Engineered murine IL-21-secreting leukemia cells induce granzyme B+ T cells and CD4+CD44+CD62L- effector memory cells while suppressing regulatory T cells, leading to long-term survival.

We have explored the use of an IL-21 cell-based anti-leukemia treatment in a mouse model of acute lymphoblastic leukemia. 70Z/3 leukemia cells, engineered to secrete IL-21 and injected into the peritoneum of syngeneic mice, induced a strong anti-leukemia response resulting in 100% survival. Mice that mounted an IL-21-induced anti-leukemia immune response were immune to the parent cell line (no IL-21) when rechallenged.Above a certain threshold, IL-21 secretion correlated with improved survival compared to mice injected with parent 70Z/3 cells. IL-21 was detected in serum with peak levels on day 7, correlating with the maximum expansion of IL-21-secreting 70Z/3 cells which subsequently were eliminated. Mice injected with IL-21-secreting leukemia cells had elevated numbers of granzyme B+ CD4+ and CD8+ T cells in the peritoneum, compared to mice injected with the parent cell line. Regulatory T cells, which increased greatly in 70Z/3-injected mice, failed to do so in mice injected with IL-21-secreting cells. Upon rechallenge, IL-21-primed mice went through a secondary immune response, primarily requiring CD4+ T cells, triggering a significant increase of CD4+CD44+CD62L- effector memory T cells. Adoptive transfer of T cells from IL21-primed/rechallenged hosts into naïve mice was successful, indicating that IL-21-primed antigen-experienced T cells convey immunity to naïve mice.Our study shows that delivery of IL-21 in a cell-based anti-leukemia protocol has the potential to induce a potent immune response leading to cancer elimination and long-term immunity-properties which make IL-21 an attractive candidate for cancer immunotherapy. Protecting against tumor antigens as well as improving cancer immunity is justified, as current strategies are limited.

Cytotoxic CD4+ T Cells in Bladder Cancer-A New License to Kill.

CD4+ T cells with cytotoxic capability are increasingly recognized as potentially key actors in anti-tumor immunity. A new report in Cell elucidates the presence and potential role of a population of cytotoxic CD4+ T cells in bladder cancer using single cell sequencing technology.

Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells.

Efficient Ca2+ flux induced during cognate T cell activation requires signaling the T cell receptor (TCR) and unidentified G-protein-coupled receptors (GPCRs). T cells express the neurokinin-1 receptor (NK1R), a GPCR that mediates Ca2+ flux in excitable and non-excitable cells. However, the role of the NK1R in TCR signaling remains unknown. We show that the NK1R and its agonists, the neuropeptides substance P and hemokinin-1, co-localize within the immune synapse during cognate activation of T cells. Simultaneous TCR and NK1R stimulation is necessary for efficient Ca2+ flux and Ca2+-dependent signaling that sustains the survival of activated T cells and helper 1 (Th1) and Th17 bias. In a model of contact dermatitis, mice with T cells deficient in NK1R or its agonists exhibit impaired cellular immunity, due to high mortality of activated T cells. We demonstrate an effect of the NK1R in T cells that is relevant for immunotherapies based on pro-inflammatory neuropeptides and its receptors.

Interleukin-15 in cancer immunotherapy: IL-15 receptor complex versus soluble IL-15 in a cancer cell-delivered murine leukemia model.

Cytokines of the common γ-chain receptor family such as IL-15 are vital with respect to activating immune cells, sustaining healthy immune functions, and augmenting the anti-tumor activity of effector cells, making them ideal candidates for cancer immunotherapy. IL-15, either in its soluble form (IL-15sol) or complexed with IL-15Rα (IL-15Rc), has been shown to exhibit potent anti-tumor activities in various experimental cancer studies. Here we describe the impact of intraperitoneal IL-15 in a cancer cell-delivered IL-15 immunotherapy approach using the 70Z/3-L leukemia mouse model. Whereas both forms of IL-15 led to significantly improved survival rates compared to the parent cell line, there were striking differences in the extent of the improved survival: mice receiving cancer cells secreting IL-15sol showed significantly longer survival and protective long-term immunity compared to those producing IL-15Rc. Interestingly, injection of leukemia cells secreting IL-15sol lead to heightened expansion of CD4+ and CD8+ T-cell populations in the peritoneum compared to IL-15Rc. Cell-secreted IL-15Rc resulted in an influx and/or expansion of NK1.1+ cells in the peritoneum which was much less pronounced in the IL-15sol model. Furthermore, IL-15Rc but not IL-15sol lead to T-cell exhaustion and disease progression. To our knowledge, this is the first study detailing a significantly different biological effect of cell-delivered IL-15sol versus IL-15Rc in a mouse cancer immunotherapy study.

c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells.

Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease.

Differential regulation of IgA+ B cells in vitro by stromal cells from distinctive anatomical compartments.

B cell development is regulated by stromal cells (SCs) that form a supportive microenvironment. These SCs along with other cell types produce cytokines, chemokines, and adhesion molecules that guide B cell commitment and differentiation. BM, spleen (Sp), and the gut lamina propria (LP) constitute distinctive anatomical compartments that support B cell differentiation. In order to characterize and compare the signals necessary to generate IgA+ B cells, we developed an in vitro system to co-culture gut LP, BM, or Sp-derived SCs with B lineage cells. Using this co-culture system, we found that gut LP SCs promote IgA+ B cell accumulation through the production of soluble stimulatory factors. In contrast to gut LP SCs, BM and splenic SCs were found to impair IgA+ B cell accumulation in vitro. Taken together, these observations provide new insights into how SCs derived from different anatomical locations shape IgA+ B cell responses.

Functional Consequences of the Postnatal Switch From Neonatal to Mutant Adult Glycine Receptor α1 Subunits in the Shaky Mouse Model of Startle Disease.

Mutations in GlyR α1 or ß subunit genes in humans and rodents lead to severe startle disease characterized by rigidity, massive stiffness and excessive startle responses upon unexpected tactile or acoustic stimuli. The recently characterized startle disease mouse mutant shaky carries a missense mutation (Q177K) in the ß8-ß9 loop within the large extracellular N-terminal domain of the GlyR α1 subunit. This results in a disrupted hydrogen bond network around K177 and faster GlyR decay times. Symptoms in mice start at postnatal day 14 and increase until premature death of homozygous shaky mice around 4-6 weeks after birth. Here we investigate the in vivo functional effects of the Q177K mutation using behavioral analysis coupled to protein biochemistry and functional assays. Western blot analysis revealed GlyR α1 subunit expression in wild-type and shaky animals around postnatal day 7, a week before symptoms in mutant mice become obvious. Before 2 weeks of age, homozygous shaky mice appeared healthy and showed no changes in body weight. However, analysis of gait and hind-limb clasping revealed that motor coordination was already impaired. Motor coordination and the activity pattern at P28 improved significantly upon diazepam treatment, a pharmacotherapy used in human startle disease. To investigate whether functional deficits in glycinergic neurotransmission are present prior to phenotypic onset, we performed whole-cell recordings from hypoglossal motoneurons (HMs) in brain stem slices from wild-type and shaky mice at different postnatal stages. Shaky homozygotes showed a decline in mIPSC amplitude and frequency at P9-P13, progressing to significant reductions in mIPSC amplitude and decay time at P18-24 compared to wild-type littermates. Extrasynaptic GlyRs recorded by bath-application of glycine also revealed reduced current amplitudes in shaky mice compared to wild-type neurons, suggesting that presynaptic GlyR function is also impaired. Thus, a distinct, but behaviorally ineffective impairment of glycinergic synapses precedes the symptoms onset in shaky mice. These findings extend our current knowledge on startle disease in the shaky mouse model in that they demonstrate how the progression of GlyR dysfunction causes, with a delay of about 1 week, the appearance of disease symptoms.

Cytokines and depression in cancer patients and caregivers.

