Advancements in the early detection of cancer coupled with improved surgery, radiotherapy, and adjuvant therapy led to substantial increase in patient survival. Nevertheless, cancer metastasis is the leading cause of death in several cancer patients. The majority of these deaths are associated with metastatic relapse kinetics after a variable period of clinical remission. Most of the cancer recurrences are thought to be associated with the reactivation of dormant disseminated tumor cells (DTCs). In this review, we have summarized the cellular and molecular mechanisms related to DTCs and the role of microenvironmental niche. These mechanisms regulate the dormant state and help in the reactivation, which leads to metastatic outgrowth. Identification of novel therapeutic targets to eliminate these dormant tumor cells will be highly useful in controlling the metastatic relapse-related death with several cancers. Metastasis is a continuous biological process consists of an orderly sequence of basic steps including local invasion, intravasation, extravasation, and colonization. These classical events of metastasis help in understanding the complex array of biological properties that are necessary for the progression of primary malignancy to overt metastasis , 2. It iTumor dormancy is a clinical process that eventually associates with local recurrences or cancer metastases. During this process, the residual disease might be present even after the treatment of primary tumors either in the forms of CTCs, DTCs, and/or micrometastases which have the capability of evading the treatment and survive in a quiescent state. Traditional chemotherapies are most effective on proliferative cells, however, ineffective toward the dormant cells . The dorSeveral signaling pathways such as RAS-MEK-ERK/MAPK and PI3K-Akt play a crucial role during the process of cancer dormancy . AdditioSeveral reports suggest that cancer cells undergo a protracted period of dormancy by the inhibitory molecular cues derived from primary tumors or restrains caused by target organ microenvironment –27. Bidi+ and bone marrow-derived CD11b+Gr1+ myeloid cells are involved in premetastatic niche formation , known as metastasis-associated macrophages (MAMs), are enriched in metastatic breast cancer as compared to primary tumors. Flt1-regulated signaling in these MAMs upregulates inflammatory gene signature which is imperative for cancer cell survival during metastatic seeding . In addiormation –36. Myelormation . CYP4A-iid cells . Other sid cells . Furtherogenesis , 39, 40.Epithelial to mesenchymal transition-driven mesenchymal features in cancer cells enable them to invade and metastasize to the distant organs. Several studies suggest that EMT-inducing transcription factors such as Twist and Snail show inhibitory effects on cancer cell proliferation, however, these factors induce migratory potential by downregulating the cadherin junctions . A reverMetastatic dormancy is a result of growth arrest either in a single DTC termed as cellular dormancy or in micrometastatic lesions called as tumor mass dormancy. Cellular dormancy marked by a quiescent state in DTCs is associated with the decline in Ki67 expression, a proliferation marker or G0/G1 cell cycle arrest. There are various cellular and molecular mechanisms through which DTCs undergo dormancy which is discussed below.Mitogen, stress signal, and other factors present in the premetastatic niche may be responsible for cell cycle arrest and dormancy. Crosstalk between mitogen and stress-induced signaling pathways are crucial for cellular dormancy. Studies have shown that a set of genes selectively affects the growth at the secondary site including MKK4, MKK6, and Nm23-H1. Interestingly, MKK4 and MKK6 are upstream activators of p38 while, Nm23-H1 indirectly downregulates ERK1/2 by inhibiting EDG2 LPA receptor, a strong activator of ERK1/2 . Hence, Several reports have shown that the upregulation of various UPR-associated genes like Grp78, Grp94, PDI, heat shock protein 47 (HSP47), and cyclophilin B in dormant cells play a crucial role in metastatic dormancy –58. RangMicroenvironmental factors like BMPs and growth arrest-specific 6 (GAS6) derived from mesenchymal cells and osteoclasts, respectively, can curb proliferation and induce dormancy in cancer cells Figure . By usinhi/DEC2hi/p27hi/TGFβ2hi population with dormant phenotype. NR2F1 and HIF1α involved in the regulation of p27 expression in posthypoxic dormant DTCs. Moreover, hormone receptor-dependent breast cancer cells exhibit higher affinity toward NR2F1-dependent dormancy  and primary breast tumor. Posthypoxic solitary DTCs in patient-derived xenografts and transgenic mice show NR2F1dormancy . Harper g Figure . Interestastasis . These dmiRNAs play an important role in the various biological process. It has been shown that miRNAs may affect the hallmarks of cancer, including sustained proliferation, blocking growth inhibition signals, resisting cell death, inducing invasion, metastasis, and angiogenesis . Ono et Autophagy is an extremely conserved self-degradation process, which has an important role in cancer stem cells (CSCs) regulation and tumor cell survival. Several reports suggest that DTCs possess CSCs properties, which prompted researchers to explore the potential role of autophagy in cancer cell dormancy and stress response. Various groups have shown that autophagy helps in the survival of DTCs for protracted periods , 69. Aut+ cells coexists with dormant cells, proposing that for the reactivation defined niches are essential (In contrast to single cell dormancy, tumor mass dormancy is governed by a balance between the rate of proliferation and apoptosis in micrometastatic lesions. The tumor mass dormancy is induced by slow proliferation, restrained blood supply and active immune response. Recent studies reveal that the frequency of osteolytic bone metastasis depends on metastatic niche environment rather than the number of cancer cells , 75. Morssential , 78. Unfin vivo conditions (in vivo dormancy while its upregulation results in dormancy exit and enhanced vascular density (Micrometastatic lesions require the higher blood supply to grow beyond 1–2 mm, which leads to the induction of vessel formation by secretion of angiogenic factors like VEGF . Therefonditions . Chapero density .Clearance of tumor cells by immune system contributes to another mechanism of tumor mass dormancy. Cancer cells coevolve in a microenvironment where the immune system is suppressed. However, DTCs do not have such support and eventually, most of these cells die due to the natural immune response. It has also been reported that immune system regulates the number of DTCs as well as the size of micrometastatic lesions . AdditioDormant cancer cells may be subjected to reactivation to initiate metastasis in response to specific signals from their specialized niche, which maintains the balance between the self-renewal and production of differentiated progeny –88. Cancin vivo preclinical and 3D in vitro model of dormancy demonstrated that targeting the Src family kinase and MEK1/2 using their specific inhibitors resulted in apoptosis in a large fraction of the dormant cells and delayed metastatic outgrowth in breast cancer (Recent achievements in cancer therapy and increased overall survival motivate the researchers to look for new diagnostics for the patients at high risk of late metastasis and therapeutic system targeting DTCs. The limitation of current conventional and adjuvant therapies to prevent relapse, is they basically target growing tumor cells rather than DTCs. The systemic nature of the metastatic disease along with the heterogeneity of metastatic tumors, complex inter-connected pathways and the resistance against therapy makes its pharmacological management very difficult. Hence, there is a need to focus on preventing metastasis , 99. Bont cancer . Inhibitt cancer . TherefoImmunotherapy is being explored extensively for cancer management. Saudemont et al. have shown NK cells-based immune therapy targets dormant cells. Their study demonstrated that NK cells activated by CXCL10 can kill dormant tumor cells which are able to resist CTL-mediated lysis . As discTumor dormancy and reactivation has become an interesting point as a key element of tumor evolution and metastatic relapse. Although metastasis-initiating cells undergo dormancy and ultimately get reactivated under the influence of microenvironment signals, various key questions are still unanswered. It will be interesting to explore the phenotypic and functional similarities between DTCs and CSCs, the role of MET, microenvironmental niches and genetic and epigenetic changes in metastasis-initiating cells in metastatic reactivation. Current approaches and models to investigate the molecular basis of metastasis have been very successful. Nevertheless, new approaches need to be discovered in order to gain an in-depth understanding of tumor dormancy and reactivation. Lineage-tracing studies utilizing newly developed reporter systems can provide critical understanding in this area by using the transgenic mouse models which mimic the natural conditions. Moreover, recently invented genetic screening strategy can be useful in quick identification of mediators involved in dormancy and reactivation. Future studies need to be conducted to assess the efficacy of screening the shRNA libraries for the recognition of regulators of dormancy and their potential use in various tumor types and clinical samples.Advance strategies for characterization of various aspects of CTCs and better access to samples of metastases will be required to complete this goal. With the current progress in the field of metastasis, these questions will be addressed rapidly by designing and implementing the improved strategies for cancer treatment.DK, AY, PP, and RB drafted the manuscript, composed the figures, and critically revised the manuscript. DK and GK conceived the manuscript and finalized the draft. NR, SR, SB, and MG have written some part and revised the manuscript. All authors read and approved the final 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.


Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells critically dependent on microenvironmental stimulation for their survival and proliferation. For decades, research focused on the neoplastic B cells, revealing the central role of the B cell receptor (BcR) in CLL pathophysiology and leading to the recent development of drugs that exhibit remarkable clinical efficacy by inhibiting B cell signalling, critically including the BcR pathway. However, besides transducing signals provided through antigenic stimulation, the BcR molecule also serves as a channel of CLL cell communication with cognate T cells. Functional studies of the immune synapse in CLL have unveiled an intricate crosstalk of CLL B cells with T cells, culminating in the induction of T-cell anergy, thus allowing the neoplastic clone to escape immune surveillance , 2. MoreThat notwithstanding, little is known regarding the architecture of the T-cell repertoire in CLL. To address this knowledge gap and to further dwell into preliminary findings obtained by low-throughput techniques suggesting antigenic selection , we condOur study revealed the restricted nature but also the dynamics of the T cell repertoire in CLL, with clonotypes that persist and expand over time, thus alluding to persistent antigenic stimulation. An important finding concerned the existence of shared clonotypes amongst different patients, especially those belonging to the same stereotyped subset. Moreover, comparison to public databases indicates that these clonotypes may be disease-specific, altogether corroborating that antigenic elements may be selecting T cells in a CLL subset-specific context. On these grounds, we hypothesized that these could be the same antigens selecting the malignant clone or tumor-derived antigens. Recent evidence of homotypic BcR interactions underlying malignant B cell selection in CLL may in fact unify these scenarios into one, where epitopes expressed on the clonotypic BcR IG are recognized by both malignant B cells, triggering downstream BcR signaling, as well as cognate T cells, which clonally expand but somehow fail to mount a truly effective anti-tumor response  Figure .+ cytotoxic population. Moreover, the PI3Kδ inhibitor idelalisib may directly affect T-cell cytokine production, reducing inflammatory cytokines such as CD40L. In addition, BcR inhibitors may impact on T cells indirectly by interfering with the ability of the malignant B cells to induce T cell tolerance. Hence, arguably, novel drugs may exert anti-tumor action also through effects on the CLL T cells.This information can prove clinically relevant in the era of new drugs designed to stimulate a sustained anti-tumor effect via the host immune system rather than directly target the malignant cells, such as immunomodulatory drugs acting on the level of B-T cell synapse and immune checkpoint inhibitors. In fact, lenalidomide has recently been tested in CLL and demonstrated clinical benefit . That noLittle information exists on the dynamics of TR repertoire in CLL as a result of treatment, especially with novel agents. In particular, the single available publication involves only ibrutinib and reports that ibrutinib therapy increases diversification of the T cell compartment in CLL patients, contributing to cellular immune reconstitution . Our groOverall, T cells emerge as a critical player in CLL pathophysiology and have thus become an enticing target, which actually coincides with the development of novel drugs that harness the lymphoma microenvironment. In-depth immunoprofiling of the T cell repertoire, paired with functional characterization, may reveal mechanisms to abrogate T cell tolerance and translate directly into clinical practice, especially in the context of boosting the host immune surveillance for sustained MRD eradication.


Immune cells can significantly predict and affect the clinical outcome of stroke. In particular, the neutrophil-to-lymphocyte ratio was shown to predict hemorrhagic transformation and the clinical outcome of stroke; however, the immunological mechanisms underlying these effects are poorly understood. Neutrophils are the first cells to invade injured tissue following focal brain ischemia. In these conditions, their proinflammatory properties enhance tissue damage and may promote ischemic incidences by inducing thrombus formation. Therefore, they constitute a potential target for therapeutic approaches and prevention of stroke. Indeed, in animal models of focal brain ischemia, neutrophils have been targeted with successful results. However, even in brain lesions, neutrophils also exert beneficial effects, because they are involved in triggering immunological removal of cell debris. Furthermore, intact neutrophil function is essential for maintaining immunological defense against bacterial infections. Several studies have demonstrated that stroke-derived neutrophils displayed impaired bacterial defense capacity. Because infections are known to impair the clinical course of stroke, therapeutic interventions that target neutrophils should preserve or even restore their function outside the central nervous system (CNS). This complex situation requires well-tailored therapeutic approaches that can effectively tackle immune cell invasion in the brain but avoid increasing poststroke infections. Stroke is one of the leading causes of death in the world. Most stroke-related deaths result from thrombotic occlusion of brain vessels. Infections are a known risk factor for acute stroke . This enThe control of inflammation at the site of the ischemic lesion is a potential therapeutic target that has resulted in promising results in experimental stroke studies and may allow for a longer therapeutic window. Cellular invasion and the resulting proinflammatory response develop within days rather than hours and contribute to secondary lesion growth, which enhances ischemic brain tissue destruction. The role of neutrophils in these central events and corresponding therapeutic approaches are discussed in Another aspect of stroke is the systemic immune suppression that predisposes patients to systemic bacterial infections. Infection by itself is an independent risk factor of an unfavorable outcome in ischemic stroke , 13. InfNeutrophils promote thrombus formation through different mechanisms, including the release of molecules involved in neutrophil extracellular trap (NET) formation, the release of proteases, and direct interactions with platelets. This prothrombotic process might (i) increase the risk of ischemic stroke and (ii) promote further thrombosis during acute stroke. The percentage of neutrophil-platelet interactions was enhanced in patients with symptomatic carotid stenosis . HoweverNeutrophils adhere to injured vessels immediately, preceding platelets, by binding to the activated endothelium through an interaction between leukocyte function-associated antigen and ICAM-1. This is an important step for the activation and accumulation of thrombocytes, and blocking this step might be an efficient strategy for reducing cerebral thrombosis. Both candesartan and dipyridamole were found to inhibit the adhesion of neutrophils to vascular endothelium in patients with ischemic stroke, but not in patients with chronic stroke or healthy individuals .The protease, cathepsin G, is released by neutrophils and acts on coagulation factors to promote clot formation. Inhibition of cathepsin G decreased thrombus formation and reduced brain injury. The result was an improvement in the neurobehavioral outcome in a mouse model of ischemic stroke . On the An additional neutrophil contribution to thrombus formation results from the prothrombotic activity of NETs . NETs haNecrotic cell death within the infarcted area leads to the release of proinflammatory cytokines and a rapid infiltration of immune cells. Neutrophils are the first cells recruited into the brain within minutes after stroke. The mechanism of neutrophil entry into the brain after stroke was investigated in permanent and transient experimental stroke models with in vivo imaging. Blood-borne neutrophils immediately migrate, even against blood flow, and then transmigrate out of blood vessels to reach the injured brain area . The zenThe impact of immune cell invasion is a controversial issue. Although these cells might play a role in the initiation of tissue repair, their detrimental effects dominate. This was demonstrated in experimental stroke settings, where invading neutrophils enhanced ischemic neurotoxicity through different actions . When neγδ T-cells. Blocking this pathway with an anti-IL-17-antibody reduced the experimental stroke lesion size [These detrimental effects of neutrophils make them a prime target in novel therapies for stroke. Indeed, in experimental focal brain ischemia models, a variety of therapeutic interventions successfully reduced lesion size. One approach was to block proinflammatory cytokines and mediators that act as chemoattractants. For example, antagonization of C-X-C motif chemokine receptor 2 (CXCR-2) prevented recruitment of neutrophils to the infarct area . Anotherion size . In addiion size .A different approach, which does not interfere with neutrophil invasion, is to block the neutrophil proinflammatory function. Oxidative stress, caused by an overload of ROS, contributes to various acute, chronic, and inflammatory diseases. Thus, this mechanism has been suggested as a target of stroke therapy. In the preclinical setting, beneficial effects were achieved by inhibiting type 4 nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4). In experimental stroke models, brain damage was also ameliorated by inhibiting myeloperoxidase oxidant (MPO) production, with N-acetyl lysyltyrosylcysteine amide or with the flavonoid, eriodictyol , 36. MorNitric oxide (NO) produced by inducible NO synthase (iNOS) contributes to ischemic brain injury. iNOS expression is predominantly found in invading neutrophils after stroke. When iNOS(+/+), but not iNOS(−/−), neutrophils were transferred into iNOS(−/−) mice, infarct volume increased. That result identified iNOS as an important mediator of secondary tissue destruction . The inhUric acid, another free radical scavenger, was thought to protect the brain from oxidative injury. Until now studies investigating the neuroprotective effect of UA after stroke remain controversial . While dNevertheless it is known that different additional factors like advanced age, increased time to treatment, the extent of ischemic injury prior to administration of therapy, higher baseline National Institutes of Health Stroke Scale (NIHSS) score, high systolic blood pressure, or diabetes mellitus increase the risk of hemorrhagic incidence after stroke . Therefoβ-adrenoceptors with propranolol or by neutralizing HMGB-1 activity with antibodies or an antagonist of its receptor, the receptor for advanced glycation end-products (RAGE) [Another molecule discussed in the modulation of poststroke immune response is the HMGB-1. This DNA-binding protein is passively released during stroke from cells undergoing necrosis. This damage-associated molecular pattern molecule can also be actively secreted by immune cells and is released and exposed by platelets promoting thrombus formation as described in s (RAGE) , 52. TheIn addition to enhancing ischemic injury and the subsequent signaling cascades, neutrophils are also involved in reperfusion injury. Risk of hemorrhagic transformation is increased by as much as tenfold after intravenous rtPA administration, largely due to reperfusion injury and the toxic effects of rtPA . High neGranulocyte colony stimulating factor (G-CSF) had a neuroprotective effect in several models of experimental stroke. Administration of G-CSF decreased infarct size and improved motor function recovery . A recenEven before the appearance of stroke, neutrophils might play a role as a predictive marker of stroke risk. The neutrophil-to-lymphocyte ratio (NLR) was directly associated with the risk of stroke in patients with atrial fibrillation. As a predictor of stroke, the NLR appeared to be important for refining the risk of stroke and for improving the management of patients with atrial fibrillation and a low CHA2DS2-VASc score, a score for atrial fibrillation stroke risk . The NLRIn peripheral blood of patients with stroke, lymphocyte number and HLA-DR expression on monocytes decline, but neutrophil numbers increase. Animal studies have described an immediate reduction in spleen volume following cerebral ischemia. The change due to cerebral ischemia in human spleens was described as a biphasic process; splenic volumes initially decreased over time, reached a nadir at 48 h after stroke onset, and then increased thereafter. This process was positively correlated to the percentage of peripheral blood neutrophils . AdditioAs previously discussed, despite an increase in granulocyte numbers, infections can occur in patients within days after a stroke . A diagnThe role of neutrophils in infections after stroke is controversial. In the Enlimomab study protocol, patients with stroke were treated with anti-ICAM-1 antibodies to diminish neutrophils; however, those patients experienced an increased rate of infection, particularly pneumonia on day 5 after Enlimomab administration (2.2% versus 1.6% in placebo patients) as serious adverse event. Moreover, the infections were associated with a worse outcome . In thatNeutrophils obtained from patients that underwent neurosurgical interventions for hemorrhagic stroke showed significantly lower levels of oxygen species than neutrophils from healthy controls . An earlIn patients with stroke, NETs were impaired in the early phase of stroke (day 1 of admission) and recovered function on day 5 after admission. Because these phagocyte dysfunctions were present upon admission to the stroke unit, they might contribute to the susceptibility to stroke-associated infections .β-blockade at the time of stroke could reverse most effects of stroke-induced immune alterations observed in an experimental stroke model [According to our current understanding of stroke-induced immune alterations, immediately after a stroke, a “storm” of stress hormones, particularly catecholamines, are released by the adrenal gland and via direct sympathetic innervation of the lymphoid organs ke model . It is cke model .Neutrophils are a promising target in stroke therapy. However, the development of novel, neutrophil-based therapies must take into account the opposing effects that immune suppression has in the CNS and in the periphery. Although bacterial defense must be maintained or enhanced in the periphery, the immune response in the brain is largely detrimental and should be inhibited. This quandary has been reflected in clinical trial results that could not reproduce preclinical experimental successes.


