor-deficient mice.

Cytotoxic innate lymphoid cells sense cancer cell-expressed interleukin-15 to suppress human and murine malignancies.

Malignancy can be suppressed by the immune system. However, the classes of immunosurveillance responses and their mode of tumor sensing remain incompletely understood. Here, we show that although clear cell renal cell carcinoma (ccRCC) was infiltrated by exhaustion-phenotype CD8+ T cells that negatively correlated with patient prognosis, chromophobe RCC (chRCC) had abundant infiltration of granzyme A-expressing intraepithelial type 1 innate lymphoid cells (ILC1s) that positively associated with patient survival. Interleukin-15 (IL-15) promoted ILC1 granzyme A expression and cytotoxicity, and IL-15 expression in chRCC tumor tissue positively tracked with the ILC1 response. An ILC1 gene signature also predicted survival of a subset of breast cancer patients in association with IL-15 expression. Notably, ILC1s directly interacted with cancer cells, and IL-15 produced by cancer cells supported the expansion and anti-tumor function of ILC1s in a murine breast cancer model. Thus, ILC1 sensing of cancer cell IL-15 defines an immunosurveillance mechanism of epithelial malignancies.

Estradiol suppresses psoriatic inflammation in mice by regulating neutrophil and macrophage functions.

BACKGROUND: Psoriasis is a common inflammatory skin disease resulting from dysregulation of the IL-23/TH17 immune axis. The prevalence and severity of psoriasis is higher in men than in women, although the underlying reasons for this are unclear. OBJECTIVE: We studied whether estradiol, a female hormone, plays protective roles in imiquimod-induced psoriatic inflammation in mice by regulating neutrophil and macrophage functions. METHODS: Wild-type mice and conditional knockout mice were ovariectomized, supplemented with placebo or estradiol pellets, and an imiquimod-containing cream applied. RESULTS: Mice without endogenous ovarian hormones exhibited exacerbated psoriatic inflammation including increased production of IL-17A and IL-1ß, which was reversed by exogenously added estradiol. The suppressive effect of estradiol on the production of IL-1ß and IL-17A was abolished in mice lacking estrogen receptors in neutrophils and macrophages (Esr1f/fEsr2f/fLysM-Cre+ mice). IL-1ß, which is required for production of IL-17A in the psoriasis model, was mainly produced by neutrophils and inflammatory macrophages. Estradiol suppressed IL-1ß production from neutrophils and macrophages in mice both in vivo and in vitro and from human neutrophils in vitro. CONCLUSION: Our results suggest a novel mechanism for sex-dependent differences in psoriasis clinical phenotypes that may shed new light on the pathology of psoriasis.

IRF4 deficiency vulnerates B-cell progeny for leukemogenesis via somatically acquired Jak3 mutations conferring IL-7 hypersensitivity.

The processes leading from disturbed B-cell development to adult B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) remain poorly understood. Here, we describe Irf4-/- mice as prone to developing BCP-ALL with age. Irf4-/- preB-I cells exhibited impaired differentiation but enhanced proliferation in response to IL-7, along with reduced retention in the IL-7 providing bone marrow niche due to decreased CXCL12 responsiveness. Thus selected, preB-I cells acquired Jak3 mutations, probably following irregular AID activity, resulting in malignant transformation. We demonstrate heightened IL-7 sensitivity due to Jak3 mutants, devise a model to explain it, and describe structural and functional similarities to Jak2 mutations often occurring in human Ph-like ALL. Finally, targeting JAK signaling with Ruxolitinib in vivo prolonged survival of mice bearing established Irf4-/- leukemia. Intriguingly, organ infiltration including leukemic meningeosis was selectively reduced without affecting blood blast counts. In this work, we present spontaneous leukemogenesis following IRF4 deficiency with potential implications for high-risk BCP-ALL in adult humans.

High-throughput identification and quantification of single bacterial cells in the microbiota.

The bacterial microbiota works as a community that consists of many individual organisms, i.e., cells. To fully understand the function of bacterial microbiota, individual cells must be identified; however, it is difficult with current techniques. Here, we develop a method, Barcoding Bacteria for Identification and Quantification (BarBIQ), which classifies single bacterial cells into taxa-named herein cell-based operational taxonomy units (cOTUs)-based on cellularly barcoded 16S rRNA sequences with single-base accuracy, and quantifies the cell number for each cOTU in the microbiota in a high-throughput manner. We apply BarBIQ to murine cecal microbiotas and quantify in total 3.4 × 105 bacterial cells containing 810 cOTUs. Interestingly, we find location-dependent global differences in the cecal microbiota depending on the dietary vitamin A deficiency, and more differentially abundant cOTUs at the proximal location than the distal location. Importantly, these location differences are not clearly shown by conventional 16S rRNA gene-amplicon sequencing methods, which quantify the 16S rRNA genes, not the cells. Thus, BarBIQ enables microbiota characterization with the identification and quantification of individual constituent bacteria, which is a cornerstone for microbiota studies.

The Roles of IL-7 and IL-15 in Niches for Lymphocyte Progenitors and Immune Cells in Lymphoid Organs.

Lymphoid organs consist of immune cells and stromal cells. The stromal cells produce various cytokines that support the development, maintenance, and response of the immune cells. IL-7 and IL-15 are the major cytokines produced by stromal cells and are essential for the development and maintenance of lymphocytes and innate lymphoid cells (ILCs). In addition, IL-7 is indispensable for the organogenesis of lymphoid organs. However, because the amount of these two cytokines is relatively low, it has been difficult to directly detect their expression. Recently, several groups succeeded in establishing IL-7 and IL-15 reporter mouse lines. As expected, IL-7 and IL-15 were detected in mesenchymal stromal cells in the bone marrow and lymph nodes and in epithelial cells in the thymus. Furthermore, IL-7 and IL-15 were differentially expressed in lymphatic endothelial cells and blood endothelial cells, respectively. In addition to their expression, many groups have analyzed the local functions of IL-7 and IL-15 by using cell-type-specific knockout mice. From these experiments, CXCL12-expressing mesenchymal stromal cells were identified as the major niche for early B cell precursors. Single-cell RNA sequencing (scRNA-seq) analysis has revealed different subpopulations of stromal cells in the lymphoid organs, including those that express both IL-7 and IL-15. Future research is still needed to elucidate which stromal cells serve as the niche for the early precursors of ILCs and NK cells in the bone marrow.

Lymph Node Stromal Cells: Diverse Meshwork Structures Weave Functionally Subdivided Niches.

Lymph nodes (LNs) are secondary lymphoid organs that function as the first line of defense against invasive foreign substances. Within the LNs, different types of immune cells are strategically localized to induce immune responses efficiently. Such a sophisticated tissue structure is a complex of functionally specialized niches, constructed by a variety of fibroblastic stromal cells. Elucidating the characteristics and functions of the niches and stromal cells will facilitate comprehension of the immune response induced in the LNs. Three recent studies offered novel insights into specialized stromal cells. In our discussion of these surprisingly diverse stromal cells, we will integrate information from these studies to improve knowledge about the structure and niches of LN.