Metastasis of cancer cells to the brain occurs frequently in patients with certain subtypes of breast cancer. In particular, patients with HER2-positive or triple-negative breast cancer are at high risk for the development of brain metastases. Despite recent advances in the treatment of primary breast tumors, the prognosis of breast cancer patients with brain metastases remains poor. A better understanding of the molecular and cellular mechanisms underlying brain metastasis might be expected to lead to improvements in the overall survival rate for these patients. Recent studies have revealed complex interactions between metastatic cancer cells and their microenvironment in the brain. Such interactions result in the activation of various signaling pathways related to metastasis in both cancer cells and cells of the microenvironment including astrocytes and microglia. In this review, we focus on such interactions and on their role both in the metastatic process and as potential targets for therapeutic intervention. Cancer metastasis to the brain occurs predominantly in cases of lung adenocarcinoma, breast carcinoma, and melanoma . Brain mMetastatic brain tumors develop when cancer cells from a primary tumor elsewhere in the body reach the brain via the bloodstream and begin to proliferate. Metastases have been detected in the frontal, temporal, parietal, and occipital lobes, the cerebellum, and other locations in the brain. Brain metastases are diagnosed by contrast-enhanced magnetic resonance imaging or computed tomography. The neurological symptoms of brain metastases are largely the result of compression of the brain tissue at the site of tumor formation and of increased intracranial pressure, and they include headache, nausea, and vomiting, epileptic seizures, dizziness, limb paralysis, convulsions, impaired vision, and speech problems. There are three therapeutic options for metastatic brain tumors: radiation therapy, surgery, and treatment with anticancer drugs including molecularly targeted agents. Serious neurological symptoms often disturb the treatment of tumors with anticancer drugs. On the basis of the number of metastases and the predicted prognosis, patients are treated so as to control tumor growth, to ameliorate neurological symptoms, or to improve quality of life.− ER− PR−) breast cancer (TNBC) have a higher risk of brain metastasis compared with those with the luminal subtype (ER+ or PR+) of breast cancer. The frequency of brain metastasis is thus as high as 20% to 30% in HER2-positive breast cancer and TNBC but is < 10% in luminal breast cancer [As mentioned above, brain metastases commonly arise in patients with lung cancer, breast cancer, or melanoma and are associated with a poor survival outcome . In addit cancer .p < 0.001 for both scores) [Despite recent advances in the treatment of primary breast tumors, the prognosis for breast cancer patients with brain metastases remains poor overall—although the prognosis of such patients is actually heterogeneous, with some individuals achieving a better survival outcome than others . Several scores) , and modCancer metastasis is a multistep process that includes local invasion of a primary tumor into the surrounding tissue, intravasation of tumor cells and their dissemination in the circulation, extravasation of the circulating cancer cells at distant sites, and the colonization by these cells of such sites, giving rise to the formation of micro- and then macroscopic metastases , 12. OrgThe percentage of intratumoral lymphocytes has been found to be an independent predictor of a pathological complete response to neoadjuvant chemotherapy for breast cancer . Tumor-iAstrocytes are abundant cells in the brain and play a role in tissue homeostasis, including maintenance of the blood-brain barrier. Circulating cancer cells need to pass through this barrier (extravasation) before they can colonize and proliferate in the brain. Although most extravasated cancer cells die, the surviving cells bind to the external surface of brain capillaries and grow as a sheath around the vessels . AstrocyBoth primary and metastatic brain tumors are influenced by the distinct biology of the brain microenvironment characterized by its unique cell types, anatomic structures, metabolic constraints, and immune properties. Given the important role of the tumor microenvironment in both the metastatic process and response to treatment, characterization of the relation between tumor cells and their microenvironment in the brain is likely to inform the development of new approaches to the prevention or therapy of primary brain tumors and brain metastases. We will describe several signal transduction molecules and their regulatory mechanisms that are associated with both tumor cells and brain microenvironmental cells.+ reactive astrocytes blocked the access of CD8+ cytotoxic T cells to cancer cells  and increased binding to CD74+ microglia –κB signaling pathway that promotes the development of brain metastases [The Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway is a key signaling mechanism activated by the interaction of cytokines and growth factors with their receptors. The induction of A2 astrocytes by ischemia is associated with scar formation , and thetastases . –Akt pathway is a key intracellular signaling pathway that promotes various cellular processes including proliferation, survival, metabolism, and angiogenesis in response to extracellular signals that activate receptor tyrosine kinases or G protein–coupled receptors. The PI3K-Akt signaling pathway has also been implicated as a major regulator of brain metastasis Fig. . The act+ T cells in mouse models of breast and cervical cancer [PI3K class I enzymes include four catalytic subunit isoforms, with the α and β isoforms often being overexpressed in breast cancer cells  and the l cancer .+ cancer cells recruited macrophages expressing the receptor for CCL2 (CCR2) , a negative regulator of PI3K-Akt signaling, has been detected in 25% to 71% of brain metastases in breast cancer patients and occurs preferentially in TNBC , 35, 41.Mammalian target of rapamycin (mTOR) is a downstream effector of the PI3K-Akt pathway, and its activity in breast cancer has been shown to mediate resistance to PI3K inhibition. Combined inhibition of PI3K and mTOR was able to overcome resistance to a PI3K inhibitor in an orthotopic model of brain metastasis by HER2-positive breast cancer .HER2-amplified breast cancer cell line (BT-474) after implantation of the cells into the mouse brain [. Immunohistochemistry revealed that HER3 was overexpressed in 60% of brain metastases in breast cancer patients [+ and HER2− breast cancer, lung cancer, and colon cancer—were positive for phosphorylated (activated) HER3 [HER2-positive breast cancer shows a susceptibility to brain metastasis similar to that of TNBC. Formation of HER2-HER3 heterodimers results in marked activation of PI3K-Akt signaling in breast cancer cells . The expse brain . Immunohpatients , whereased) HER3 . Resista line BT-4 after i+ breast cancer patients with brain metastases. Neratinib is a pan-HER tyrosine kinase inhibitor (TKI) that binds irreversibly to HER1, HER2, and HER4, and the combination of neratinib plus capecitabine showed a CNS response rate of 49% in such patients, compared with a value of only 8% for neratinib monotherapy [+ breast cancer and brain metastases.Although HER2-targeted agents including trastuzumab, pertuzumab, and trastuzumab plus emtansine (T-DM1) have failed to prevent brain metastasis in breast cancer patients –50, variotherapy . Tucatinotherapy . In thatAn inhibitor of cyclin-dependent kinases (CDKs) 4 and 6 has also shown promise for the treatment of brain metastases in breast cancer patients. The combination of this inhibitor, abemaciclib, with endocrine therapy was thus found to be effective in patients with hormone receptor–positive, HER2-negative breast cancer and brain metastases, with 38% of patients showing a decrease in the metastatic tumor load .Given that cross talk between metastatic cancer cells and their microenvironment is implicated in the development of brain metastases of breast cancer, therapy targeted to the microenvironment or to such cross talk is also under investigation. The STAT3 inhibitor silibinin, which crosses the blood-brain barrier , has thuImmune checkpoint inhibitors have been approved for the treatment of lung cancer and melanoma. The immune checkpoint protein PD-L1 is expressed on the surface of cancer cells and induces exhaustion or apoptosis in tumor-infiltrating cytotoxic T cells through interaction with programmed cell death–1 (PD-1) expressed on their surface . Pembrol+ regulatory T cells has been found to increase in association with the progression of brain metastases [The mechanisms underlying the effectiveness of immunotherapy for brain metastases are under investigation. The number of FOXP3tastases . Immune tastases .Tph2, Sspo, Ptprq, and Pole was specifically upregulated in brain tissue harboring metastases, whereas that of the human genes CXCR4, PLLP, TNFSF4, VCAM1, SLC8A2, and SLC7A11 was specifically upregulated in brain-metastasizing cancer cells. Further characterization of such novel metastasis-associated genes and their interactions may eventually lead to advances in therapy