OBJECTIVE: A better understanding of the biobehavioral mechanisms underlying depression in cancer is required to translate biomarker findings into clinical interventions. We tested for associations between cytokines and the somatic and psychological symptoms of depression in cancer patients and their healthy caregivers. PATIENTS AND METHODS: The GRID Hamilton Rating Scale for Depression (Ham-D) was administered to 61 cancer patients of mixed type and stage, 26 primary caregivers and 38 healthy controls. Concurrently, blood was drawn for multiplexed plasma assays of 15 cytokines. Multiple linear regression, adjusted for biobehavioral variables, identified cytokine associations with the psychological (Ham-Dep) and somatic (Ham-Som) subfactors of the Ham-D. RESULTS: The Ham-Dep scores of cancer patients were similar to their caregivers, but their Ham-Som scores were significantly higher (twofold, p=0.016). Ham-Som was positively associated with IL-1ra (coefficient: 1.27, p≤0.001) in cancer patients, and negatively associated with IL-2 (coefficient: -0.68, p=0.018) in caregivers. Ham-Dep was negatively associated with IL-4 (coefficient: -0.67, p=0.004) in cancer patients and negatively associated with IL-17 (coefficient: -1.81, p=0.002) in caregivers. CONCLUSION: The differential severity of somatic symptoms of depression in cancer patients and caregivers and the unique cytokine associations identified with each group suggests the potential for targeted interventions based on phenomenology and biology. The clinical implication is that depressive symptoms in cancer patients can arise from biological stressors, which is an important message to help destigmatize the development of depression in cancer patients.

Bone marrow basophils provide survival signals to immature B cells in vitro but are dispensable in vivo.

Immature B cells are the first B cell progenitors to express a fully formed B cell receptor and are therefore subject to extensive selection processes that act to mitigate the emergence of autoreactive clones. While it is well appreciated that most B cell generation in the bone marrow is highly dependent on access to molecules present in the local milieu, the existence of extrinsically provided factors that modulate immature B cell biology is ambiguous. Nonetheless, a population of CD49b+CD90lo cells has demonstrated in vitro potential to promote immature B cell survival. Using a mouse basophil reporter strain we confirmed the identity of these CD49b+CD90lo supportive cells as basophils. However, analysis of bone marrow B cell populations following lineage specific basophil depletion demonstrates that basophils do not have a significant role in vivo in modulating immature B cell biology during steady-state conditions.

Disruption of a Structurally Important Extracellular Element in the Glycine Receptor Leads to Decreased Synaptic Integration and Signaling Resulting in Severe Startle Disease.

Functional impairments or trafficking defects of inhibitory glycine receptors (GlyRs) have been linked to human hyperekplexia/startle disease and autism spectrum disorders. We found that a lack of synaptic integration of GlyRs, together with disrupted receptor function, is responsible for a lethal startle phenotype in a novel spontaneous mouse mutant shaky, caused by a missense mutation, Q177K, located in the extracellular ß8-ß9 loop of the GlyR α1 subunit. Recently, structural data provided evidence that the flexibility of the ß8-ß9 loop is crucial for conformational transitions during opening and closing of the ion channel and represents a novel allosteric binding site in Cys-loop receptors. We identified the underlying neuropathological mechanisms in male and female shaky mice through a combination of protein biochemistry, immunocytochemistry, and both in vivo and in vitro electrophysiology. Increased expression of the mutant GlyR α1Q177K subunit in vivo was not sufficient to compensate for a decrease in synaptic integration of α1Q177Kß GlyRs. The remaining synaptic heteromeric α1Q177Kß GlyRs had decreased current amplitudes with significantly faster decay times. This functional disruption reveals an important role for the GlyR α1 subunit ß8-ß9 loop in initiating rearrangements within the extracellular-transmembrane GlyR interface and that this structural element is vital for inhibitory GlyR function, signaling, and synaptic clustering.SIGNIFICANCE STATEMENT GlyR dysfunction underlies neuromotor deficits in startle disease and autism spectrum disorders. We describe an extracellular GlyR α1 subunit mutation (Q177K) in a novel mouse startle disease mutant shaky Structural data suggest that during signal transduction, large transitions of the ß8-ß9 loop occur in response to neurotransmitter binding. Disruption of the ß8-ß9 loop by the Q177K mutation results in a disruption of hydrogen bonds between Q177 and the ligand-binding residue R65. Functionally, the Q177K change resulted in decreased current amplitudes, altered desensitization decay time constants, and reduced GlyR clustering and synaptic strength. The GlyR ß8-ß9 loop is therefore an essential regulator of conformational rearrangements during ion channel opening and closing.