The contribution of the decline in thymic activity for the emergence of autoimmunity is still debatable. Immune-competent adults submitted to complete thymectomy early in life provide a unique model to address this question. We applied here strict criteria to identify adults lacking thymic activity based on sjTREC levels, to exclude thymic rebound and/or ectopic thymuses. In agreement, they featured severe naïve CD4 T-cell depletion and contraction of T-cell receptor diversity. Notwithstanding this, there was neither increased incidence of autoimmune disease in comparison with age-matched controls nor significant changes in their IgG/IgA/IgM/IgE autoreactivity profiles, as assessed through extensive arrays. We reasoned that the observed relative preservation of the regulatory T-cell compartment, including maintenance of naïve regulatory CD4 T-cells, may contribute to limit the emergence of autoimmunity upon thymectomy. Our findings have implications in other clinical settings with impaired thymic activity, and are particularly relevant to studies of autoimmunity in ageing. The thymus is essential to the establishment of the “peripheral” T-cell compartment before birth and during the accelerated somatic growth of childhood, and contributes to its continuous replenishment until at least the sixth decade of life. Thymus Autoimmunity and allergy are controlled by a subset of regulatory CD4 T-cells (Tregs), defined by FoxP3 expression–13. TregWe recently reported that naïve-Tregs are preserved in adults more than 18y (median 21y) after complete thymectomy early in infancy, despite the marked contraction of conventional naïve CD4 T-cells, 16. ImpHere we investigated the possibility that the maintenance of naïve-Tregs limits the development of autoimmunity and/or allergy likely associated with the skewed conventional T-cell repertoire upon complete thymectomy.We compared our cohort of adults with strictly defined complete thymus removal in early infancy with age-matched healthy individuals . ThymectAll subjects gave written informed consent for blood sampling. The study was approved by the Ethical Boards of Faculdade de Medicina da Universidade de Lisboa, Centro Hospitalar Lisboa Norte, and Hospital de Santa Cruz, Portugal.An extensive structured questionnaire was applied to all individuals by the same investigator to assess clinical manifestations of autoimmunity and allergy, in parallel with the evaluation of serum reactivity to large panels of autoantigens and allergen components. Modules of ISAAC Questionnaire validated for Portuguese speaking adults were included in this evaluation. RegardiEx-vivo phenotypic analysis of lymphocyte subsets was performed in freshly-collected whole blood by 8-color flow cytometry, as previously described, 16.Autoantigen microarrays were used to detect IgG/IgM or IgA/IgE autoantibodies in serum to a panel of 125 autoantigens. The samIn order to expose a putative increase in the frequency of IgE sensitizations (sIgE) upon complete thymectomy, we used ImmunoCAP ISAC® (TermoFischer scientific), a miniaturized immunoassay platform, to assess serum specific IgE (sIgE) to 112 allergen components from 51 aeroallergen or food sources. This test was performed in the same day for all individuals , followiAdults with complete thymectomy early in infancy featured very low naïve CD4 and CD8 T-cell counts , as expeP>0.05). Moreover, the clustering analysis showed a clear inability to correctly assign thymectomized and controls on the basis of antigen reactivity profiles . Their targets included nuclear antigens , cytoplasmic/membrane proteins , cell matrix proteins (fibronectin), glomeruli-specific proteins (glomerular basement membrane) and circulating proteins . Interestingly, these autoantigens have been identified in systemic lupus erythematosus (SLE) patients[In spite of the lack of significant differences after applying the multiple test correction, when analysing IgG reactivity to distinct autoantigens, we identified 18 autoantibodies with higher expression within complete thymectomized than in age-matched healthy subjects, . There was no statistical difference between healthy and thymectomized. We also found 4 IgE autoantibodies, not previously reported[P<0.05, before multiple testing correction), but in contrast to IgG, these differences did not feature a consistent trend between the two cohorts. These findings reinforce the need for further investigation on IgE autoreactivity[Recent data recall attention for a role of IgE reactivity against self-peptides in autoimmunity although their significance is still debatable. We were reported, for whieactivity.Regarding allergy, we found no significant differences in the prevalence of clinical manifestations in complete thymectomyzed and age-matched control individuals . AllergiOur study, that was strictly designed to include only adults without evidence of thymic activity upon thymus removal in infancy, revealed no increase of clinical/subclinical manifestations of allergy or of autoimmune diseases, as compared with a group of age matched healthy individuals. This occurred despite these individuals featured marked contraction of the conventional naïve T-cell pool and a restricted T cell-receptor diversity. It is lNevertheless, beyond this well acknowledged role of the Treg compartment in the containment of autoimmunity, other factors may control the emergence of autoimmunity . AutoimmCohorts of adults submitted to thymectomy in infancy are expanding due to generalization of the access to early corrective/palliative cardiac surgery, though the eldest individuals are still in the fourth decade of life. TherefoThere are several previous studies addressing whether thymectomy performed early in life is associated with the subsequent development of autoimmunity and allergy–8. An inOur study applied strict criteria to exclude individuals with remaining thymic activity due to incomplete thymus removal and/or ectopic thymus. Of note, cardiothoracic surgeries have revealed a high frequency of ectopic thymic tissue . MoreoveIndividuals submitted to thymectomy in infancy have been considered a privileged model to address the contribution of the thymus and of peripheral mechanisms to control autoimmunity and allergy. Our data are consistent with the possibility that in the absence of thymic activity, peripheral tolerance limits the emergence of autoimmunity possibly through relative Treg preservation. These findings have implications for other clinical settings with impaired thymic activity. Particularly, they may steer the development of new strategies targeting peripheral immunoregulatory systems and thymus regeneration to control autoimmunity in the context of ageing or disease-associated thymus involution.S1 TableSamples and antigens are in the same hierarchical order as shown in the corresponding heatmap figure.(XLS)Click here for additional data file.S2 TableSamples and antigens are in the same hierarchical order as shown in the corresponding heatmap figure.(XLS)Click here for additional data file.S3 TableSamples and antigens are in the same hierarchical order as shown in the corresponding heatmap figure.(XLS)Click here for additional data file.S4 TableSamples and antigens are in the same hierarchical order as shown in the corresponding heatmap figure.(XLS)Click here for additional data file.