that improve the prognosis of cancer patients. Investigation of the influences of immune cells on brain metastasis will require the development of improved experimental models with an intact immune system.To identify novel and specific mechanisms of brain metastasis and thereby provide insight into prevention or treatment of this condition, we developed mouse xenograft models of brain metastasis based on intracardiac injection of human breast cancer or melanoma cell lines and performed RNA-sequencing analysis of both brain metastases and matched primary tumors . The seqSLC7A11 is of particular interest. The SLC7A11 gene encodes the xCT subunit of system xc(−), a sodium-independent cystine-glutamate antiporter that mediates the uptake of cystine into cells in exchange for intracellular glutamate [Among the human genes whose expression was specifically upregulated in brain metastatic tumor cells of our mouse xenograft models, lutamate . Expresslutamate . Expresslutamate , 65. Furlutamate . Togethe+ cancer stemlike cells, and the combination treatment with a significant increase in progression-free survival. Whether the promise of such xCT-targeted therapy may extend to the prevention or treatment of breast cancer metastasis warrants further investigation.Sulfasalazine, a drug that has been administered for the treatment of inflammatory bowel disease and rheumatoid arthritis , has alsIn addition to the treatment of patients with brain metastases, prevention of brain metastasis from the primary tumor is an important clinical goal. Such prevention will require the detection of circulating brain-tropic cancer cells before their extravasation. Liquid biopsy is a potential screening tool for the detection of such cells in the circulation . The genTraditional cytotoxic agents and anti-HER2 agents have a limited role in the management of brain metastases in breast cancer patients. Intrathecal injection of methotrexate is adopted in the clinic as a therapeutic option for leptomeningeal metastasis in breast cancer. A case report showed that intrathecal trastuzumab was also a safe and effective therapy for HER2-positive breast cancer with leptomeningeal metastasis . Such inWe previously found that the heterogeneity of HER2 expression among breast cancer cells is associated with poor survival in mice with brain metastases . Further+ or luminal breast cancer have other therapeutic options, given that anti-HER2 agents and endocrine therapy can be administered concurrently with corticosteroids and radiation therapy. Immune checkpoint inhibitors are a promising therapeutic option for TNBC patients with brain metastases before radiation therapy, although further studies are required to support this option.TNBC has the worst prognosis among breast cancer subtypes as a result of its rapid progression and lack of a conventional therapeutic target. TNBC with symptomatic brain metastasis is preferentially treated with radiation therapy rather than systemic therapy. Patients with brain metastases manifest various symptoms including headache (61.9%), nausea and vomiting (45.7%), visual disorders (26.3%), seizures (30.4%), and motor dysfunction (46.6%) . Cortico


Pi is often a limiting nutrient in the environment, particularly for plants and microorganisms. At the same time, the need for phosphate varies, establishing conflicts of goals. Cells experience strong peaks of Pi demand, e.g., during the S-phase, when DNA, a highly abundant and phosphate-rich compound, is duplicated. While cells must satisfy these Pi demands, they must safeguard themselves against an excess of Pi in the cytosol. This is necessary because Pi is a product of all nucleotide-hydrolyzing reactions. An accumulation of Pi shifts the equilibria of these reactions and reduces the free energy that they can provide to drive endergonic metabolic reactions. Thus, while Pi starvation may simply retard growth and division, an elevated cytosolic Pi concentration is potentially dangerous for cells because it might stall metabolism. Accordingly, the consequences of perturbed cellular Pi homeostasis are severe. In eukaryotes, they range from lethality in microorganisms such as yeast (i homeostasis is thus not only a fundamental topic of cell biology but also of growing interest for medicine and agriculture.Cells face major changes in demand for and supply of inorganic phosphate (Pas yeast , severe as yeast  to neuroas yeast . Intrace To achieve this, they may use signaling networks that sense extra- and intracellular Pi and a buffering system for cytosolic Pi. In this review, we do not provide a global overview of phosphate homeostasis in all model organisms studied in this respect because this adds significant complexity and detail, resulting for example from multicellularity, tissue differentiation, or the complexity of their life cycles. We will rather focus on the yeast Saccharomyces cerevisiae because it is a unicellular model and the eukaryotic model system in which relevant pathways and mechanisms have been best characterized. Focusing on a single well-characterized and simple model provides the best basis for our effort to develop a conceptual framework for intracellular phosphate homeostasis, which may provide leads to dissect this crucial homeostatic system also in other eukaryotic organisms   , which sic state . Interese kidney .i regulation at the organismal level has been intensely studied, particularly in humans  transcriptional Pi starvation programs. But cells may also experience a sudden excess of Pi and may need highly reactive mechanisms to protect themselves against the potentially lethal consequences. In this situation, posttranslational signaling becomes important, allowing to rapidly and simultaneously regulate multiple systems for import, export, and storage of Pi and to thus dampen cytosolic Pi peaks.Whereas Pn humans , it is psumption . Cells rrategies , in whici is accumulated to similarly high concentrations in the form of polyphosphate (polyP), a polymer of up to a thousand Pi units linked through phosphoric anhydride bonds. PolyP is stored in the acidocalcisome lumen, where polyphosphatases are also located. It is assumed that these enzymes can hydrolyze polyP, which may allow re-export of the liberated Pi into the cytosol  lumen contains many chaperones that hydrolyze ATP  and low (Pho87 and Pho90) affinity and a putative Pi exporter in the plasma membrane   (i transporter (Pho91)  . An ATP-nd Ppn2) , 2017. T (Pho91) , which mi homeostasis are expressed through a transcriptional control mechanism, the phosphate-responsive signal transduction (PHO) pathway (i-responsive kinase (Pho80/Pho85/Pho81), which phosphorylates the transcription factor Pho4, thereby keeping it inactive. Pi starvation favors the dephosphorylation of Pho4 and allows it to activate a wide spectrum of genes dedicated not only to Pi import and storage but also to foraging for extracellular Pi and the recycling of intracellular phosphate (i importers (Pho84) and the polyP polymerase VTC are induced, whereas proteins for Pi foraging are upregulated only upon further Pi limitation  proposed to sense extracellular Pi in this manner and to regulate intracellular responses, such as the protein kinase A (PKA) pathway , reacts to intracellular rather than extracellular Pi, arguing against a general role of Pho84 or Pho87 in Pi sensing  domain. The fact that all of the 10 yeast proteins that contain an SPX domain are involved in Pi homeostasis strongly suggests a role of this domain in coordinating Pi signaling   . They bi factors . A varie factors , but it  factors . Availabntration , 2008, lation . We postulate that InsPPs address these proteins in a coordinated fashion in order to maintain cytosolic Pi in a suitable range. Then, we must expect that SPX domains bound to inositol pyrophosphates can have either activating or inactivating effects, depending on the proteins that they associate with. Since the levels of inositol pyrophosphates increase with the level of available Pi, proteins exporting Pi, either across the plasma membrane or into intracellular storage places such as the acidocalcisome-like vacuole, should be activated by inositol pyrophosphate-bound SPX. Likewise, we should expect that the activation of the transcriptional phosphate starvation response, the PHO pathway, should be repressed by InsPPs. This scheme is partially consistent with existing data because the SPX-containing polyP polymerase VTC is activated through the interaction between its SPX domains and the inositol pyrophosphate 5-InsP7 . The deletion of all five transporters together is lethal, but the quintuple mutant can be rescued by the overexpression of any one of these five importers  with a Km of 8–40 μM , but becomes transferred to the vacuole for degradation in the absence of Pi or at high excess of Pi  or to the Pi-replete state (stabilizing the low-affinity transporters and repressing the PHO pathway). 