Node Cells Research Articles

Microbial synthesis of Prussian blue for potentiating checkpoint blockade immunotherapy

Cancer immunotherapy is revolutionizing oncology. The marriage of nanotechnology and immunotherapy offers a great opportunity to amplify antitumor immune response in a safe and effective manner. Here, electrochemically active Shewanella oneidensis MR-1 can be applied to produce FDA-approved Prussian blue nanoparticles on a large-scale. We present a mitochondria-targeting nanoplatform, MiBaMc, which consists of Prussian blue decorated bacteria membrane fragments having further modifications with chlorin e6 and triphenylphosphine. We find that MiBaMc specifically targets mitochondria and induces amplified photo-damages and immunogenic cell death of tumor cells under light irradiation. The released tumor antigens subsequently promote the maturation of dendritic cells in tumor-draining lymph nodes, eliciting T cell-mediated immune response. In two tumor-bearing mouse models using female mice, MiBaMc triggered phototherapy synergizes with anti-PDL1 blocking antibody for enhanced tumor inhibition. Collectively, the present study demonstrates biological precipitation synthetic strategy of targeted nanoparticles holds great potential for the preparation of microbial membrane-based nanoplatforms to boost antitumor immunity.

IL-1β Derived Th17 Immune Responses Are a Critical Factor for Neutrophilic-Eosinophilic Airway Inflammation on Psychological Stress-Induced Immune Tolerance Breakdown in Mice

Introduction: Asthma is an inflammatory reaction mediated by type 2 helper T (Th2) cells and is known to increase eosinophil levels. Our previous study showed that stress-related asthma can cause neutrophilic and eosinophilic airway inflammation by suppressing immune tolerance. However, the mechanism of stress-induced neutrophilic and eosinophilic airway inflammation remains unclear. Therefore, to elucidate the cause of neutrophilic and eosinophilic inflammation, we investigated the immune response during the induction of airway inflammation. In addition, we focused on the relationship between immune response modulation immediately after stress exposure and the development of airway inflammation. Methods: Asthmatic mice were induced by three phases using female BALB/c mice. During the first phase, the mice were made to inhale ovalbumin (OVA) to induce immune tolerance before sensitization. Some mice were exposed to restraint stress during the induction of immune tolerance. In the second phase, the mice were sensitized with OVA/alum intraperitoneal injections. In the final phase, onset of asthma was induced through OVA exposure. Asthma development was evaluated based on airway inflammation and T-cell differentiation. Microarray and qPCR analyses were used to enumerate candidate factors to investigate the starting point of immunological modification immediately after stress exposure. Furthermore, we focused on interleukin-1β (IL-1β), which initiates these immune modifications, and performed experiments using its receptor blocker interleukin-1 receptor antagonist (IL-1RA). Results: Stress exposure during immune tolerance induction increased eosinophil and neutrophil airway infiltration. This inflammation was associated with decreased T regulatory cell levels and increased Th2 and Th17 levels in bronchial lymph node cells. Microarray and qPCR analyses showed that the initiation of Th17 differentiation might be triggered by stress exposure during tolerance induction. IL-1RA administration during stress exposure suppressed neutrophilic and eosinophilic airway inflammation via Th17 reduction and Treg increase. Conclusions: Our results show that psychological stress causes both eosinophilic and neutrophilic inflammatory responses due to the breakdown of immune tolerance. Furthermore, stress-induced inflammation can be abolished using IL-1RA.