Hemokinin-1 mediates anxiolytic and anti-depressant-like actions in mice.

The tachykinin NK1 receptor was suggested to be involved in psychiatric disorders, but its antagonists have failed to be effective as antidepressants in clinical trials. Hemokinin-1 (HK-1), the newest tachykinin, is present in several brain regions and activates the NK1 receptor similarly to substance P (SP), but acts also through other mechanisms. Therefore, we investigated the roles of the Tac4 gene-derived HK-1 in comparison with SP and neurokinin A (NKA) encoded by the Tac1 gene, as well as the NK1 receptor in anxiety and depression-like behaviors in mice. Mice lacking SP/NKA, HK-1 or the NK1 receptor (Tac1-/-, Tac4-/-, Tacr1-/-, respectively) compared to C57Bl/6 wildtypes (WT), and treatment with the NK1 antagonist CP99994 were used in the experiments. Anxiety was evaluated in the light-dark box (LDB) and the elevated plus maze (EPM), locomotor activity in the open field (OFT) tests. Hedonic behavior was assessed in the sucrose preference test (SPT), depression-like behavior in the tail suspension (TST) and forced swim (FST) tests. FST-induced neuronal responsiveness was evaluated with Fos immunohistochemistry in several stress-related brain regions. In the LDB, Tac4-/- mice spent significantly less, while Tacr1-/- and CP99994-treated mice spent significantly more time in the lit compartment. In the EPM only Tac4-/- showed reduced time in the open arms, but no difference was observed in any other groups. In the OFT Tac4-/- mice showed significantly reduced, while Tac1-/- and Tacr1-/- animals increased motility than the WTs, but CP99994 had no effect. NK1-/- consumed markedly more, while Tac4-/- less sucrose solution compared to WTs. In the TST and FST, Tac4-/- mice showed significantly increased immobility. However, depression-like behavior was decreased both in cases of genetic deletion and pharmacological blockade of the NK1 receptor. FST-induced neuronal activation in different nuclei involved in behavioral and neuroendocrine stress responses was significantly reduced in the brain of Tac4 -/- mice. Our results provide the first evidence for an anxiolytic and anti-depressant-like actions of HK-1 through a presently unknown target-mediated mechanism. Identification of its receptor and/or signaling pathways might open new perspectives for anxiolytic and anti-depressant therapies.

Pro- and anti-inflammatory cytokine associations with major depression in cancer patients.

OBJECTIVE: Cytokines may be linked to depression, although it has been challenging to demonstrate this association in cancer because of the overlap between depressive symptoms and other sickness behaviors. This study investigates the relationship between cytokines and depression in cancer patients, accounting for confounding clinical and methodological factors. METHODS: The GRID Hamilton Rating Scale for Depression and Neurotoxicity Rating Scale (NRS) for cytokine-induced sickness behaviors were administered to 61 cancer patients and 38 healthy controls. The cancer group was of mixed type and largely of late stage, with a recruitment rate of 35% and completion rate of 47%. Major depression was diagnosed in 19 of 61 (31%) cancer patients. Multiplexed cytokine assays for inflammatory and anti-inflammatory cytokines were conducted in plasma samples using electrochemiluminescence. RESULTS: All cancer patients had high NRS scores and elevated levels of most cytokines. Cancer patients with major depression had higher NRS scores than those without major depression. IL-1rα was positively associated with the GRID scores of depressive symptoms (regression coefficient, 3.52 ± 1.18; P = .004), but not with major depression. Major depression was negatively associated with the anti-inflammatory cytokine IL-4 (regression coefficient, -0.65 ± 0.26; P = .013), but not with IL-1rα. CONCLUSIONS: Depressive symptoms in cancer patients may represent sickness behaviors, which may have distinct cytokine associations from major depression. Sickness behaviors may be associated with an increase in inflammatory cytokines, whereas major depression may be induced by a failure to adequately resolve inflammation. Our findings suggest that cytokine-mediated interventions may be of value to treat depression in this population.

Preclinical validation: LV/IL-12 transduction of patient leukemia cells for immunotherapy of AML.