Autologous and allogeneic adult mesenchymal stem/stromal cells (MSCs) are increasingly being investigated for treating a wide range of clinical diseases. Allogeneic MSCs are especially attractive due to their potential to provide immediate care at the time of tissue injury or disease diagnosis. The prevailing dogma has been that allogeneic MSCs are immune privileged, but there have been very few studies that control for matched or mismatched major histocompatibility complex (MHC) molecule expression and that examine immunogenicity in vivo. Studies that control for MHC expression have reported both cell-mediated and humoral immune responses to MHC-mismatched MSCs. The clinical implications of immune responses to MHC-mismatched MSCs are still unknown. Pre-clinical and clinical studies that document the MHC haplotype of donors and recipients and measure immune responses following MSC treatment are necessary to answer this critical question.This review details what is currently known about the immunogenicity of allogeneic MSCs and suggests contemporary assays that could be utilized in future studies to appropriately identify and measure immune responses to MHC-mismatched MSCs. Mesenchymal stem cells (MSCs) are currently defined as plastic-adherent cells with a fibroblast-like morphology that are capable of differentiating into bone, cartilage, and fat in vitro and that express a defined set of surface markers, which vary slightly by species , 2. The Secretion of paracrine factors is now recognized as the primary mechanism by which MSCs promote a regenerative environment conducive to healing with healthy tissue , althoug2, which contribute to the ability of MSCs to modulate immune responses .–35.29–35+ and CD8+ splenocytes with a memory phenotype (CD122+CD44+CD62Llow), but not mice injected with MHC-matched MSCs [Cell-mediated responses to MSCs are induced when T cells become activated following recognition of foreign donor MHC molecules expressed on the surface of the MSCs. Significant increases in circulating T cells and natural killer cells were detected in rhesus macaques as early as 10 days after intracranial injection with MHC-mismatched MSCs, but not those injected with autologous MSCs . Cytotoxhed MSCs . In sepahed MSCs , 32. ThePre-existing antibodies crossreactive for donor MHC molecules or alloantibodies produced following activation of B cells by cognate alloantigens can also contribute to rejection of allogeneic cells. A significant increase in total serum immunoglobulin (Ig)G was reported in rhesus macaques injected with MHC-mismatched MSCs, but not in macaques injected with autologous MSCs . AlloantIn-vivo rejection of MSCs has been measured both directly using bioluminescent imaging and in-vivo cytotoxicity assays and indirectly by measuring hematocrit following injection with erythropoietin-expressing MSCs. In each of these studies, MHC-mismatched MSCs survived for a significantly shorter period of time than MHC-matched MSCs in immunocompetent mice and were rejected more quickly in previously sensitized animals , 30, 33.A number of standard immunological assays and techniques are available to measure the immunogenicity of MSCs. For these assays, the MHC haplotype of donors, recipients, stimulators, and responders should be determined to understand if donor or stimulator MSCs are full or partial mismatches to recipients or responder cells. Control cells should include donor or target cells of the same MHC haplotype as recipients or responders to control for MHC-specific immune responses. Modified one-way in-vitro MLRs, where responder splenocytes or PBLs are cocultured with stimulator allogeneic MSCs, have traditionally been used to measure the immunogenicity of MSCs, but several studies have demonstrated that in-vitro MLR assays are poor predictors of in-vivo immunogenicity , 35, 43.Measuring heat, swelling, or the infiltration of immune cells or lack thereof into the site where MSCs were injected or the tissue of interest is also not sufficient to determine if donor MSCs have induced an immune response. The absence of a local immune response does not rule out a systemic response  and doeEx-vivo MLRs can be useful for estimating sensitization of recipient T cells to donor MHC molecules postadministration with allogeneic MSCs. Splenocytes or PBLs from recipients are collected and used as responders in a standard one-way MLR using stimulator splenocytes or PBLs of the same MHC haplotype as the MSC donor . Prolife+ or CD4+ effector and memory cells against donor MSCs [Interferon (IFN)-γ and interleukin (IL)-4 enzyme-linked immunospots (ELISPOTs) can measure functional responses of T cells upon restimulation with donor MHC molecules as well as predict the in-vivo immunogenicity. Similar to the ex-vivo MLR, following in-vivo administration of MSCs, recipient splenocytes or PBLs can be isolated and restimulated using splenocytes or PBLs of the same MHC haplotype as donor MSCs. Secretion of IFN-γ or IL-4 above baseline indicates expansion of CD8nor MSCs . Preformnor MSCs  and may Cytotoxicity assays can be used to measure direct lysis of MSCs by MHC-specific cytotoxic T lymphocytes (CTLs). As MSCs inhibit formation of CTLs in MLRs , effectoSeveral assays are available for detecting alloantibodies and identifying the specificity and function of alloantibodies. Microcytotoxicity assays, also called lymphocytotoxicity assays, were originally developed for tissue typing, but can also be used to detect cytotoxic anti-MHC alloantibodies in serum. Standard one- or two-stage microcytotoxicity assays use eosin or fluorescent dye to detect antibody-mediated complement-dependent cytotoxicity (CDC) following incubation of sera from animals injected with MHC-matched or MHC-mismatched MSCs with donor PBLs or MSCs and rabbit complement , 52. FloDonor MSC-specific antibodies can also be detected by incubating donor MSCs or PBLs of the same MHC haplotype with sera from recipients and staining with anti-IgG or anti-IgM secondary antibodies . Single In-vivo imaging can also be used to track the survival of injected MSCs. MSCs from transgenic mice that constitutively express luciferase or fluorescent proteins allow for long-term tracking of cells and estimations of survival in vivo . For larAllogeneic MSC therapy holds significant promise for treating numerous diseases, but further studies are needed to assess the potential of allogeneic MSCs for widespread clinical use. In-vitro and in-vivo studies designed with appropriate MHC controls and thorough immune response analyses will help answer under what conditions immune responses to allogeneic MSCs occur and if these immune responses affect the safety and efficacy of MSC therapy. Additionally, as animal studies support that allogeneic MSCs are rejected in vivo, strategies to reduce the immunogenicity and increase the ability of MSCs to avoid immune responses should be investigated to enhance the survival of allogeneic MSCs.


The quest to characterize the neural signature distinctive of different basic emotions has recently come under renewed scrutiny. Here we investigated whether facial expressions of different basic emotions modulate the functional connectivity of the amygdala with the rest of the brain. To this end, we presented seventeen healthy participants  with facial expressions of anger, disgust, fear, happiness, sadness and emotional neutrality and analyzed amygdala’s psychophysiological interaction (PPI). In fact, PPI can reveal how inter-regional amygdala communications change dynamically depending on perception of various emotional expressions to recruit different brain networks, compared to the functional interactions it entertains during perception of neutral expressions. We found that for each emotion the amygdala recruited a distinctive and spatially distributed set of structures to interact with. These changes in amygdala connectional patters characterize the dynamic signature prototypical of individual emotion processing, and seemingly represent a neural mechanism that serves to implement the distinctive influence that each emotion exerts on perceptual, cognitive, and motor responses. Besides these differences, all emotions enhanced amygdala functional integration with premotor cortices compared to neutral faces. The present findings thus concur to reconceptualise the structure-function relation between brain-emotion from the traditional one-to-one mapping toward a network-based and dynamic perspective. Affective neurosciences are concerned with understanding how emotions are represented in brain activity and how this translates into behaviour, thereby characterizing structure-to-function relations. Traditionally, lesion studies informed early localization approaches by outlining the deficits in emotion processing that follow focal brain damageThese functional magnetic resonance imaging (fMRI) investigations are improving our understanding of which individual brain regions respond to specific emotions. Moreover, the sizable fMRI literature accumulating in recent years has enabled quantitative meta-analyses to assess the consistency and specificity of the neural signature of emotion processing beyond idiosyncrasies of individual studies. Yet results are mixed, with some original studies and meta-analyses supporting the existence of discrete and non-overlapping neural correlates for different emotions3456729Common to most of these approaches scrutinizing the neural signatures of basic emotions is the reliance on methods that characterize the static response of individual areas, rather than their dynamic connectional changes. This focus on ‘disconnected’ brain structures, however, can be incomplete in representing how complex functions like emotion processing map into brain activity. In fact, the role of a given structure is partly determined by the dynamic interactions it entertains with other regions, thus shifting the focus toward inter-regional connectivity patterns111314171923The amygdala is indeed a key structure in the perception and response to emotional signals and has been classically linked to fear processing252627293132343536Although this heterogeneity of functions in the amygdala has been initially taken as suggesting that emotions do not have a characteristic or unique neural signature313238A host of techniques can be used to examine how spatially remote brain regions interact during emotion processing by gauging brain activity across time. One of the most popular and successful approaches for examining dynamic and task-related interactions between one region and the rest of the brain is to assess psychophysiological interactions (PPI)4043F = 3.05, p = 0.001]. Post-hoc comparisons revealed that amygdala activity increased significantly in response to all 5 emotional expressions, as compared to the neutral expression , whereas there was no difference in amygdala response amidst emotions (p ≥ 0.39).A preliminary analysis assessed the percentage of blood oxygen level dependent (BOLD) signal change in the amygdala in response to the different stimulation conditions. The PPI analysis of fMRI data revealed that neural activity in multiple cortical and subcortical areas co-varied, and was therefore functionally correlated, with the activity in the amygdala during observation of basic emotions compared with neutral expressions. All five basic emotions dynamically modulated amygdala interactions in a distinctive and category-specific manner, thereby carving a functionally specialized and spatially distributed network for each emotion studied, as displayed in The emotion-dependent connectivity pattern of the amygdala can be broadly grouped in five sets, based on their anatomical locations. The first set comprises anterior cortical midline regions, including the dorso-medial prefrontal cortex (dmPFC) and anterior cingulate cortex (ACC). Enhanced amygdala integration with these anterior midline regions characterizes the processing of happy expressions. The second set of inter-regional amygdala connectivity engages posterior area in the medial surface, such as the posterior cingulate cortex (PCC) and the precuneus (PCUN), which are uniquely associated with exposure to angry expressions. The third cluster consists of lateral prefrontal regions, anatomically connected with the amygdala via the uncinate fasciculusX = −44, Y = 2, X = 37) and rostral premotor cortex , known to be involved in motor resonance48Finally, we investigated the possible presence of voxels that increase their synchrony with the amygdala in response to all five basic emotions, thus constituting