14-3-3 proteins  were proposed to influence this commitment  family. Both Pho87 and Pho90 harbor an SPX domain at their N-terminus. Removal of this SPX domain leads to phenotypes suggesting an enhanced, unrestricted flux of Pi across these transporters: up to an eightfold increased Pi uptake activity, higher total phosphate, and higher polyP content. Cells expressing Pho90 without an SPX domain also become sensitive to high Pi concentrations in the media, and they show an enhanced leakage of cellular Pi on low-Pi media  domain. The function of the EXS domain is still unknown. Although the transport activity for Syg1 itself has not yet been shown, its human homolog XPR1 does facilitate Pi export across the plasma membrane  linked by energy-rich phosphoric anhydride bonds. PolyP efficiently chelates ions such as Ca2+, Mg2+, and Mn2+. Yeast cells can accumulate up to a quarter of their dry weight in the form of polyphosphate  superfamily of metallophosphatases that resides in the vacuolar lumen. The enzyme depends on Znactivity . Ppn1 ani becomes limiting, the cells use the PHO pathway to induce first the high-affinity transporter Pho84. They secrete phosphatases to recover Pi from external sources and induce VTC expression to maximize their polyP stores , in which cells arrest growth and become more resistant to stresses  . These eer InsP6 . The simer InsP6 , has imper InsP6 . This pri in the low millimolar range—a concentration range that is commonly found in the cytosol—and their kinase activity is boosted by an increasing ATP concentration P2 P2 , are not6 or InsP5, creating 5-InsP7 or 5PP-InsP4, respectively P5 into InsP6 and, hence, does not offer InsP6, which is the far more abundant substrate of Kcs1 in a wild-type cell, where it probably outcompetes InsP5. Kcs1 shares a PxxxDxKxG motif in its catalytic site with other members of the family. The ablation of Kcs1 activity leads to the constitutive activation of the PHO transcription pathway even on high-Pi media, to an overaccumulation of Pi and ATP, and to a complete absence of polyP synthesis . In marked contrast to the low selectivity in binding, the agonist properties of different isomers vary considerably. The VTC complex, for example, is strongly activated by 1,5-InsP8, but very poorly by 5-PPP-InsP5, which carries the same number of phosphate groups  binding site for inositol polyphosphates and pyrophosphates. Purified SPX domains show a relatively low discrimination in binding different inositol polyphosphates or pyrophosphate isomers, and their affinity decreases in parallel to the net charge of the compound  has no significant impact on polyP synthesis in vivo. The ablation of 5-InsP7 synthesis (Kcs1), in contrast, eliminates polyP synthesis, strongly suggesting this isomer as the relevant controller of the SPX domains of VTC in vivo. Whether 5-InsP7 is the principal regulator also for the other SPX-controlled proteins in the cell is an important issue that remains to be explored. The mammalian Pi exporter XPR1 provides an important example in this respect because this SPX-controlled transporter is opened by 1,5-InsP8 rather than by 1-InsP7 or 5-InsP7  are also controlled by the PHO pathway  . AICAR bi limitation, where such self-inactivation might occur, because the measures by which the cells seek to improve Pi acquisition may completely rectify the initial decline in cytosolic Pi that triggered the starvation response, thereby leading to an oscillation of the PHO pathway activation and inactivation. This cannot happen upon complete depletion of Pi from the media. A decisive difference is thus whether the cells experience only moderate Pi limitation or profound Pi starvation. Upon moderate limitation of Pi, a first wave of transcription induces a subset of genes: for the high-affinity Pi importer Pho84; Pho86, which is involved in its biogenesis; the secreted acid phosphatases Pho12 and Pho11  reveal other features. They induce the same set of immediately activated genes as cells in intermediate Pi. However, due to the absence of Pi in the media, the induction of these genes cannot suffice to stabilize their cytosolic Pi. The more pronounced and stable decline in cytosolic Pi is believed to trigger a second wave of gene inductions 2 h after the first wave ; the high-affinity Pi importer Pho89; and the secreted phosphatase Pho5. This second wave of transcription coincides with the induction of the environmental stress response and a decline in the growth rate. The partial dephosphorylation of the transcription factor Pho4 may be the trigger for the first transcriptional wave  and the major regulator of Pho85/80 kinase and the PHO pathway . Pho81 bnditions . Pho81 ctabolism . PHO81 gmitation , and Phomitation . This cri starvation can induce the PHO pathway. The central part of the protein contains six ankyrin repeats. Part of these repeats and an adjacent region form a 141-amino acid piece that is sufficient to maintain some Pi-dependent regulation of the PHO pathway in vivo. The resulting inducibility is limited, however, because this central domain yields only a fivefold induction of the PHO5 gene on Pi-free media, and this only upon overexpression, whereas full-length Pho81, expressed at normal levels from its native promoter, yields an almost 200-fold induction  from these two genes (PHO5 transcription (PHO84 promoter, it recruits the histone deacetylase Hda1, which downregulates transcription (KCS1 locus, antisense and intragenic transcripts are produced, and their production depends on the transcription factor Pho4 and on Pi starvation (Yeast cells also perform pervasive transcription, which produces non-coding RNAs . Antisenwo genes  and affecription . On the cription . At the arvation . These ti-dependent manner (Phosphate-dependent regulation of gene expression appears to act also on steps subsequent to the initiation of transcription. For example, Pho92 regulates the degradation of Pho4 mRNA by binding to its 3′-UTR in a Pt manner . The noni homeostasis is an essential and complex aspect of metabolism. Even a simple unicellular organism such as yeast dedicates a significant percentage of its only 6,000 genes on it, underlining its fundamental importance. Many components involved in the Pi homeostasis in yeast are conserved in other organisms, suggesting that there are common approaches to the problem. Others appear functionally conserved, but realized in a quite organism-specific manner. For example, Pi-dependent transcriptional control is a widespread phenomenon which is conserved in that it employs SPX domains and InsPPs, and we expect that the way in which SPX domains interact with their target proteins may reveal common features. However, in other organisms, the mediators transmitting this control to the transcription factors can differ from the PHO pathway components acting in yeast. Work in a variety of different model organisms will thus contribute essential information necessary to grasp the full spectrum of strategies underlying Pi homeostasis.Pi are known and important elements of the signaling cascades regulating their expression and activity have begun to emerge. A major challenge lies in the high degree of redundancy that cells use in order to regulate their internal phosphate store. In order to study the regulatory mechanisms behind Pi homeostasis, it will therefore be necessary to establish reduced and simplified systems, both in vivo and in vitro, which will allow isolating individual aspects of the complex regulatory network and exploring them without too much interference by redundant mechanisms. The potent and convenient methods for manipulating the yeast genome provide the necessary tools to tackle this task.Yeast cells are a prime model to study phosphate homeostasis in a eukaryotic cell system because many of the proteins immediately implicated in stabilizing cytosolic PBoth authors wrote all parts of the review together.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mn2+ is a calcium ion analog and can enter excitable cells through voltage-gated calcium channels. Mn2+ can be transported along the axons of neurons via microtubule-based fast axonal transport. Based on these properties, MEMRI is used to describe neuroanatomical structures, monitor neural activity, and evaluate axonal transport rates. The application of MEMRI in preclinical animal models of central nervous system (CNS) diseases can provide more information for the study of disease mechanisms. In this article, we provide a brief review of MEMRI use in CNS diseases ranging from neurodegenerative diseases to brain injury and spinal cord injury.Manganese-enhanced magnetic resonance imaging (MEMRI) relies on the strong paramagnetism of Mn Central nervous system (CNS) lesions are serious diseases affecting human health. Many imaging methods are currently available for the diagnosis and treatment of CNS diseases, and magnetic resonance imaging (MRI) is the most widely used imaging method. In addition to conventional T1-weighted imaging (T1WI) and T2-weighted imaging (T2WI), the continuous development of multiple MRI functional imaging methods  has enabled a better understanding and investigation of the occurrence, development, and prognosis of CNS diseases. Manganese enhanced magnetic resonance imaging (MEMRI) is a special MR functional imaging method, which is different from other imaging methods. For example, DWI has a limited ability to detect complex fiber connections and has low sensitivity in tracking subcortical fibers, leading to underestimation of nerve fiber connections, whereas MEMRI can produce results comparable to histological results . BOLD im2+ has strong paramagnetism, significantly