Circulating NK cells establish tissue residency upon acute infection of skin and mediate accelerated effector responses to secondary infection.

Natural killer (NK) cells are present in the circulation and can also be found residing in tissues, and these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigate whether circulating conventional NK (cNK) cells can develop into long-lived tissue-resident NK (trNK) cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi trNK cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK cells arose from interferon (IFN)-γ-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK cell formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissue residency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny.

Lymph node medulla regulates the spatiotemporal unfolding of resident dendritic cell networks.

Unlike macrophage networks composed of long-lived tissue-resident cells within specific niches, conventional dendritic cells (cDCs) that generate a 3D network in lymph nodes (LNs) are short lived and continuously replaced by DC precursors (preDCs) from the bone marrow (BM). Here, we examined whether specific anatomical niches exist within which preDCs differentiate toward immature cDCs. In situ photoconversion and Prtn3-based fate-tracking revealed that the LN medullary cords are preferential entry sites for preDCs, serving as specific differentiation niches. Repopulation and fate-tracking approaches demonstrated that the cDC1 network unfolded from the medulla along the vascular tree toward the paracortex. During inflammation, collective maturation and migration of resident cDC1s to the paracortex created discontinuity in the medullary cDC1 network and temporarily impaired responsiveness. The decrease in local cDC1 density resulted in higher Flt3L availability in the medullary niche, which accelerated cDC1 development to restore the network. Thus, the spatiotemporal development of the cDC1 network is locally regulated in dedicated LN niches via sensing of cDC1 densities.

Type 1 conventional dendritic cells maintain and guide the differentiation of precursors of exhausted T cells in distinct cellular niches.

Reinvigoration of exhausted CD8+ T (Tex) cells by checkpoint immunotherapy depends on the activation of precursors of exhausted T (Tpex) cells, but the local anatomical context of their maintenance, differentiation, and interplay with other cells is not well understood. Here, we identified transcriptionally distinct Tpex subpopulations, mapped their differentiation trajectories via transitory cellular states toward Tex cells, and localized these cell states to specific splenic niches. Conventional dendritic cells (cDCs) were critical for successful αPD-L1 therapy and were required to mediate viral control. cDC1s were dispensable for Tpex cell expansion but provided an essential niche to promote Tpex cell maintenance, preventing their overactivation and T-cell-mediated immunopathology. Mechanistically, cDC1s insulated Tpex cells via MHC-I-dependent interactions to prevent their activation within other inflammatory environments that further aggravated their exhaustion. Our findings reveal that cDC1s maintain and safeguard Tpex cells within distinct anatomical niches to balance viral control, exhaustion, and immunopathology.

Lymphatic migration of unconventional T cells promotes site-specific immunity in distinct lymph nodes.

Lymphatic transport of molecules and migration of myeloid cells to lymph nodes (LNs) continuously inform lymphocytes on changes in drained tissues. Here, using LN transplantation, single-cell RNA-seq, spectral flow cytometry, and a transgenic mouse model for photolabeling, we showed that tissue-derived unconventional T cells (UTCs) migrate via the lymphatic route to locally draining LNs. As each tissue harbored a distinct spectrum of UTCs with locally adapted differentiation states and distinct T cell receptor repertoires, every draining LN was thus populated by a distinctive tissue-determined mix of these lymphocytes. By making use of single UTC lineage-deficient mouse models, we found that UTCs functionally cooperated in interconnected units and generated and shaped characteristic innate and adaptive immune responses that differed between LNs that drained distinct tissues. Lymphatic migration of UTCs is, therefore, a key determinant of site-specific immunity initiated in distinct LNs with potential implications for vaccination strategies and immunotherapeutic approaches.

Diversification of memory B cells drives the continuous adaptation of secretory antibodies to gut microbiota.

Secretory immunoglobulin A (SIgA) shields the gut epithelium from luminal antigens and contributes to host-microbe symbiosis. However, how antibody responses are regulated to achieve sustained host-microbe interactions is unknown. We found that mice and humans exhibited longitudinal persistence of clonally related B cells in the IgA repertoire despite major changes in the microbiota during antibiotic treatment or infection. Memory B cells recirculated between inductive compartments and were clonally related to plasma cells in gut and mammary glands. Our findings suggest that continuous diversification of memory B cells constitutes a central process for establishing symbiotic host-microbe interactions and offer an explanation of how maternal antibodies are optimized throughout life to protect the newborn.