the common core of amygdala functional network shared across emotions. As anticipated, 126 voxels matched this criterion and were localized in the left premotor  with normal or corrected-to-normal vision participated in this study . All subjects were screened for fMRI compatibility had no personal history of neurological or psychiatric illness, drug or alcohol abuse, or current medication. All experimental methods and protocols in the study were performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and approved by the bioethics committee of the University of Turin under the project ‘Neuropsychological bases of emotional and social perception’. All participants provided written informed consent also approved by the same committee.2.The stimuli were taken from Ekman’s seriesThe experiment consisted of an event-related design divided in four runs. In each run, lasting approximately 8 min, all 24 stimuli were presented randomly for a total of 96 trials and 16 repetitions of the same facial expression. Each trial started with a central fixation cross lasting for 1 second against a dark background, followed by stimulus presentation for 1 second. Inter-stimulus interval (ISI) varied randomly within 16–20 seconds time range during which the central cross remained present on the screen. A PC running E-prime controlled stimulus presentation.Participants lay supine in the scanner with head movements minimized by an adjustable padded head holder. A colour LCD screen projected the stimuli onto a rear projection screen in the bore of the magnet. Participants viewed the screen via an angled mirror system. They were instructed to fixate the central cross during a passive exposure paradigm. This enabled us to record neural activity related to spontaneous emotion processing that was unaffected by spurious factors such as deliberate processing, top-down attentional modulation or action execution and button press. Otherwise, the interpretation of activations in motor and somatosensory cortices, or in the dorsal fronto-parietal attentional network, as predicted from previous studies, had been problematic if they had been concomitant upon these additional task demands. To verify that participants viewed the stimuli, after each run participants were asked whether they had paid attention to the stimuli. All participants responded positively after every run.Data acquisition was performed on a 1.5 Tesla INTERA™ scanner  with a SENSE high-field, high-resolution (MRIDC) head coil that was optimized for functional imaging. The functional T2*-weighted images were acquired using echoplanar (EPI) sequences . A total of 240 volumes were acquired covering the whole brain. Two scans were added at the beginning of the functional scanning session and the data discarded to reach a steady-state magnetization before acquiring the experimental data.In the same session, a set of three-dimensional high-resolution T1-weighted structural images was acquired for each participant using a Fast Field Echo (FFE) sequence .www.fmrib.ox.ac.uk/fsl)3 using FMRIB’s Linear Image Registration Tool (FLIRT–FSL). All participants maintained head motion <3 mm for all scans.Functional data were preprocessed using FSL 5.0.9 , reflecting the mean peak of stimulus-evoked activity of all voxels in the ROI over a temporal window of 16 sec from stimulus onset.where PPI reveals how activity in a particular brain region  is differentially correlated  with that in other brain regions depending on the experimental conditions 40Z > 2, p < 0.05. A preliminary analysis did not reveal any significant gender difference in amygdala connections. Therefore, the data of male and female participants were considered together. The resulting voxel-wise contrast maps were then loaded into MATLAB environment using xjView toolbox . This enable us to localize anatomically the significant clusters via MNI-based template atlas, to identify active voxels common to all emotions (using the option “common region” in xjView), and to obtain the number of voxels in each these clusters as reported in http://www.mccauslandcenter.sc.edu/mricrogl/home).At the second level, a fixed-effects analysis combined the connectivity data of each subject across all the four runs. At the third level , the obtained contrasts  for each subject were modelled for between-subjects variance using mixed effects (FLAME1 option), and the results were cluster-corrected for multiple comparisons at the whole-brain level of How to cite this article: Diano, M. et al. Dynamic Changes in Amygdala Psychophysiological Connectivity Reveal Distinct Neural Networks for Facial Expressions of Basic Emotions. Sci. Rep.7, 45260; doi: 10.1038/srep45260 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Childhood trauma may increase the risk for psychiatric illness by its negative impact on brain development. Studies investigating the association between childhood trauma and deviations in gray matter volume have shown inconsistent findings, often restricted by a region-of interest approach with a sole focus on the amygdala and hippocampus and without controlling for the presence of psychiatric illness.First, using a whole-brain approach in a large cross-diagnostic sample (n=554) of healthy individuals and patients with a bipolar type-I or psychotic disorder, we investigated the neurobiological correlates of childhood trauma by evaluating gray matter volume. Follow-up analyses were conducted to evaluate the effect of psychiatric illness. Second, we investigated to what extent these trauma-related structural correlates could be observed in both groups separately . Participants were recruited as part of three different studies, all conducted in the University Medical Center Utrecht (the Netherlands) between 2007 and 2016. We included 554 participants: 220 healthy individuals without a psychiatric history, 250 patients with a bipolar-I disorder and 84 patients with a psychotic disorder. Childhood trauma was evaluated with the Childhood Trauma Questionnaire (CTQ-SF). Anatomical T1 MRI scans were acquired at 3T. FreeSurfer was used to assess regional brain morphology.In the total sample, childhood trauma severity was associated with bilateral reductions in frontal and insular gray matter volumes. In the right hemisphere, medial orbitofrontal and superior frontal volume reductions were related to childhood trauma. These associations remained when adjusting for psychiatric illness, with the exception of the right superior frontal subregion. However, when evaluating both groups separately, these structural correlates of childhood trauma were mainly observed in patients. Healthy controls did show trauma related reductions in right medial orbitofrontal region, while this association was not significant in the patient group.Our results suggest that gray matter reductions in the frontal and insular regions are important neurobiological correlates of childhood trauma. For future research, a whole brain approach should be applied, as cortical rather than subcortical areas may be the main correlate of childhood trauma contributing to the development of psychopathology.


Here, we combine sequencing data from mouse models of IR-induced malignancies and human IR-induced malignancies. To determine whether the signatures identified from IR-exposed subjects can be differentiated from other mutagenic signatures, we included data from an ultraviolet radiation (UV)-induced human skin cancer and from a mouse model of urethane-induced cancers. NMF distinguished all three mutagens and in the pooled analysis IR was associated with mutational signatures common to both species. These findings illustrate the utility of pooled analysis of mouse and human sequencing data.Single nucleotide variants (SNVs) identified in cancer genomes can be de-convolved using non-negative matrix factorization (NMF) into discrete trinucleotide-based mutational signatures indicative of specific cancer-causing processes. The stability of NMF-generated mutational signatures depends upon the numbers of variants available for analysis. In this work, we sought to assess whether data from well-controlled mouse models can compensate for scarce human data for some cancer types. High quality sequencing data from radiotherapy-induced cancers is particularly scarce and the mutational processes defining ionizing radiation (IR)-induced mutagenesis  Characterizations of single nucleotide variants (SNVs) and the local nucleotide contexts in which they arise reveal trinucleotide-based signatures2. Cancers harbor multiple trinucleotide-based mutational signatures that can be extracted from whole exome sequencing (WES) data using non-negative matrix factorization (NMF)4. Remarkably, different tumor histologies share discrete mutational signatures, with some mutational signatures originating with known mutagenic exposures such as tobacco, ultraviolet light and certain chemotherapies, while others are less understood or are of unknown etiology3.Analyses of thousands of human cancers have revealed cancer genomes to be complex structures bearing the marks of oncogenic germline and somatic alterations, as well as imprints of environmental exposures. The term “mutational signature” refers to the patterns of somatic mutations present in the tumor genome and which serve as a record of the mutational processes leading to cancer developmentMonte Carlo simulations2. This study is, to the best of our knowledge, the first in this area to explore pooling human sequencing data with data from appropriate mouse models to overcome analysis limitations secondary to scarce human cancer samples.In NMF trinucleotide-based mutational signature analysis, six possible substitutions are considered, based on the pyrimidine in the reference position and the proximal sequence context (one nucleotide 5′ and 3′). SNVs together with their 5′ and 3′ neighboring bases can be organized into 96 trinucleotide-based groups. Large numbers of variants are generally used to identify and distinguish stable trinucleotide-based signatures using NMF. However the extraction of stable NMF signatures may not be possible if the numbers of samples available for study are limited, an issue that is particularly problematic for uncommon clinical samples of limited availability. Attempts have been made to examine the trade-off between number of tissue samples and number of mutations per sample by 6, the genome-wide mutational signatures of most mutagens remain uncharacterized. Clinical samples in which to study in vivo mutagenesis may, depending upon the mutagen, be limited in availability and arise after unknown and unquantifiable exposures. In contrast to the challenges inherent in studying clinical samples, multiple variables including mutagen exposure can be studied systematically and independently in mouse models, making them powerful tools for studying disease pathogenesis and characterizing mutational mechanisms promoting cancer7. Thus, well-annotated cancers from mouse models can potentially inform the analyses of human malignancies, although the utility of this type of cross-species NMF analysis has not been described.With the exception of common and well-characterized mutagens such as ultraviolet (UV) radiation and tobacco, whose mutational signatures were characterized prior to the development of next generation sequencingin vivo are poorly defined. We previously developed a mouse model utilizing anatomically localized  dose-fractionated irradiation, which more accurately models radiation-induced secondary neoplasms in human cancer survivors9. Focally irradiated mice develop IR-induced malignancies that resemble clinical IR-induced malignancies both anatomically and histologically9. Similar to the clinically-based radiation protocols utilized in our mouse model, the two human IR-induced malignancies included in this analysis were induced by focal fractionated radiation10. As a result, the mouse and human IR-induced malignancies shared key similarities in mutagenic exposure with the previously analyzed WES data from the IR-induced malignancies arising within our mouse model4.We utilized mouse and human malignancies initiated by ionizing radiation (IR) to study the utility of cross-species analysis. The genetic features of IR-induced malignancies arising 4. Using this previously described mutation data and WES data from the human IR-induced malignancies, we tested the utility of cross-species analysis by extracting trinucleotide-based mutational signatures from the exomes of IR-induced human tumors as a group and also pooled with WES data generated from a mouse