reduces the T1 relaxation time, and exhibits a high signal on T1WI, which is the basis of MEMRI. Mn2+ is a Ca2+ analog that is absorbed by excitable cells through voltage-gated calcium channels [particularly L-type Cav1.2 channels   –13. At p1 (DMT1) –18, trac1 (DMT1) , 19, 20,1 (DMT1) –26. Aftepathways –29. For pathways , 31. Thipathways . Many ty2+ administration in MEMRI are mainly local administration (LA) and systemic administration (SA). SA is performed through oral gavage or intravenous, intraperitoneal, subcutaneous, or intramuscular injection and enables observation of the scopes and boundaries of anatomical structures  and blood-cerebrospinal fluid barrier [  did not diffuse into the vitreous cavity, possibly reached the retina through the capillary circulation after iris absorption, and were then transported to the superior colliculus. This method is safe and does not damage the visual system. The authors further showed that an acidic MnCl2 solution produced more pronounced MEMRI signal intensity than a neutral solution, and the use of longer intervals between repeated local Mn2+ doses can reduce adverse effects  . Local d process . Accordi process , locally effects . In addi imaging , 88 and 2+. Researchers administer Mn contrast agents through either systemic routes or local routes to study the CNS. SA is simple and noninvasive and can be repeated as needed. However, one major disadvantage is that the dose of MnCl2 is significantly higher than the dose used for LA, and Mn2+ reaches the liver, heart, and kidney before reaching the brain, increasing the risk of acute toxicity  and intraperitoneal MnCl2 injection to detect markedly abnormal elevations in ΔR1 in the hippocampal CA1 and CA3 regions before the onset of cognitive deficits in tau protein transgenic mice (rTg4510) ; this increased intensity was associated with impaired learning and memory in behavioral tests  and the formation of neuritic amyloid-β (Aβ) plaques. The brains of patients with AD contain a large amount of hyperphosphorylated tau protein. Under these conditions, the normal functions of the tau protein are either inhibited or lost, and paired helical filaments or NFTs associated with synaptic damage begin to appear . MEMRI crTg4510) . In anotal tests . The incal tests . Moreoveke model . MEMRI aodel  was normal before Aβ deposition, decreased significantly as the Aβ level increased before plaque formation, and substantially decreased after plaque formation. Aβ deposition in the olfactory bulb occurs earlier than in other brain region and is responsible for the decrease in olfactory function observed in subjects with early AD , an increase in Mn2+ uptake during the first few days was postulated to be caused by increased astrocyte reactivity due to early striatum terminal degeneration, whereas the enhancement of the MEMRI signal was reduced after anti-inflammatory treatment using vasoactive intestinal peptide receptor 2 agonists, thus facilitating neuronal protection (−1) to quantify the neuronal activity map of the entire brain, revealing the patterns and locations of changes in neuronal activity in animal models of PD. In particular, a quantitative analysis of the R1 value in the dorsal caudate-putamen determines the severity of PD and thus facilitates proper treatment (2+ into the STN) shows a decrease in the axonal transport function from the ipsilateral STN to the ventral globus pallidus  to induce dopaminergic (DAergic) neuron death in the SNc . In ratsoutcomes . In miceotection . Quantitreatment . Unilatepallidus . Some re ganglia  and redu ganglia . Based o2) was used to estimate the axonal transport rate, and anterograde axonal transport was significantly reduced in the rhinencephalon of ALS mice but returned to normal after acute treatment with davunetide (a neuroprotective compound that facilitates microtubule stabilization and repair). In addition, tau hyperphosphorylation associated with microtubule dysfunction and impaired axonal transport was observed in ALS mice, and chronic treatment with davunetide significantly reduced tau hyperphosphorylation  were significantly lower than those in the control group, and the change in axonal transport was associated with visual function and structural damage  and then gradually increases (diabetes for more than 5.5 months)  . Early d months) .2+ influx caused by hypoxic depolarization. The area with an enhanced Mn2+ signal is the central area of ischemia and is smaller than the area detected using the apparent diffusion coefficient (ADC). MEMRI is expected to become the main imaging method used to detect cerebral ischemia in the hyperacute phase , suggesting disruptions in the neural connections in the motor cortex  can detect early excitotoxic damage and early signs of BBB destruction before vasogenic edema; thus, MEMRI allows earlier and more sensitive detection than T2WI. In the subacute phase, MEMRI detects reactive astrocyte proliferation around the lesion (2+) has also been used to diagnose explosive blast-induced TBI, and an increase in MEMRI signal intensity serves as a biomarker for mild-to-moderate explosive blast-induced TBI  and manganese superoxide dismutase (Mn-SOD) (these two enzymes are conjugated enzymes that protect against glutamate toxicity and oxidative stress during neurodegeneration), and MEMRI detected late-stage mild HIE in damaged gray matter, which cannot be detected using conventional methods . Mn2+ enhancement occurred in the cortex, basal ganglia, and hippocampus in the medium phase (starting from day 3), which was associated with increased local Mn-SOD and GS activity. However, in the late stage, the enhanced region was mainly confined to the basal ganglia and areas around the cortex, and the signal gradually weakened  showed no increase in enhancement on days 1 and 3, but Mn2+ enhancement increased in the cortex, hippocampus, and thalamus on day 7. Histology revealed delayed neuronal death and inflammation in these areas, and a large number of activated microglia were present in these regions with high enhancement. Based on these findings, late-stage Mn2+ enhancement may be associated with inflammatory processes rather than cell death  and decreased cell density after prenatal radiation exposure -, pilocarpine-, and pentylenetetrazol-induced epilepsy models, which are divided into an acute phase and a latent phase. Different stages of disease development are associated with different neurobiological changes, such as hippocampal sclerosis, mossy fiber sprouting, inflammation, and neurodegeneration .2+ through the tail vein) study showed an increase in the enhancement of the CA3 pyramidal cell layer after KA treatment that was associated with astrocyte proliferation, and the enhancement on MEMRI was reduced after treatment with the L-type calcium channel blocker diltiazem due to focal edema and decreased neuronal swelling , and this reduction was associated with cerebral edema . The changes in the MEMRI signal in the hippocampus were attributed to an increase in axonal density following mossy fiber sprouting rather than neurodegeneration or proliferation of astrocytes or microglia  is an important health issue among society. MEMRI is sensitive to neuronal activities affected by drug abuse and is a very useful for understanding the mechanism and treatment of drug abuse. Dudek et al. demonstrated the extensive brain activation associated with voluntary alcohol drinking in rats using MEMRI, which may be a useful imaging tool for investigating modulation of alcohol-related brain activation by drugs . They al2+ signal intensity is significantly correlated with motor function, and no enhancement is observed at the injury site or at the caudal end (in vivo at different stages (2 injection) was positively correlated with the myelin load in the acute phase of SCI (2 injection) showed an interruption in spinal cord enhancement in the thoracic region, and histology confirmed a more significant reduction in the number of neurons in the ventral horn than that in the dorsal horn (2 injection) was significantly higher in rats with acute SCI than that in the control group after short-term erythropoietin treatment. The intensity of the MEMRI signal was significantly correlated with the recovery of function in rats, which is potentially useful for early monitoring and treatment  is another serious CNS disease with high mortality and disability rates. MRI has important application value in SCI. MEMRI has been used to assess the type, extent, and dysfunction conditions of SCI and to track the integrity of the corticospinal tract , 59, 64.udal end . By compt stages . MEMRI we of SCI . In the sal horn . MEMRI hreatment . Similarreatment , 69.2+ is a calcium ion analog that enters cells and represents a unique functional imaging tool used to visualize changes in vivo, allowing the detection of subtle, early changes in the body. MEMRI has been used to assess cellular/structural integrity, functional activity, and neural connectivity, thus enabling early detection of neuronal function, the intracellular ion balance, and axonal transport. MEMRI also helps determine disease severity and evaluate treatment efficacy.This review describes the applications of MEMRI in CNS diseases and provides a visual approach to study the pathophysiological processes of