T cell and dendritic cell interactions in lymphoid organs: More than just being in the right place at the right time.

The initiation of T lymphocyte responses within secondary lymphoid organs involves interactions with different subsets of dendritic cells (DCs). Recent studies have revealed the complexity of microanatomical organization within lymphoid organs. Exactly how T cells and DCs locate each other and the type of cellular interactions required for optimal priming of effector and memory T cell responses are beginning to be unraveled. Here we review advances in our understanding of how T cell priming is choreographed during infections, highlight the importance of cell positioning in this process and discuss how a spectrum of cellular interactions shapes T cell activation and differentiation.

Resident regulatory T cells reflect the immune history of individual lymph nodes.

Regulatory T cells (Tregs) are present in lymphoid and nonlymphoid tissues where they restrict immune activation, prevent autoimmunity, and regulate inflammation. Tregs in nonlymphoid tissues are typically resident, whereas those in lymph nodes (LNs) are considered to recirculate. However, Tregs in LNs are not a homogenous population, and circulation kinetics of different Treg subsets are poorly characterized. Furthermore, whether Tregs can acquire memory T cell properties and persist for extended periods after their activation in LNs is unclear. Here, we used in situ labeling with a stabilized photoconvertible protein to uncover turnover rates of Tregs in LNs in vivo. We found that, whereas most Tregs in LNs recirculate, 10 to 20% are memory-like resident cells that remain in their respective LNs for weeks to months. Single-cell RNA sequencing revealed that LN-resident cells are a functionally and ontogenetically heterogeneous population and share the same core residency gene signature with conventional CD4+ and CD8+ T cells. Resident cells in LNs did not actively proliferate and did not require continuous T cell receptor (TCR) signaling for their residency. However, resident and circulating Tregs had distinct TCR repertoires, and each LN contained exclusive clonal subpopulations of resident Tregs. Our results demonstrate that, similar to conventional T cells, Tregs can form resident memory-like populations in LNs after adaptive immune responses. Specific and local suppression of immune responses by resident Tregs in draining LNs might provide previously unidentified therapeutic opportunities for the treatment of local chronic inflammatory conditions.

Evidence that membrane-bound G protein-coupled melatonin receptors MT1 and MT2 are not involved in the neuroprotective effects of melatonin in focal cerebral ischemia.

Melatonin is synthesized and released by the pineal gland in a circadian rhythm, and many of its peripheral actions are mediated via membrane MT1 and MT2 receptors. Apart from its metabolic functions, melatonin is a potent neuroprotective molecule owing to its antioxidative actions. The roles of MT1 and MT2 in the neuroprotective effects of melatonin and cell signaling after cerebral ischemia remain unknown. With the use of MT1 and MT2 knockout (mt1/2(-/-) ) mice treated with melatonin, we evaluated brain injury, edema formation, inducible nitric oxide synthase (iNOS) activity, and signaling pathways, including CREB, ATF-1, p21, Jun kinase (JNK)1/2, p38 phosphorylation, resulting from ischemia/reperfusion injury. We show that the infarct volume and brain edema do not differ between mt1/2(-/-) and wild-type (WT) animals, but melatonin treatment decreases infarct volume in both groups and brain edema in WT animals after middle cerebral artery occlusion. Notably, melatonin's neuroprotective effect was even more pronounced in mt1/2(-/-) animals compared to that in WT animals. We also demonstrate that melatonin treatment decreased CREB, ATF-1, and p38 phosphorylation in both mt1/2(-/-) and WT mice, while p21 and JNK1/2 were reduced only in melatonin-treated WT animals in the ischemic hemisphere. Furthermore, melatonin treatment lowered iNOS activity only in WT animals. We provide evidence that the absence of MT1 and MT2 has no unfavorable effect on ischemic brain injury. In addition, the neuroprotective effects of melatonin appear to be mediated through a mechanism independent of its membrane receptors. The underlying mechanism(s) should be further studied using selective melatonin receptor agonists and antagonists.

Erythropoietin receptor-like immunostaining on human spermatozoa.

This study aimed to demonstrate the presence of erythropoietin (EPO) receptor on spermatozoa. Whole ejaculates of four healthy volunteers were incubated with polyclonal rabbit anti-EPO receptor and subsequently stained with a Cy-3 labelled secondary antibody. Four slides per subject were analysed, no staining was observed in slides incubated with either primary or secondary antibody alone. EPO receptor staining was positive in 92±8% of EPO pre-treated and 91±4% of non-treated sperm cells. The results suggest that spermatozoa express EPO receptor on plasma membrane, which might act to protect these cells from damage after ejaculation.