model of IR-induced malignancies.In prior work we analyzed whole exome sequencing (WES) data from IR-induced malignancies arising within our mouse model7. In addition, two samples were included from a single UV-induced human skin cancer, a known non-IR malignancy control. The trinucleotide mutational signature for UV is defined, thereby providing the opportunity to determine whether our methods and data could reproduce the previously reported UV-signature.To test the ability of our procedures to discriminate between different mutagens, we included sequencing data from 22 mouse tumors induced by the chemical mutagen urethaneUsing WES data for three patients combined with previously published WES data from mouse IR-induced and urethane-induced malignancies, we tested the utility of cross-species analysis by comparing trinucleotide-based mutational signatures from the human tumors as a group with mutational signatures extracted from pooled human and mouse data. Trinucleotide-based signatures from IR, UV or urethane-induced malignancies were robustly extracted by NMF. In addition, the pooled analysis enabled the resolution of an additional IR-related mutational signature that was not detectable prior to pooling but was present in both IR-induced human and IR-induced mouse cancers. This cross-species analysis illustrates an opportunity for using sequencing data from mouse models to uncover insights into human cancer genetics.NMF, which utilizes somatic SNVs as input, can be used in the study of cancer genomes to extract patterns reflecting mutagenic processes. However, those patterns may be obscured if NMF is applied to pooled data for which data types harbor inherent differences in trinucleotide frequency .Similarly, it would make little sense to directly compare trinucleotide-based signatures separately extracted by NMF from datasets of different types for which there are inherent trinucleotide frequency differences.et al.3, and we present two corresponding sets of signatures that differ only in the normalization applied. For the first of these two sets, the signature coefficients were normalized on the basis of the actual trinucleotide frequency of the whole human genome. These signatures are referred to hereafter using the published numbering, as Wellcome Trust Sanger Institute (WTSI) signatures 1A, 1B, 2, …, 21. Updated versions of these 22 WTSI signatures  are given on the COSMIC site (at http://cancer.sanger.ac.uk/cosmic/signatures), and are referred to hereafter as COSMIC signatures 1–30. . For Patient 3, two tumor samples were taken at one year apart (designated as pre- and post-) and were known not to have received radiotherapy. All tumor samples were analyzed and compared to the matched germline. Each of Patient 3’s tumor samples possessed significantly more SNVs than those of either Patient 1 or 2 . Prior descriptions of UV-associated skin cancers indicate that these malignancies typically bear higher mutational loads as compared to other, non-UV associated malignancies3. The mutational loads occurring in our skin cancer samples compare similarly to previously published rates in human skin cancers.In the human-only NMF analysis, we included tumor samples initiated by either IR or UV. Two IR-induced malignancies from patients were analyzed by whole exome sequencing , revealing a total of 194 single nucleotide variants (SNVs) for Patient 1, and a total of 361 SNVs for Patient 2 (202 in sample A and 159 in sample B)4, the normalized mutational frequency data from the somatic SNVs in the five human tumor samples for the three patients (the P123 dataset) were found to support the extraction of two stable signatures .Performing NMF analysis as previously describedres Fig. . Signatures Fig. . The mut7 to test the robustness of our approach. This alternative dataset introduced an additional mutational signature found in mouse non-small cell lung cancers resulting from isolated exposure to urethane7.Analyzing the UV-induced human tumor exomes with IR-induced human tumor exomes demonstrated that two distinct mutational signatures could be distinguished in a small cohort of samples Fig. . In asseet al.)7. NMF analysis of this pooled P123+IRmse+Uremse dataset permitted the extraction of four stable signatures, shown in Fig. We pooled the normalized data for Patients 1, 2 and 3 (the P123 dataset) with the normalized data for the IR-induced mouse malignancies (the IRmse dataset), and with normalized data for 22 samples from urethane-induced mouse malignancies  and the IRa signature (56.3%), though with different nucleotide contexts. The IRb signature has more T → G (25.7%) than C → T (21%) substitutions. The urethane signature has more C → A substitutions than any other type (30.5%), followed by T → C (29.2%) substitutions.4, and without normalization. Differences between the signatures obtained in that prior study and those identified here are potentially due to the use of mouse-only versus pooled mouse-human data, the use of un-normalized versus normalized data, and the number of mutational types considered.In prior work, IR-associated signatures were identified using mouse data for 192-mutational types Cell Reports (CellR) paper4. Rows 1–2 give correlations when two signatures are extracted, and rows 3–5 give correlations when three signatures are extracted from the IRmse dataset.Rows 1–5 in Table 4 represents a process also operating in cancers induced by UV radiation. This possibility is consistent with the statistically significant correlation of 0.649  between signature 1/2 extracted from the P12+IRmse dataset (containing no data from the skin cancer of Patient 3), and the UV signature  consisted of deletions and balanced inversions, as opposed to the SNVs that are our focus. To examine this independent dataset for the presence of the IR-associated signatures identified above, we pooled the SNV data for the 12 IR-induced cancers examined in the Behjati et al. study (included as a supplementary file in that work11), suitably normalized and referred to hereafter as the IRB dataset, with our normalized data for Patients 1, 2 and 3 and for the IR-exposed mice. The pooled P123+IRmse+IRB12 dataset allows extraction of seven signatures before the stability falls steeply.In this analysis we sought to also determine whether the IR signatures we identified were present in the sequencing data of other known IR-associated human malignancies to date. Behjati Table 12. The challenge, then, is to de-convolve the totality of somatic abnormalities present in cancer genomes into interpretable motifs that provide insights into cancer origin and evolution. As a statistical approach, NMF is sensitive to variant numbers and large numbers of variants are generally used to obtain mutational signatures with high stability2. However, apart from common cancers and the most common environmental exposures, the mutational imprints produced by many known and suspected carcinogens remain poorly identified.Analyses of thousands of diverse human malignancies have shown that cancer genomes reflect multiple influences and pathogenetic events4.As a result, most mutational signatures described to date are produced by poorly defined mechanisms as compared to specific, well-characterized mutagens such as ultraviolet radiation and tobacco that produce characteristic mutational signatures. IR is one such mutagen whose trinucleotide-based mutational signature has only recently been elucidatedHuman cancers arise in diverse individuals after variable and often undocumented exposures, which complicates efforts to analyze discrete mechanisms of genotoxicity and cancer promotion. Furthermore, in certain patient populations germline variants are increasingly detected and recognized as facilitating tumor formation, which further complicates tumor analyses depending on the genetic variant under study. When attempting to isolate mutational motifs related to discrete genetic events, analyzing clinical samples may be limiting. To create datasets of sufficient size to extract stable, replicable NMF signatures and for carrying out other sorts of statistical analysis where the size of the dataset can matter, researchers sometimes pool samples from subjects reported to have been exposed to the same mutagen , but lacking relevant specifics of those exposures and details regarding other potentially confounding factors.in vivo experimental systems in which to study cancer pathogenesis and mutational mechanisms underlying human cancers7. Mouse models have multiple specific advantages for complementing clinical samples and can overcome some of the limitations faced when clinical samples alone are analyzed by sequencing. These advantages include the homogeneity of the genetic background and the ability to control environmental exposures precisely.Mouse models are invaluable This work demonstrates that exomic data from well-controlled experimental systems can be pooled with human data to facilitate insights into the mutational profiles of human cancers. NMF, when based on mutation frequency data normalized according to the appropriate reference genome trinucleotide frequencies, represents the mutation incidence so that the probability of a purely random mutation at any one trinucleotide is equalized. This work reports for the first time that the same trinucleotide-based mutational signatures characterize both IR-induced human malignancies and IR-induced mouse malignancies. These findings argue in favor of mouse models as relevant tools with which to study mechanisms of human cancer pathogenesis.These findings also suggest that mutation spectra are not necessarily exclusive. The UV and urethane-induced malignancies harbored single signatures with little mixing of additional signatures. In contrast, the known IR-induced malignancies from both humans and mice harbored signatures shared with the known UV and urethane-induced malignancies. Because neither the human and mouse IR-induced malignancies were exposed to UV or urethane the presence of what we identified as the UV and urethane signatures in these malignancies as well suggests that IR or other mutational processes not yet identified can recapitulate these signatures.13. As secondary events, reactive oxygen species interact with DNA through a variety of mechanisms, producing base damage, crosslinking and single strand breaks15. The relative diversity of free radicals and molecular interactions induced by IR may help to explain the complexity of IR-associated mutational signatures, why multiple signatures are present in IR-induced malignancies, and why some of these signatures may be present in malignancies produced by other non-IR mutagens. This is an important area for further research.Ionizing radiation interacts directly with DNA, producing double-strand breaks, but also produces chemical changes in DNA through the production of water-derived free radicals also known as reactive oxygen species3. This would indicate that a large number of mutations from a very small number of samples can yield reliable signatures, a finding that holds particular significance for mutational analyses of data from small numbers of highly mutated clinical samples.In contrast to IR, the UV signature, which was extracted from only two tissue samples from one patient, closely matched the signature previously establishedIn this work, an unsupervised analysis demonstrates that multiple mutagenic processes can be simultaneously distinguished in human and mouse cancers. Previously reported IR-related trinucleotide signatures were found in IR-induced human malignancies, using the same NMF extraction methods and normalized mutational input data. This is an important step forward, though much future work remains to improve the precision of the estimated signatures coefficients and to explore their generality.16 utilizes previously identified mutational signatures previously identified in comprehensive analyses of human cancers, and presents an approach that uses this information to enable mutational signature analysis of clinical samples on an individual basis. This method determines the contributions of each of the specified previously identified mutational signatures to a single tumor sample16. Operationally, this methodology involves an a priori assumption of the mutational signatures expected to be present. In contrast, we show an unsupervised analysis and demonstrate that multiple mutagenic processes can be simultaneously distinguished in human and mouse cancers. Our findings suggest that the deconstructSigs approach could use results from well-controlled mouse models where human data are scarce.The deconstructSigs method18. Our findings suggest that other mouse models of cancer formation or mutagenesis may similarly be used to extract and clarify mutational motifs where limited numbers of human samples are available. Experimental mouse models may also be particularly effective for de-convolving, or extracting, mutational signatures when cancers arise from exposures that are imprecisely defined, as is frequently the case for human malignancies.Cancer mouse models demonstrating fidelity to human cancers enable detailed studies of pathogenesisAll work was performed under a research protocol approved by the UCSF Committee on Human Research (IRB protocol 11–07304). All work was performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects.4.Genomic DNA was isolated from fresh frozen tumor samples and fresh peripheral blood samples using previously described techniques19. All tumor samples were analyzed and compared to the matched germline.Whole exome sequencing was performed using the NimbleGen Human exome v3.0 kit. Captured material was indexed and sequenced on the Illumina GAII and HiSeq. 