such diseases. Mn2 limit the use of MEMRI in humans, and only a few studies have used the Food and Drug Administration (FDA)-approved agent mangafodipir in humans. When conditions permit, local administration of a low dose is the preferred method due to reduced toxic effects. The sensitivity of PET is much higher than that of MRI, and PET uses a lower dose of manganese for imaging. In particular, the emergence of Mn-PET/MRI will be beneficial for clinical applications of Mn2+ (2+, these contrast agents will provide broad prospects and endless possibilities for MEMRI clinical applications.As in-depth studies are conducted, MEMRI will have increasing applications in preclinical research on the nervous system, which may reveal the relationship between neurological function and injury and between plasticity and repair. The combination of MEMRI with other imaging methods will provide complementary techniques to reveal the pathophysiological processes of diseases. However, the toxic effects of MnCl of Mn2+ , 181. FuJY proposed the topic and wrote the manuscript. QL edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Therefore, combining both aging research and appropriate interrogation of animal disease models towards the understanding of the disease and age-related stem cell failure is imperative to the discovery of new therapies. This review article will highlight the main intrinsic and extrinsic regulators of neural stem cell (NSC) aging and discuss how these factors impact normal homeostatic functions within the adult brain. We will consider established in vivo animal and in vitro human disease model systems, and then discuss the current and future trajectories of novel senotherapeutics that target aging NSCs to ameliorate brain disease.The adult central nervous system (CNS) contains resident stem cells within specific niches that maintain a self-renewal and proliferative capacity to generate new neurons, astrocytes, and oligodendrocytes throughout adulthood. Physiological aging is associated with a progressive loss of function and a decline in the self-renewal and regenerative capacities of CNS stem cells. Also, the biggest risk factor for neurodegenerative diseases is age, and current  NSCs are mitotic cells characterized by symmetric divisions  during early development. They gradually shift to asymmetrical division to generate differentiated cells and maintain a multipotent reservoir Gage, . AlthougIn adult mice, NSCs residing within the specialized niches are known to play important roles in maintaining cognitive functions, such as learning and memory formation, and contributing to repair and regeneration of injured tissue, which includes their neurogenic capability  response and initiates the secretion of pro-inflammatory cytokines, proteases, and immune modulators inhibiting NSC proliferation and leading to age-associated chronic inflammation  are DNA elements that have been copied and pasted from one location to another and can also give rise to genomic instability through mutations and double-strand breaks which in turn impacts gene regulation  within the FOXO3 gene that is associated with extreme longevity in humans DNA. A higher incidence of mtDNA mutations is known to be associated with physiological brain aging and in age-related neurodegenerative diseases such as AD and PD , a DNA repair protein that restores damaged mtDNA. This repair machinery is important in maintaining NSC multipotency as evidenced in NSCs from Ogg1 knockout-mice which spontaneously accumulate mtDNA damage, shifting their differentiation pattern towards an astrocytic lineage vs. neuronal  in mice produces histological and pathological hallmarks of aging in the hippocampus of mice, including impaired proliferation and neurogenesis in NSCs  are a family of proteins that form channels within the nuclear envelope, thus regulating the flow of macromolecules, and have emerged as epigenetic regulators of NSC differentiation  occurs to meet the new energy demands of the cell  are transcription factors that regulate the cellular response to changes in oxygen levels, of which HIF1α plays a prominent role in NSCs. Conditional deletion of HIF1α in NSCs of adult mice resulted in a loss of NSC proliferation within the SVZ, highlighting its importance in maintaining the adult NSC niche , and lymph which can affect the NSC niche . The bloDue to the SVZ being uniquely situated above the lateral ventricles, exposure to blood and CSF factors routinely occurs. Systemic factors have been identified in aged mice that promote age-induced dysfunction, such as C-C motif chemokine 11 (CCL11), a chemokine increased in the blood of aged mice which directly correlates with a decrease in neurogenesis. Systemic exposure of young mice to recombinant CCL11 impaired hippocampal neurogenesis, which is rescued with a CCL11 blocking antibody. Interestingly, CCL11 is increased in aged human plasma and CSF  is comprised of a microvasculature unit of brain endothelial cells (BECs), which are exposed to circulating factors on their luminal side and brain cells on their abluminal side. BECs in aged mice express an inflammatory transcriptional profile, including upregulation of vascular cell adhesion molecule 1 (VCAM1). Soluble VCAM1 is increased in the plasma of both aged humans and mice. Plasma collected from aged mice and infused into young mice induced a significant increase in VCAM1 expression in the BEC population, which caused microglial activation and decreased the amount of proliferating NSCs. Genetic deletion of Aging and neurodegenerative diseases are associated with impaired integrity of the BBB, which creates a more permissive environment for the entry of peripheral immune cells into the brain parenchyma  directly impacting local cell networks  to reach a similar expression level to that of wild-type mice at 24 months of age. NSC proliferation and maturation were significantly reduced in BubR1H/H mice impairing hippocampal neurogenesis despite having no impact on neuronal survival , rescued NSC proliferation in the hippocampal DG in these mice  showed a complete loss of Terc expression and telomerase activity , a catalytic subunit of the telomerase complex, induces aging phenotypes similar to that observed in Terc−/− mice, such as impaired spatial memory, dendritic development, and neurogenesis  somatic telomerase reactivation reversed these observed deficits through the restoration of proliferating Sox2+ NSCs and new-born neurons, consistent with the integral role of SVZ NSCs in the generation and maintenance of mature neurons in adulthood  and the main source of NSC NAD+ required for cell-cycle progression, reduced the NSC pool and their proliferation in adult mice , associated with reductions in quiescent NSCs and a loss of self-renewal in the adult SVZ , familial PD (mutations in Parkin and PINK1), and HD (HTT mutation), yet many sporadic forms of these and other diseases occur with increasing age, such as sporadic PD, ALS, and progressive MS  rather than somatic aged brain cells, which are rare and difficult to obtain. Additionally, human post-mortem samples reflect cell states at the end stage of the disease, hindering the study of disease pathogenesis. To generate iPSCs, primary human cells, such as fibroblasts or blood cells, are reprogrammed to an embryonic-like state, similar to embryonic stem cells (ESCs), through the expression of Yamanaka transcription factors. iPSCs are then given cues to direct differentiation towards the mature cell of interest to model disease. Typically, iPSCs are used to model diseases with genetic origins, such as ALS  were reset, and cells retained a youthful phenotype , retains 5–30% of the original aged methylation pattern according to the Horvath model, making them an ideal candidate to study aging both in the context of health and disease , which are further differentiated Induced motor neurons (iMNs) from primary fibroblasts or iPS-NSCs retain many of the aging hallmarks of the old donors, including increased DNA damage foci (γH2AX), SA-β-galactosidase activity, and loss of heterochromatin and nuclear organization  gene, which was identified in HGPS. Modification of the LMNA transcript results in a truncated form of lamin A called progerin. Progerin acts to shorten telomeres, inflict DNA damage, and encourage cellular senescence. Interestingly, iPSCs from patients with HGPS do not show an accumulation of progerin and lack the cellular alterations associated with accelerated aging, however, this phenotype is reinstated following iPSC differentiation into smooth muscle cells  robustly induces cellular senescence identified by increased p53 and p16Ink4a expression, SA-β-galactosidase staining, and downregulation of DNA repair proteins, which has also been replicated in human NSCs in vitro , used for the treatment of leukemia, disrupts cellular proliferation, and has been found to induce senescence in human cells  protein  to inhibit mTOR signaling  signaling and suppressed IL-1RA levels  phenotype in a presenilin mouse model of AD-related tauopathy  ligand 3 (CCL3) are correlated with chronological age and cognitive decline , allowing researchers to study in detail how changes alter cellular and organismal aging. In vitro models using novel stem cell technologies also hold potential for