Prevotella intermedia Aggravates Subclinical Hypothyroidism

Subclinical hypothyroidism (SCH) has been shown to be associated with microbiota. However, the association between SCH and oral microbiota has not yet been elucidated. The results of our previous clinical studies showed that Prevotella intermedia was abundant in the oral microbiota of SCH patients. This study aimed to investigate the relationship between SCH and oral microbiota, verify the pathogenicity of P. intermedia in SCH, and preliminarily explore the possible mechanism. The SCH mouse model with oral application of P. intermedia was established, and the variance in the mouse oral microbiota and changes in thyroid function and metabolism were detected in mice. Student’s t test and analysis of variance were used for statistical analysis. Oral application of P. intermedia changed the composition of the oral microbiota of SCH mice, which enhanced the damage to the thyroid and decreased the expression of functional genes of the thyroid. Moreover, P. intermedia decreased oxygen consumption and aggravated glucose and lipid metabolism disorders in SCH mice. Glucose tolerance and insulin tolerance decreased, and the triglyceride content of the liver and inflammatory infiltration in adipose tissue increased in SCH mice after P. intermedia stimulation. Mechanistically, P. intermedia increased the proportion of CD4+ T cells in cervical lymph nodes and thyroids in SCH mice. Th1 cells were suggested to play an important role in the pathogenesis of SCH involving P. intermedia. In conclusion, P. intermedia aggravated SCH manifestations, including thyroid dysfunction and glucose and lipid metabolism disorders, by causing immune imbalance in mice. This study sheds new light on the pathogenesis of SCH from the perspective of oral microbiota.

CTLA-4 suppresses hapten-induced contact hypersensitivity in atopic dermatitis model mice

Atopic dermatitis (AD) patients with skin barrier dysfunction are considered to be at a higher risk of allergic contact dermatitis (ACD), although previous studies showed that attenuated ACD responses to strong sensitizers in AD patients compared to healthy controls. However, the mechanisms of ACD response attenuation in AD patients are unclear. Therefore, using the contact hypersensitivity (CHS) mouse model, this study explored the differences in CHS responses to hapten sensitization between NC/Nga mice with or without AD induction (i.e., non-AD and AD mice, respectively). In this study, ear swelling and hapten-specific T cell proliferation were significantly lower in AD than in non-AD mice. Moreover, we examined the T cells expressing cytotoxic T lymphocyte antigen-4 (CTLA-4), which is known to suppress T cell activation, and found a higher frequency of CTLA-4+ regulatory T cells in draining lymph node cells in AD than in non-AD mice. Furthermore, the blockade of CTLA-4 using a monoclonal antibody eliminated the difference in ear swelling between non-AD and AD mice. These findings suggested that CTLA-4+T cells may contribute to suppressing the CHS responses in AD mice.

Harnessing NK cell-based immunotherapy to prevent the high-dose radiotherapy-inducing tumor survival recurrence

Natural killer cells play crucial roles in tumor immunosurveillance and serve as first responders to recognize abnormal cells. Radiotherapy is the mainstay of cancer treatment. However, the effect of high-dose radiotherapy on NK cells remains elusive. Here, we used tumor-bearing mice in the murine colorectal cancer cell line, MC38. The function of NK cells in tumor-draining lymph nodes and tumors was explored after the mice were treated using radiotherapy with 20 Gy and/or blocking antibody αTIGIT at the indicated time. High-dose radiotherapy shaped an immunosuppressive tumor microenvironment to support tumor growth, showing a decreased anti-tumor immunity phenotype in which effector T cells were reduced significantly. Furthermore, the production of functional cytokines and markers in NK cells, including CD107a, granzyme B, and IFN-γ, also remarkably decreased after radiotherapy, while the inhibitory receptor TIGIT was significantly upregulated by FACS analysis. The effect of radiotherapy was significantly elevated after treatment with the combination of radiotherapy and TIGIT inhibition. Moreover, this combination significantly decreased tumor recurrence. Our findings reported that local single high-dose radiotherapy shaped the immunosuppressive microenvironment and inhibited the function of NK cells. Our study revealed compelling evidence suggesting that the enhancement of NK cell function through TIGIT targeting is an effective strategy to mitigate immune suppression caused by high-dose radiotherapy, thereby promoting the inhibition of tumor recurrence.