Interleukin-12 (IL-12) is a potent cytokine that may be harnessed to treat cancer. To date, nearly 100 IL-12-based clinical trials have been initiated worldwide. Yet systemic administration of IL-12 is toxic. Different strategies are being developed to reduce such toxicities by restricting IL-12 distribution. Our previous studies employed lentivector-mediated expression of murine IL-12 in tumor cells and demonstrated effective protection in both mouse leukemia and solid tumor challenge models. In this study, we carried out preclinical validation studies using a novel lentivector to engineer expression of human IL-12 in acute myeloid leukemia blast cells isolated from 21 patients. Acute myeloid leukemia cells were transduced with a bicistronic lentivector that encodes the human IL-12 cDNA as a fusion, as well as a LNGFR (ΔLNGFR)/mutant thymidylate kinase cassette as a marking and cell-fate control element. A range of 20-70% functional transduction efficiencies was achieved. Transduced acute myeloid leukemia cells produced bioactive IL-12 protein and displayed dose-dependent sensitivity to the prodrug 3'-azido-3'-deoxythymidine. In vitro immortalization assays using transduced mouse hematopoietic stem cells demonstrated minimal genotoxic risk from our IL-12 vector. Scale-up transduction and cell processing was subsequently validated in a GMP facility to support our (now approved) Clinical Trial Application (CTA).

Tumor-secreted products repress B-cell lymphopoiesis in a murine model of breast cancer.

Growing cancers are known to modify immune responses through suppressive mechanisms manifested within the local tumor microenvironment. Accumulating evidence indicates that secreted tumor products can also influence on distant immunological compartments, including myelopoiesis in the bone marrow. However, it is unknown if a similar effect can occur to regulate B-cell lymphopoiesis in breast cancer. Examining the MMTV-PyMT murine model of breast cancer, we show a complete block in bone marrow B-cell lymphopoiesis, which is dependent on tumor burden. We also observed an increase in the total number of splenic B cells and an elevated frequency of marginal zone B cells. By using in vitro assays of B-cell lymphopoiesis, we show that tumor-secreted molecules directly inhibit B-cell progenitor proliferation and favor maturation. These data demonstrate a profound sensitivity of B-cell lymphopoiesis to the accumulation of ectopically produced molecules during tumor growth in PyMT.

Inflammation rapidly reorganizes mouse bone marrow B cells and their environment in conjunction with early IgM responses.

Systemic inflammation perturbs the bone marrow environment by evicting resident B cells and favoring granulopoiesis over lymphopoiesis. Despite these conditions, a subset of marrow B cell remains to become activated and produce potent acute immunoglobulin M (IgM) responses. This discrepancy is currently unresolved and a complete characterization of early perturbations in the B-cell niche has not been undertaken. Here, we show that within a few hours of challenging mice with adjuvant or cecal puncture, B cells accumulate in the bone marrow redistributed away from sinusoid vessels. This response correlates with enhanced sensitivity to CXC chemokine ligand 12 (CXCL12) but not CXCL13 or CC chemokine ligand 21. Concurrently, a number of B-cell survival and differentiation factors are elevated to produce a transiently supportive milieu. Disrupting homing dynamics with a CXC chemokine receptor 4 inhibitor reduced the formation of IgM-secreting cells. These data highlight the rapidity with which peripheral inflammation modifies the marrow compartment, and demonstrate that such modifications regulate acute IgM production within this organ. Furthermore, our study indicates that conversion to a state of emergency granulopoiesis is temporally delayed, allowing B cells opportunity to respond to antigen.

CD4+ T cell plasticity engenders robust immunity in response to cytokine therapy.

CD4+ T cells represent an entire arm of the immune system that has hitherto been incompletely understood, but their potential to act as both helper and effector may make them optimal protagonists in immunotherapeutic approaches to treat cancer. Cytokine therapy can activate this population in a manner that ensures maximal diversification of effector function for a robust immune response.

Hemokinin-1 is an important mediator of endotoxin-induced acute airway inflammation in the mouse.