studying NSC aging in humans as they retain aging and disease markers. Defining the mechanisms that trigger modifications in this cell population with age and in disease will allow for a more directed approach to developing preventative or curative treatments.There is no single mechanism to aging, and multiple processes likely contribute to declining. Therefore, using models in which we can parse these details is important to understanding the fundamentals of this process. Interrogating how aging may differ from diseases associated with aging, both transcriptionally and proteomically, would allow us to better understand the discrepancy between those who do and do not develop the disease, but also identify what facets of aging portend disease. This includes AN designed review outline, wrote manuscript, and designed figures. CW and SC contributed sections of manuscript. CW, SC, and SP critically reviewed manuscript.SP is co-founder and CSO at CITC Limited and iSTEM Therapeutics, and co-founder and Non-executive Director at Asitia Therapeutics. AN is an advisor for iSTEM Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antigen receptor recognition is a key event that both initiates lymphocyte activation and impacts the fate of immune responses. Co-receptors and co-stimulatory molecules coordinate with antigen receptors to amplify and transduce the initial signals initially triggered at the cell surface, and integrate subsequent signals required to sustain responses. Equally important are the molecular mechanisms that control and terminate activation responses, which act at multiple levels to attenuate activation, modulate signaling thresholds for activation, and shutdown responses in the face of chronic antigen receptor engagement. A very fine line separates activation-mediated clonal expansion from cell death by apoptosis at all stages of lymphocyte development and inhibitory receptors and their associated signaling molecules can determine the difference between life and death. Lymphocyte differentiation and acquisition of effector functions is continuously modulated by a variety of receptors and signaling pathways which balance quiescent, activated and exhausted phenotypes as well as cytolytic activity and cytokine production patterns. Inhibitory receptors and signaling molecules collectively have critical functions to integrate multiple environmental inputs to modulate immune cell activation states in a variety of tissues. As discussed in the reviews in this Topic, inhibitory receptor function can tip the balance between health and disease in a variety of contexts involving chronic lymphocyte activation.De Sousa Linhares et al. provide an excellent overview, summarizing current evidence regarding the unique features and clinical significance of many of these receptors, as well as discussing the remaining open questions. Yasuma-Mitobe and Matsuoka discuss studies implicating these receptors in retroviral infections such as HIV. Morris et al. discuss the function of these inhibitory receptors in the context of CD8+ T cell memory, recall responses and terminal differentiation. Brunner-Weinzierl and Rudd discuss the roles of PD-1 and CTLA-4 in tumor immunotherapy, focussing on their roles in controlling T cell migration via impact on integrin activation and chemokine receptor expression and signaling. Gianchecchi and Fierabracci comprehensively review evidence regarding the roles of PD-1 and its ligand PD-L1 in regulatory T cell (Treg) development and functional activity, and address how this may relate to development of autoimmunity. Paluch et al. address the role of both PD-1 and CTLA-4 in autoimmunity, discuss the rationale for checkpoint receptor agonists as treatment for this class of diseases and outline several different approaches being used in the design of checkpoint agonists.Several reviews in this Topic address the biological functions and clinical applications of Ig superfamily “checkpoint receptors” PD-1, CTLA-4, BTLA, TIGIT, 2B4, Tim3, and LAG-3. Georgiev et al. provides a focused review of CD96, another type 1 transmembrane glycoprotein of the Ig superfamily. CD96, together with TIGIT and other receptors, form a distinct sub-group of regulatory Ig superfamily receptors that are relatively less studied, but likely play regulatory roles in T cells and NK cells. Kim and Kim also address the role of CD96 in NK cell function and discuss the role of established checkpoint receptors such as PD-1, CTLA-4, TIM-3, and LAG-3 in NK cells. These authors also address the role of killer inhibitory receptors (KIR) and CD94/NKG2A in mediating the unique regulatory mechanisms of NK cells.The article by Gonçalves et al. and Voisinne et al. address the scavenger receptor cysteine-rich (SRCR) glycoproteins CD5 and CD6 and their differential roles in T cell development and responses. The significance of ligand binding in signal modulation is explored, as well as the inhibitory signaling mechanisms. The therapeutic potential of targeting these receptors for immunotherapy is discussed.Tsubata has reviewed data regarding the roles of the Siglec receptors CD22, CD72, and SiglecG in development and function of B cells, focusing on how distinct ligand-recognition properties of these receptors determine their functional roles. Clark and Giltiay comprehensively review literature regarding the roles of CD22 in B cell activation, migration, tolerance and autoimmunity, as well as evidence regarding therapeutic targeting of CD22.Sialic acid-binding Ig-like lectins (Siglecs) are another important family of inhibitory receptors in lymphocytes. Pike and Tremblay review the roles of protein tyrosine phosphatases such as PTPN22 in controlling CD4+ T cell activation and function, particularly in the context of intestinal inflammation and inflammatory bowel disease. The role of SHP-1 protein tyrosine phosphatase recruitment via receptor ITIM motifs is touched on by several other reviews, while some authors indicate that early implication of this phosphatase turned out to be incorrect . The role of cbl family ubiquitin ligases in CD5/6 function is explored by Voisinne et al. and Gonçalves et al.. These authors also touch on the roles of the inhibitory tyrosine kinase Csk which controls the activity of Src kinases.Different classes of signaling molecules mediate the inhibitory functions of these receptors. Rodríguez-Galán et al. uniquely explore the interesting topic of immune regulation mediated by miRNA networks. They discuss evidence that miRNAs can control not only expression of inhibitory receptors such as PD-1 and CTLA-4 in lymphocytes, but also inhibitory protein phosphatases and phosphoinositide phosphatases, critical cell survival and cell cycle regulators as well as co-stimulatory receptors and cytokines.Together, the excellent reviews collected under this Topic only begin to capture some of the breadth and depth of research in this fascinating and important area. It is clear that “Inhibitory receptors” is somewhat of an over-simplification to describe the nuances of how these receptors function to maintain homeostasis within the immune system. Their roles extend beyond modulation of initial lymphocyte activation signals, with perhaps their greatest impact being in balancing the needs of host defense, avoiding excessive immunopathology, and maintaining normal physiology during infection and chronic inflammation. Future studies will no doubt address how these receptors each function within different tissue contexts and in different contexts of physiological and metabolic stress. This will require better understanding of individual receptor/ligand dynamics and how expression of receptors and ligands are modulated in different locations and contexts.Therapeutic targeting of inhibitory circuits in lymphocytes will no doubt continue to be a major focus over the coming years. Creative approaches to targeting at the level of receptors, ligands and signaling molecules will be guided by improved understanding of the relevant molecular and cell biology. One issue in therapeutic targeting is the clear functional redundancy among some receptors and ligand systems, which may require targeting multiple molecules to achieve biological impact. Targeting at the level of inhibitory signaling molecules may in some cases offer opportunities to bypass receptors and manipulate lymphocyte functions via small molecule inhibitors or activators of signaling molecules, or perhaps miRNAs. A limitation that has become very apparent with therapeutic targeting of checkpoint receptors is that disturbing the immune ecosystem by blocking inhibitory pathways can have unintended consequences. Freeing the immune system from control networks that have evolved to maintain homeostasis may cure one disease, but create another. This underlines the need for a deeper understanding of how these inhibitory networks function at the molecular, cellular, tissue and whole organism level. Ultimately the goal is to more selectively release or restore these powerful regulatory systems to reset the ecosystem for maximum therapeutic benefit.AC, PL, and AM served as co-editors of this topic and wrote the editorial. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a shared affiliation, though no other collaboration, with one of the authors PL at time of review.