Robo4 contributes to the turnover of Peyer's patch B cells.

All leukocytes can get entrance into the draining lymph nodes via the afferent lymphatics but only lymphoid cells can leave the nodes. The molecular mechanisms behind this phenomenon have remained unknown. We employed genome wide microarray analyses of the subcapsular sinus and lymphatic sinus (LS) endothelial cells and found Robo4 to be selectively expressed on LS lymphatics. Further analyses showed high Robo4 expression in lymphatic vessels of Peyer's patches, which only have efferent lymphatic vessels. In functional assays, Robo4-deficient animals showed accumulation of naïve  B cells (CD19+/CD62Lhi/CD44lo) in Peyer's patches, whereas no difference was seen within other lymphocyte subtypes. Short-term lymphocyte homing via high endothelial venules to peripheral and mesenteric lymph nodes and Peyer's patches was also slightly impaired in Robo4 knockout animals. These results show for the first time, selective expression of Robo4 in the efferent arm of the lymphatics and its role in controlling the turnover of a subset of B lymphocytes from Peyer's patches.

Lymph node γδ and αß CD8+ T cells share migratory properties.

During immune responses, T cells differentiate into subsets with different functions and migratory properties. Here we characterize migratory behavior of endogenous αß CD8+ and γδ T cells in lymph nodes by long-term tracking following in vivo photoconversion. We identified subsets of γδ T cells with distinct circulation kinetics that closely mirrored migratory subsets of αß CD8+ T cells. Notably, αß CD8+ and γδ T cells both comprised resident populations which stayed in lymph nodes for 4 weeks without circulation or proliferation. Furthermore, in contrast to the common conception, we observed that central memory αß CD8+ T cells circulate with slower kinetics than naïve cells. Our results show that, similar to αß T cells, γδ T cells can acquire distinct migratory properties during their development and differentiation and reveal unexpected intricacies of T cell migratory patterns.

Resident CD4+ T cells accumulate in lymphoid organs after prolonged antigen exposure.

Effector and memory CD4(+) T cells acquire distinct migratory properties depending on the type and location of the immune responses. Due to the highly dynamic nature of T cell circulation, the comprehensive analysis of these migratory routes requires dedicated experimental approaches. Here, we analyse the migration of effector/memory CD4(+) T cells by long-term in vivo cell tracking. We identify a resident population of antigen-experienced CD4(+) T cells that resides in lymph nodes and Peyer's patches without circulation or proliferation. Resident CD4(+) T cells constitute up to 50% of all effector/memory cells, including, but not limited to, follicular helper T cells. Furthermore, these non-circulating T cells possess a distinct T cell receptor repertoire and accumulate in Peyer's patches after continuous oral antigen exposure. Our results provide the first direct evidence for a resident population of effector/memory CD4(+) T cells that is retained in lymphoid tissues.

Role of Nogo-A in neuronal survival in the reperfused ischemic brain.

Nogo-A is an oligodendroglial neurite outgrowth inhibitor, the deactivation of which enhances brain plasticity and functional recovery in animal models of stroke. Nogo-A's role in the reperfused brain tissue was still unknown. By using Nogo-A(-/-) mice and mice in which Nogo-A was blocked with a neutralizing antibody (11C7) that was infused into the lateral ventricle or striatum, we show that Nogo-A inhibition goes along with decreased neuronal survival and more protracted neurologic recovery, when deactivation is constitutive or induced 24 h before, but not after focal cerebral ischemia. We show that in the presence of Nogo-A, RhoA is activated and Rac1 and RhoB are deactivated, maintaining stress kinases p38/MAPK, SAPK/JNK1/2 and phosphatase-and-tensin homolog (PTEN) activities low. Nogo-A blockade leads to RhoA deactivation, thus overactivating Rac1 and RhoB, the former of which activates p38/MAPK and SAPK/JNK1/2 via direct interaction. RhoA and its effector Rho-associated coiled-coil protein kinase2 deactivation in turn stimulates PTEN, thus inhibiting Akt and ERK1/2, and initiating p53-dependent cell death. Our data suggest a novel role of Nogo-A in promoting neuronal survival by controlling Rac1/RhoA balance. Clinical trials should be aware of injurious effects of axonal growth-promoting therapies. Thus, Nogo-A antibodies should not be used in the very acute stroke phase.