2000 platform at the Institute for Human Genetics at UCSF. Successfully sequenced reads were then mapped to the human reference genome (GRCh37) using GATK best practices and MuTect was used for somatic mutation detectionhttp://www.agilent.com). Trinucleotide frequencies were calculated by Python code. Supplementary Table Differences in the trinucleotide frequencies between species can influence the relative weighting of SNVs. Hence either separate sets of NMF signatures must be extracted from single-species datasets with the resulting NMF signature coefficients then being compatibly normalized before clustering or other analyses, or the mutational data for each of the species must be comparably normalized prior pooling or other analysis of the NMF results. In this work, all datasets were normalized prior to application of NMF (except as indicated). Exome regions in the reference genomes, GRCh37 (hg19) for humans and NCBIM37 (mm9) for mice, were obtained from bed files downloaded from the Agilent web site (2.Non-negative matrix factorization (NMF) was performed following protocols developed by WTSIReferences with locations for previously published data  are provided in the body of the manuscript when the datasets are introduced and discussed. Patient 3’s data is available from the corresponding author.Supplementary InformationSupplementary Tables


Stimuli that varied along a neutral-to-fearful continuum were presented before conditioning to assess baseline responses, and after conditioning to assess experience-dependent changes in neural activity. Compared with trauma-exposed controls, PTSD patients exhibited greater post-study memory distortion of the fear-conditioned stimulus toward the stimulus expressing the highest fear intensity. PTSD patients exhibited biased neural activation toward high-intensity stimuli in fusiform gyrus (P<0.02), insula (P<0.001), primary visual cortex (P<0.05), locus coeruleus (P<0.04), thalamus (P<0.01), and at the trend level in inferior frontal gyrus (P=0.07). All regions except fusiform were moderated by childhood trauma. Amygdala–calcarine (P=0.01) and amygdala–thalamus (P=0.06) functional connectivity selectively increased in PTSD patients for high-intensity stimuli after conditioning. In contrast, amygdala–ventromedial prefrontal cortex (P=0.04) connectivity selectively increased in trauma-exposed controls compared with PTSD patients for low-intensity stimuli after conditioning, representing safety learning. In summary, fear generalization in PTSD is biased toward stimuli with higher emotional intensity than the original conditioned-fear stimulus. Functional brain differences provide a putative neurobiological model for fear generalization whereby PTSD symptoms are triggered by threat cues that merely resemble the index trauma.Fear conditioning is an established model for investigating posttraumatic stress disorder (PTSD). However, symptom triggers may vaguely resemble the initial traumatic event, differing on a variety of sensory and affective dimensions. We extended the fear-conditioning model to assess generalization of conditioned fear on fear processing neurocircuitry in PTSD. Military veterans ( Although fear conditioning and extinction in PTSD have received widespread attention,6 fear generalization in PTSD has not been well-studied,7 particularly with neuroimaging. Our goal was to extend the fear-conditioning model to assess generalization of conditioned fear on fear processing neurocircuitry and behavior in PTSD.Individuals with posttraumatic stress disorder (PTSD) exhibit anxiety-related behaviors based on reminders of past trauma, have difficulty extinguishing fear associations, and display frequent reawakening of fear associations.9 Similarly, animal conditioning studies have revealed asymmetrical generalization towards unreinforced stimuli of higher physical intensity than a CS along dimensions of loudness or brightness, an effect referred to as intensity generalization.10 Here, we extend the concept of intensity generalization to examine whether PTSD patients are sensitive to the emotional intensity of unreinforced stimuli after an aversive experience with a stimulus of moderate intensity. Our goal was to investigate experience-dependent changes in fear neurocircuitry, behavior, memory, and correlations with lifetime trauma exposure and clinical symptoms before and after fear conditioning in PTSD as compared with trauma-exposed veterans without PTSD.We adopted a validated fear-conditioning paradigm that incorporates faces of the same identity expressing various intensities of fear . We have11 Critically, neural activity to each stimulus was obtained pre- and post-fear conditioning with functional magnetic resonance imaging (fMRI) to observe experience-dependent changes resulting from fear conditioning. This pre–post training design is commonly used in animal neurophysiology to examine representational plasticity in sensory cortex following Pavlovian fear conditioning12 and, in the current study, to control for potential baseline differences in neural response between PTSD and non-PTSD controls.11Conditioning paradigms using fear-relevant stimuli, and testing for generalization gradient asymmetries as a function of CS intensity, serve as an appropriate model for PTSD, given that these stimuli exhibit resistance to extinction learning and that many symptom triggers involve stimuli with some inherent threat value.13 as well as fMRI investigations of fear generalization in healthy adults14 and patients with generalized anxiety disorder.16 Specifically, we expected PTSD patients to exhibit enhanced generalization of neural activity following fear conditioning in regions of the ‘central autonomic-interoceptive network' commonly identified in fMRI investigations of human fear conditioning.17 This network includes the thalamus, based on its role in sensory integration of information for the amygdala during fear learning,18 and the striatum, based on its role in continuously updating the amygdala with expectancy information based on aversive prediction errors.19 Likewise, the dorsal anterior cingulate cortex is a part of key neurocircuitry involved in appraisal and expression of learned fear.20 Asymmetric stimulus-intensity generalization of neural activity was also predicted along the ventral visual stream, including the fusiform gyrus, via sensory enhancement of visual representations by the amygdala and the thalamus.21 Finally, the locus coeruleus is the site of synthesis of noradrenergic neurotransmitters released in response to acute stress or threat, and activity in this region has been linked to stress-induced overgeneralization of memory representations.22We hypothesized that PTSD patients would exhibit robust experience-dependent changes in fear neurocircuitry, and retrospective memory biases for faces that expressed higher fear intensity, as compared with trauma-exposed control subjects. Predictions about the neurocircuitry mediating fear generalization were based on cross-species evidence of brain systems involved in acquiring and expressing learned fear,23We hypothesized that each of these regions would show greater pre- to post-fear conditioning increases in neural activity in PTSD patients compared with trauma-exposed controls for stimuli of higher intensity than a learned threat (CS+). We also hypothesized that fear generalization in PTSD would be associated with changes in functional connectivity with the amygdala, given its central role in conditioning-induced changes in brain plasticity. Finally, we predicted that childhood trauma exposure would enhance generalization biases in fear neurocircuitry, in line with prior evidence that lifetime trauma exposure is strongly correlated with PTSD severity.n=67) were recruited from February 2011 through April 2014, from a repository24 of 3500 US military veterans. All the participants served since 11 September 2001, and most were deployed to Iraq and/or Afghanistan military conflicts. All the participants provided written informed consent to participate in procedures approved by the Institutional Review Boards at Duke University Medical Center and Durham VA Medical Center. Participants underwent screening for inclusion in the study and subsequent clinical assessment of PTSD symptoms, trauma exposure, psychiatric comorbidities and medication use described in the The participants  were gradations of a single facial identity morphed incrementally between neutral and fearful end points  to offset the effects of extinction over the extended testing session .28 Stimulus duration was 4 s, and subjects were not informed of any CS–US contingencies . Hypothesis testing was conducted in each ROI with a 2 × 3 × 2 repeated-measures design that included diagnosis, stimulus-intensity and time (image volumes). The diagnosis × stimulus-intensity interaction, reflecting generalization differences in the PTSD compared with control group, was the key outcome of interest  and primary visual cortex as defined in the hypothesis-generating step , but no effect of diagnosis , nor a stimulus-intensity × diagnosis interaction . Repeated-measures analysis of variance with diagnosis as the between-group factor  and stimulus-intensity with low  or high  intensity as within-group factors . There was a significant stimulus-intensity × diagnosis interaction in the right fusiform , and at trend level in the right thalamus . However, planned comparisons of the fusiform revealed that high-intensity stimuli had lower activation in the PTSD group than the trauma-exposed control group. The remaining ROIs  showed no stimulus-intensity × diagnosis interaction . These results established that there was no bias before fear conditioning toward high-intensity stimuli in the PTSD group (12Baseline ratings before acquisition (preconditioning) revealed a  signific factors  found thSD group . Neverth1,58=107.0; P<0.0001), indicating successful fear learning.29 There was no difference between groups  or stimulus-type × group interaction . Specifically, there were no between-group differences for S1  or S3  during conditioning. (Shock expectancy ratings were significantly higher for the CS+ than the CS− during fear-acquisition runs (Ftioning. .Across the entire sample (PTSD and controls combined), fear learning-related activation (CS+>CS−) was found in primary visual cortex , inferior frontal gyrus (IFG), insula, locus coeruleus and thalamus . The neu9,53=38.25; P<0.0001), demonstrating generalization of shock expectancy ratings from the CS+ to the faces that expressed higher-intensity stimuli . However, there was no effect of diagnosis , nor a stimulus-intensity × diagnosis interaction . Specifically, there were no between-group differences during generalization for the S1  and S3 .Shock expectancy ratings during generalization  exhibiteBaseline corrected ROI activation results relative to the S1 response ; Table 1P-values >0.15), except antidepressant effects on activation in the fusiform and calcarine at trend level , locus coeruleus and thalamus. Combat exposure interacted with stimulus intensity to influence regional activation in the calcarine, locus coeruleus and fusiform (trend level). The medication covariates were found to have nonsignificant interactions with stimulus intensity , with a similar trend between the right amygdala and the thalamus , indicating greater connectivity in the PTSD group to faces expressing higher