Developing predictive biomarkers indicating response or resistance to therapy paved the way for broad clinical implementation of personalized therapies by tyrosine kinase inhibitors  in many DNA excision repair protein ERCC1 together with ERCC4 forms the ERCC1-XPF enzyme complex that participates in DNA nucleotide excision repair (NER) and DNA recombination . MeasuriIn this context, the manuscript of Takashima and colleagues in this issue  providesIn their landmark study, Takashima and colleagues used a reliable antibody and established a semi-quantitative scoring system of SLFN11 to assess SLFN11 expression in approximately 700 malignant tumors as well as in adjacent non-tumorous tissues across 16 different human adult organs. Thereby, the authors provide a reference repository of SLFN11 expression in human cancers and tissues. In addition, they assess the expression of SLFN11 in The Cancer Genome Atlas (TCGA) repository and unravel important discrepancies. Assessing SLFN11 by whole tissue-gene expression transcriptomes largely overrates SLFN11-negative tumors as TCGA samples are a mixture of tumor cells and infiltrating immune cells, including T cells, B cells, and macrophages, that have strong SLFN11 expression. In summary, the study of Takashima and colleagues in this issue will certainly trigger further studies of clinically annotated tumor samples and their extent of regression after chemotherapy. Ultimately, establishing such a clinically useful and easy assay of prediction of chemoresponse should also push forward concepts of neoadjuvant therapies. It is rewarding to see that immunohistochemistry, the good old working horse of diagnostic pathologist, leads the way to improve precision of chemotherapies and has a role even in times of large cancer genome and transcriptome studies.


MHC class I polymorphism requires their ligands either to interact with conserved epitopes, as is utilized by the NKG2A receptor family, or to co-evolve with the MHC class I allelic variation, which task is taken up by the killer cell immunoglobulin-like receptor (KIR) family. Multiple molecular mechanisms are responsible for the diversification of the KIR gene system, and include abundant chromosomal recombination, high mutation rates, alternative splicing, and variegated expression. The combination of these genetic mechanisms generates a compound array of diversity as is reflected by the contraction and expansion of KIR haplotypes, frequent birth of fusion genes, allelic polymorphism, structurally distinct isoforms, and variegated expression, which is in contrast to the mainly allelic nature of MHC class I polymorphism in humans. A comparison of the thoroughly studied human and macaque KIR gene repertoires demonstrates a similar evolutionarily conserved toolbox, through which selective forces drove and maintained the diversified nature of the KIR gene cluster. This hypothesis is further supported by the comparative genetics of KIR haplotypes and genes in other primate species. The complex nature of the KIR gene system has an impact upon the education, activity, and function of NK cells in coherence with an individual’s MHC class I repertoire and pathogenic encounters. Although selection operates on an individual, the continuous diversification of the KIR gene system in primates might protect populations against evolving pathogens.The activity and function of natural killer (NK) cells are modulated through the interactions of multiple receptor families, of which some recognize MHC class I molecules. The high level of  MHC class I genes and alleles requires the NK cell receptors to co-evolve to maintain a functional relation with their ligands. The recognition of MHC class I molecules by NK cells involves two receptor families: the conserved CD94:NKG2A receptors and the highly polymorphic and diverse killer cell immunoglobulin-like receptors (KIR). Both receptor families consist of inhibitory and activating members. Their engagement with MHC class I molecules calibrates the responsiveness of NK cells through a continuous educational process, which largely controls subsequent NK cell activity  and Old World monkeys, and how this might influence their NK cell response.The innate and adaptive arms of the immune system are interconnected, and feature several effector functions that provide efficient and specific protection against infection and tumor formation. Major components of the adaptive arm comprise T and B lymphocytes characterized by rearranging antigen receptors, which exert cytotoxic and humoral immunity, respectively. The cytotoxicity mediated by T lymphocytes highly depends on the presentation of intracellular antigen segments derived from pathogens by MHC class I molecules and subsequent clonal expansion of cells with specific receptors. A third type of lymphocytes bridge the innate and adaptive immune response, and comprises natural killer (NK) cells, which participate, for instance, in the recognition and elimination of aberrant cells that down-regulate their MHC class I expression to evade detection by T lymphocytes . Withoutactivity , 4. The activity  5). Bas. BasMHC utations . DiffereKIR gene system requires a comprehensive nomenclature guideline for the different genes and allotypes in order to distinguish the corresponding receptors by their structure and signaling potential (KIR3DP). In addition, a four-character species designation is included in front of the KIR acronym .The complex otential –9. RecepKIR genes originate from two progenitor gene lineages: KIR3DX and KIR3DL. The KIR3DX lineage is represented by a single gene copy located in the center of the LILR gene cluster  is defined by the specificity for subtypes of HLA-A and -B in humans; lineage III includes receptors with D1-D2 and D0-D1-D2 domain configurations; and lineage V (D0-D1-D2) is represented by human KIR3DL3 and its orthologs. In the primate species studied, at least one KIR gene was discovered for each lineage, which indicates that gene duplication and diversification predates primate speciation. The subsequent lineage expansions are, however, species specific ..KIR geneMHC-A and -B genes, as opposed to the fixed number of MHC class I genes in hominoids , or may be pseudogenes. The differential transcription suggests a more classical function for the major MHC molecules, such as antigen presentation, whereas the minors might exert more specialized functions . Its presence on 22% and 82% of the rhesus and cynomolgus macaque KIR haplotypes, respectively, suggests a balancing selection for this structurally modified receptor, which might execute a function other than conventional MHC recognition .–18.MHC-Aunctions , 20. Onlunctions  21–25).–25.MHC-Aectively  7). Lik. LikMHC-pertoire –28. Lineognition .MHC class I genes in most hominoids and the specialization of MHC-C as ligand for lineage III KIR is in line with their modest KIR gene expansion  and KIR1D (lineage III). These recombination events are not conserved in macaque populations, which implies the relatively recent formation of novel gene entities propelled by ancient recombination hotspots , whereas the number of reported alleles for different non-human primate species ranges from 521 KIR alleles in rhesus macaques to 5 KIR alleles in Bornean orangutans . These allele numbers may give a distorted view of the actual levels of polymorphism within a species due to the differential number of individuals studied. The high level of allelic polymorphism appears to be at least comparable in humans and macaques. The thoroughly documented allelic polymorphism in humans and macaques reveals a varying number of alleles per KIR gene, with most nucleotide variation exhibited by the framework genes  and the highly frequent inhibitory KIR genes in macaques . An expansion of the allele numbers for the frequently expressed KIR genes might indicate a continuous role in co-evolution with particular pathogens. The less common KIR genes, which include mostly activating KIR, vary in gene content rather than allelic polymorphism and therefore seem to execute more specialized functions and/or might be involved in the recognition of conserved ligands and peptides  , and this number is likely to be underestimated  , which mstimated . Sequence genome . For exaactivity . For HLAnfection .KIR genes are evenly distributed over the coding regions. The high concentration of CpG islands located in the KIR gene cluster might contribute to an elevated mutation rate, as these islands are in general more prone to promote nucleotide transitions .