OBJECTIVE: Hemokinin-1, the newest tachykinin encoded by the preprotachykinin C (Tac4) gene, is predominatly produced by immune cells. Similarly to substance P, it has the greatest affinity to the tachykinin NK1 receptor, but has different binding site and signaling mechanisms. Furthermore, several recent data indicate the existence of a not yet identified own receptor and divergent non-NK1-mediated actions. Since there is no information on its functions in the airways, we investigated its role in endotoxin-induced pulmonary inflammation. METHODS: Acute pneumonitis was induced in Tac4 gene-deleted (Tac4(-/-)) mice compared to C57Bl/6 wildtypes by intranasal E. coli lipopolysaccharide (LPS). Airway responsiveness to inhaled carbachol was measured with unrestrained whole body plethysmography 24h later. Semiquantitative histopathological scoring was performed; reactive oxygen species (ROS) production was measured with luminol bioluminescence, myeloperoxidase activity with spectrophotometry, and inflammatory cytokines with Luminex. RESULTS: All inflammatory parameters, such as histopathological alterations (perivascular edema, neutrophil/macrophage accumulation, goblet cell hyperplasia), myeloperoxidase activity, ROS production, as well as interleukin-1beta, interleukin-6, tumor necrosis factor alpha, monocyte chemoattractant protein-1 and keratinocyte chemoattractant concentrations were significantly diminished in the lung of Tac4(-/-) mice. However, bronchial hyperreactivity similarly developed in both groups. Interestingly, in LPS-treated Tac4(-/-) mouse lungs, bronchus-associated, large, follicle-like lymphoid structures developed. CONCLUSIONS: We provide the first evidence that hemokinin-1 plays a crucial pro-inflammatory role in the lung by increasing inflammatory cell activities, and might also be a specific regulator of lymphocyte functions.

Murine splenic CD4⁺ T cells, induced by innate immune cell interactions and secreted factors, develop antileukemia cytotoxicity.

Inciting the cellular arm of adaptive immunity has been the fundamental goal of cancer immunotherapy strategies, specifically focusing on inducing tumor antigen-specific responses by CD8(+) cytotoxic T lymphocytes (CTL). However, there is an emerging appreciation that the cytotoxic function of CD4(+) T cells can be effective in a clinical setting. Harnessing this potential will require an understanding of how such cells arise. In this study, we use an IL12-transduced variant of the 70Z/3 leukemia cell line in a B6D2F1 (BDF1) murine model system to reveal a novel cascade of cells and soluble factors that activate anticancer CD4(+) killer cells. We show that natural killer T cells play a pivotal role by activating dendritic cells in a contact-dependent manner; soluble products of this interaction, including MCP-1, propagate the activation signal, culminating in the development of CD4(+) CTLs that directly mediate an antileukemia response while also orchestrating a multipronged attack by other effector cells. A more complete picture of the conditions that induce such a robust response will allow us to capitalize on CD4(+) T-cell plasticity for maximum therapeutic effect.

Pten deletion in RIP-Cre neurons protects against type 2 diabetes by activating the anti-inflammatory reflex.

Inflammation has a critical role in the development of insulin resistance. Recent evidence points to a contribution by the central nervous system in the modulation of peripheral inflammation through the anti-inflammatory reflex. However, the importance of this phenomenon remains elusive in type 2 diabetes pathogenesis. Here we show that rat insulin-2 promoter (Rip)-mediated deletion of Pten, a gene encoding a negative regulator of PI3K signaling, led to activation of the cholinergic anti-inflammatory pathway that is mediated by M2 activated macrophages in peripheral tissues. As such, Rip-cre(+) Pten(flox/flox) mice showed lower systemic inflammation and greater insulin sensitivity under basal conditions compared to littermate controls, which were abolished when the mice were treated with an acetylcholine receptor antagonist or when macrophages were depleted. After feeding with a high-fat diet, the Pten-deleted mice remained markedly insulin sensitive, which correlated with massive subcutaneous fat expansion. They also exhibited more adipogenesis with M2 macrophage infiltration, both of which were abolished after disruption of the anti-inflammatory efferent pathway by left vagotomy. In summary, we show that Pten expression in Rip(+) neurons has a critical role in diabetes pathogenesis through mediating the anti-inflammatory reflex.