stimulus intensity . Task-modulated connectivity between the right amygdala and vmPFC showed a significant diagnosis × stimulus-intensity interaction , with greater connectivity in the control group to the lowest stimulus intensity (S2) that most closely resembled the CS− . Functional connectivity between the left amygdala and all the target regions was nonsignificant.Task-modulated functional connectivity between the right amygdala and calcarine cortex showed a significant diagnosis × stimulus-intensity interaction . S5 was selected most often by PTSD patients , indicating a strong memory bias to the face expressing the most fear, whereas controls selected the correct stimulus (S3) most often . There was no significant difference between the percentage of patients and controls who misidentified the other generalized stimuli ; S2 ).In the post-study recognition memory test, PTSD patients misidentified the face with the highest intensity stimulus (S5) as the CS+  more freThis study investigated generalization biases in fear neurocircuitry in military veterans with PTSD. Activity in a number of brain regions traditionally implicated in associative fear learning exhibited biased generalization towards stimuli of high emotional intensity after, but not before, fear conditioning in PTSD patients relative to controls. Activity in the amygdala broadly generalized to all stimuli in PTSD patients, and task-modulated functional connectivity of the amygdala with primary visual cortex and thalamus was biased in PTSD toward faces expressing high fear. Controls exhibited amygdala–vmPFC connectivity to a low-intensity stimulus that resembled the CS−, representing generalization of safety learning, whereas PTSD patients failed to exhibit similar patterns of amygdala–vmPFC connectivity. Neuroimaging results in PTSD were accompanied by a retrospective memory bias to falsely identify the highest intensity stimulus as the CS+. Collectively, these results provide new insights into how veterans with PTSD generalize fear from a single fear-learning episode to cues that have higher-intensity threat values than the original stimulus.32 Our functional connectivity results showed enhanced calcarine–amygdala and thalamo–amygdala functional coupling during fear generalization in PTSD. Fear generalization in PTSD was also facilitated by the locus coeruleus, which supports physiological and attentional components of the fight-or-flight response elicited by aversively conditioned stimuli through norepinephrine release.33 Finally, fear generalization in PTSD selectively recruited IFG and insula, which are part of a ventrolateral PFC circuit that integrates limbic responses with goal-directed actions, including holding affective material in working memory, directing attention to affectively salient information, and integrating somatic responses with decision-making processes.35 In contrast to regions showing selective fear generalization, the amygdala exhibited a broader generalization gradient in PTSD, including enhanced responses to faces with low-threat values. Given that the amygdala activation showed a broader generalization pattern than its functional connectivity, additional neural interactions unidentified in the present analysis must constrain the pattern of functional connectivity with calcarine cortex and thalamus.Brain regions showing intensity-based generalization in PTSD included areas along the ventral visual stream, such as primary visual cortex and fusiform gyrus, as well as sensory-integration components such as the thalamus, which have strong connections with the amygdala.36 and vmPFC disruption in extinction recall6 in PTSD. Given that extinction is widely considered a form of new inhibitory learning rather than erasing an established fear memory,37 shared neural mechanisms have been proposed for safety-signal learning and extinction learning.38 Combat veterans with PTSD lack effective safety signal learning as seen by poor modulation of fear in response to safety cues.39 Our connectivity results between vmPFC and amygdala are noteworthy in light of the essential role that vmPFC has in extinction, recall of learned extinction,26 and safety-signal learning through inhibition of the amygdala.26 Further research will be needed to explore amygdala–vmPFC connectivity associated with discriminating the safety signal from perceptually related stimuli, which is an important feature of resilience.3Our finding that controls exhibited a strong amygdala–vmPFC connectivity bias towards generalized cues that resemble the safety signal, whereas PTSD patients did not, builds on previous reports of behavioral deficits in safety learning40 One possible mechanism is that enhanced fMRI activity to high threat-value faces alters memory consolidation in PTSD to yield a retrospective memory bias. Indeed, diagnostic criteria for PTSD includes an inability to recall key features of the traumatic event, whereas clinical evidence often describes delayed recall41 or a vivid central memory of the trauma that is strongly colored by emotional and sensory impressions.42 We previously reported that patients with PTSD rely on gist memory for the recognition of similar content across trauma-related images.43 Importantly, as baseline fear-expression ratings were matched across groups, we can rule out perceptual bias as an explanation for the memory differences. Future studies are warranted to determine whether the memory biases in PTSD are reversible and to identify neural mechanisms that contribute to this memory bias.The PTSD patients also exhibited a retrospective memory bias to falsely identify the face expressing the highest fear value as the CS+. Thus, evidence of group differences in generalization was found at the neural level and, with retrospective memory, there was no evidence for group differences in behavioral generalization assessed with shock expectancy. The lack of group differences in expectancy ratings during the fear-acquisition stage provides evidence that associative fear learning remains intact in PTSD. Likewise, the behavioral ratings of faces, as well as the neural activity during preconditioning, suggests that perceptual discrimination of emotion is also intact in PTSD. The apparent inconsistency in behavioral and neural findings could be explained by a lack of sensitivity of the behavioral measures to underlying neural mechanisms working to produce generalization. Indeed, these changes do not manifest at the behavioral level until later in the experiment, but only by probing a related cognitive construct that assessed retrospective memory for the CS+. This memory distortion is consistent with a co-variation bias prevalent in anxiety disorders.44 which in turn is linked to aberrant functional connectivity in adulthood.45 Interestingly, both the groups in our sample experienced high rates of childhood trauma, albeit significantly higher in the PTSD group. It is well known that mild-to-moderate stress in childhood is required for healthy brain development, whereas more extreme low-stress and high-stress environments have negative consequences.47 To the extent that maltreatment is a form of stress, it is possible that mild exposure could result in greater resilience in adulthood. We found that childhood trauma predicted the generalization response in components of the fear neurocircuitry. These findings provide neurobiological support for findings from many studies, including a large sample (n=2181) of individuals who experienced multiple violent assaults in childhood resulting in greater likelihood of PTSD following trauma in adulthood.23 Our results extend prior evidence that exposure to child abuse is a major environmental factor, which further interacts with genetic factors that contribute significant risk for severity and chronicity of adult PTSD.48Our results indicate that exposure to childhood trauma predicted activation in the calcarine, IFG, insula, locus coeruleus and thalamus. It is well established that childhood maltreatment is associated with childhood poverty,11 there was no evidence of baseline differences in neural activity  or subjective ratings of emotional intensity in the present sample  uses low-intensity triggers for the patient to process through techniques such as corrective learning. Early treatment success then leads to titrating exposure to high-intensity triggers .56 Intensity-based fear generalization is thus a novel technique to examine fear generalization in trauma and other stress-related disorders, and may provide additional insights into the neurocircuitry of PTSD that complements perceptual similarity-based techniques.58We used fearful faces because prior research has demonstrated that fearful facial expressions tend to potentiate conditioned-fear responses, retard extinction learning and serve as a threat signal.59 Moreover, we found no significant association between generalization bias in fear neurocircuitry and depressive symptoms, alcohol use and common psychotropic medications.60 Our PTSD group had greater depressive symptoms than the trauma-exposed control group, but a PTSD group without depressive symptoms has limited clinical relevance, and new evidence calls into question whether PTSD and depression are distinct entities among trauma-exposed individuals.61 Usable skin conductance data were unavailable owing to technical challenges with reliable recording in the MRI environment; future studies should confirm our behavioral findings with psychophysiological measures. Our sample consisted mostly of male veterans from the Iraq and Afghanistan conflicts, urging caution when generalizing these results to other demographic groups.Although exclusion of participants taking psychotropic medication has been the accepted orthodoxy, leaders in PTSD neuroimaging have argued for their inclusion.8 indicates that generalization in this paradigm is asymmetric and is driven by threat intensity present in S4 and S5. That is, a group for whom the S5 served as the CS− did not show a reverse gradient skewed towards the S1.8 To fully clarify fear generalization in PTSD, future studies should incorporate other intensity dimensions  as well as non-intensity dimensions . Further research will also help relate findings in PTSD patients to forms of fear generalization found in other anxiety disorders.16Our design had two generalized stimuli of greater intensity than CS+, but only one generalized stimulus of lower intensity than the CS+. An additional stimulus with the lowest intensity served as the CS−. This design feature, which oversamples generalization toward the higher end of the spectrum, is a minor concern, given that the same design was used for both the groups and did not differentially affect our ability to detect generalization to higher or lower intensity stimuli in a particular group . Moreove57 Given that our analyses used a CS− as a nonassociative control stimulus and we found evidence for asymmetric intensity-based generalization gradients in shock expectancy ratings in many of the brain ROIs, we rule out a purely nonassociative account of the findings. Another potential interpretation is ‘selective sensitization',62 in which mere shock presentations result in sensitivity to high-intensity stimuli selectively.8 Given that we also found altered functional connectivity results indicative of impaired safety learning in PTSD, selective sensitization does not fully account for the pattern of results, making associative learning a parsimonious mechanistic interpretation. The relative contribution of associative and nonassociative mechanisms supporting overgeneralization in PTSD merits further theoretical and empirical work.Finally, we frame fear generalization results in PTSD in terms of an associative learning mechanism, such that shocks predicted selectively by the CS+ induce asymmetric intensity-based gradients. An alternative, nonassociative account is that shocks alone are sufficient to induce generalization .63 are subject to modifications that take place beyond the initial fear learning episode to make fear memories more resistant to extinction, less contextually specific, and overgeneralized. These functional brain changes may contribute to symptoms of PTSD, which are frequently triggered by trauma cues that merely resemble, but are not identical to cues in the index trauma.Fear neurocircuitry and memory in PTSD are biased toward stimuli that possess greater emotional intensity than the original conditioned-fear stimulus. This study contributes to a growing appreciation that fear-conditioning processes in PTSD