–83.KIR gsplicing  84). Th. ThKIR gsplicing  85). In. InKIR gKIR genes, and implicate the existence of conserved splice events that generate structurally and functionally distinct isoforms. For example, exon 4 (coding for the D1 domain) is frequently skipped from KIR3DL20 transcripts in macaques, thereby generating transcripts that encode both the complete receptor and receptors with a D0-D2 domain configuration  additional to KIR2DL04. The most frequent KIR splice event in humans involved the skipping of exon 6, which encodes the stem region. Other frequent events included the skipping of exon 5 (D2 domain) and partial deletions in exons 4 and 5. These events result in isoforms that are likely to display altered binding properties, but their exact activity and localization remains elusive. Another common splice event in humans might function as a regulatory switch for expression of the 9A and 10A KIR2DL4 alleles by restoring or disrupting the open reading frame (ORF)  . Less frme (ORF) . Data onKIR genes, for example, requires the constitutive skipping of exon 3 to maintain an ORF. This exon contains a deletion of 5 bp at the genomic DNA level, which would shift the reading frame that introduces an early stop codon , could be expressed in a mono- and multi-allelic manner. This may generate NK cell subsets that transcribe two or more allelic copies of a certain KIR gene  protein , and thereby induce the expression of formerly silenced KIR genes  residue present at this position, whereas in general this position is occupied by threonine (−21T) in MHC-B molecules. The -21M peptides strongly bind to MHC-E molecules and promote cell surface expression of MHC-E complexes . The preKIR gene system. We think that the primary function of macaque KIR is focused on the recognition and elimination of infected or malignant cells. This defense mechanism relies on the recognition of Bw4 and Bw6 epitopes, but KIR interactions are also sensitive to non-self peptides that can be presented by MHC class I molecules (KIR genes provides a broader repertoire to scan all the variable MHC class I allotypes in combination with their peptides originating from pathogens. It has been proposed that up to seven distinct KIR receptors are required for successful peptide recognition (KIR gene system. Considering their KIR-independent NK cell education, KIR expansion in macaques might be exempted from potential negative selection on large KIR gene repertoires.In primate species with a KIR-dependent NK cell education, one can envision that an expanded KIR repertoire may compromise NK cell activity. This might drive selection for a limited KIR expansion, as we will discuss in the next section. If this reasoning is true, the KIR-independent education of NK cells in macaques might result in an extensive expansion of their olecules –138. A lognition . This opKIR haplotypes contain a large number of genes, and they even display indications for contraction by chromosomal recombination events. The formation of novel gene entities by the shuffling of head- and tail-encoding exons is achieved by recombination events, which are coherently accompanied both by contractions and expansions of KIR haplotypes. There might be a trade-off between the expansion of the overall KIR repertoire in a population by generating fusion genes and the contraction of KIR haplotypes in individuals. Rapid expansion and diversification generate a highly plastic macaque KIR gene system that appears to be maintained by selection to militate against rapidly evolving pathogens.However, not all macaque KIR gene content per haplotype.As compared to macaques, hominoids appear to have a more limited haplotype content and overall KIR repertoire . These lA large KIR repertoire is likely to provide a broad array of MHC class I specificities that may result in the education of an increased fraction of NK cells . MoreoveKIR gene cluster. As far as we know, only few human and no non-human primate individuals are documented that were homozygous for their KIR haplotypes at an allele level (KIR-homozygous according to segregation. However, more detailed analysis illustrated that one KIR gene copy appeared to have gained point mutations that resulted in the haplotypes diverging at an allele level (KIR-heterozygosity is not vital. However, KIR haplotype diversity might compensate for limited KIR haplotypes and improve the immune surveillance, as is also described for MHC heterozygosity (In contrast, individuals that have a limited KIR haplotype rely on only one or few self-specific KIR receptors to educate their NK cells . Even thle level . In a rhle level . This inzygosity –152.MHC class I specificity.In contrast to non-self-specific T lymphocytes, which are depleted upon a failed positive or negative selection in the thymus, uneducated NK cells are present in the peripheral blood. The relatively high level of uneducated NK cells in individuals with small or large non-self-specific KIR repertoires could affect their immune surveillance, but does not preclude an efficient immune response during infection or tumor formation. In fact, unlicensed NK cells appear to be more efficient at eradicating infected or malignant cells that persistently express MHC class I molecules or viral mimic ligands through their reactivation by cytokines or NKG2D receptors –155. TheThere could be another factor, however, that limits expansion of the KIR haplotypes and gene repertoire, in addition to their role in NK cell education. In orangutans, MHC-B allotypes contain a -21M leader peptide, which would suggest education via the conserved CD94:NKG2A pathway . In contKIR haplotype content and overall gene repertoire in hominoids and Old World monkeys are likely to affect the education, activity, and function of their NK cells, but the precise effect of the haplotype expansions and contractions remains ambiguous. The equal distribution of both small and large KIR repertoires in humans and macaques indicates a balancing selection, which might be an ongoing process to achieve a haplotype equilibrium that serves differential functions, such as fighting infections and promoting successful pregnancy.Nevertheless, the diverse KIR gene system is well studied in humans, and reveals multiple mechanisms that contribute to the plasticity of this immunogenetic cluster (KIR gene cluster in macaques exceeds that observed in hominoids, with a prominent expansion of the lineage II KIR genes, which is largely mediated by recombination events. The rapid evolution of the KIR gene cluster may counteract the adaptive nature of pathogens. The species-specific diversification of the KIR gene cluster might be largely driven by co-evolution with their diversified MHC class I repertoire and thereby indirectly by the arms race with pathogens. In addition, a KIR-dependent or -independent NK cell education might impact the variable haplotype content and the extent of KIR gene expansion. Nevertheless, the different molecular mechanisms responsible for diversification of the KIR gene cluster are shared in Old World monkeys and hominoids, which suggests an evolutionary effort to diversify the KIR gene system.The  cluster . In otheJB wrote the manuscript. NG and RB edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.