Sirtuin 6 is a negative regulator of the anti-tumor function of natural killer cells in murine inflammatory colorectal cancer

The immune system plays a crucial role in controlling colorectal cancer (CRC) development. Natural killer (NK) cells are tumoricidal but undergo exhaustion in CRC patients. The current research aims to understand the role of sirtuin 6 (SIRT6) in CRC-associated NK cell exhaustion in a murine inflammatory colorectal cancer model. To this end, inflammatory CRC was induced by treating mice with azoxymethane plus dextran sulfate sodium. The expression of SIRT6 in NK cells in murine mesenteric lymph nodes (mLNs) and the CRC tissue was characterized by Immunoblotting. SIRT6 knockdown was achieved by lentiviral transduction of murine splenic NK cells, followed by evaluation of NK cell proliferation and the expression of cytotoxic mediators using flow cytometry. NK cell cytotoxicity was measured by cytotoxicity assays. Adoptive transfer of murine NK cells was applied to analyze the effect of SIRT6 knockdown in vivo. We found that SIRT6 was up-regulated in infiltrating NK cells in the murine CRC tissue, especially NK cells with an exhausted phenotype and impaired cytotoxicity. SIRT6 knockdown significantly boosted murine splenic NK cell functionality, as evidenced by accelerated proliferation, increased production of cytotoxic mediators, and higher tumoricidal activity both in vitro and in vivo. Furthermore, the adoptive transfer of SIRT6-knockdown NK cells into CRC-bearing mice effectively suppressed CRC progression. Therefore, SIRT6 up-regulation is essential for murine NK cell exhaustion in CRC because it impedes the tumoricidal activity of murine NK cells. Artificial SIRT6 down-regulation could boost the function of infiltrating NK cells to oppress CRC progression in mice.

Clonal Spreading of Tumor-Infiltrating T Cells Underlies the Robust Antitumor Immune Responses.

The repertoire of tumor-infiltrating T cells is an emerging method for characterizing effective antitumor T-cell responses. Oligoclonal expansion of the tumor T-cell repertoire has been evaluated; however, their association with antitumor effects is unclear. We demonstrate here that the polyclonal fraction of the tumor-reactive T-cell repertoire, consisting of relatively minor clones, increased in tumor-bearing mice treated with monoclonal anti-programmed death-ligand 1 (PD-L1) or anti-CD4, which correlated with antitumor effects. Meanwhile, the size of the oligoclonal fraction consisting of major clones remained unchanged. Moreover, the polyclonal fraction was enriched in progenitor exhausted T cells, which are essential for a durable antitumor response, and was more dependent on CCR7+ migratory dendritic cells, which are responsible for priming tumor-reactive T cells in the tumor-draining lymph nodes. These results suggest that the expansion of diverse tumor-reactive clones ("clonal spreading") represents characteristics of antitumor T-cell responses induced by anti-CD4 and anti-PD-L1 treatment.

A single species bacterial therapy reduces type 2 immune responses in the gut to prevent food allergy

Abstract The rising prevalence of food allergy is strongly correlated with dysbiosis of the gut microbiome. Rescuing this dysbiosis by reintroducing specific bacteria is a promising method to prevent or treat food allergy. We have shown that monocolonization with Anaerostipes caccae, a species representative of the healthy infant microbiota, protects against an allergic response to the milk protein b-lactoglobulin (BLG). Recently we developed a synbiotic bacterial therapy (A. caccaeplus prebiotic lactulose) which rescues the allergic phenotype of gnotobiotic mice colonized with the fecal bacteria of a dysbiotic, cow’s milk allergic (CMA) infant. Synbiotic treatment minimizes the anaphylactic response to intragastric BLG challenge and reduces circulating BLG-specific IgE and IgG1. To understand how a single bacterium regulates mucosal immunity during the onset of IgE-mediated allergy we first examined the alarmin response. CMA colonized mice express high transcript levels of both IL25and TLSPin ileal epithelial cells after sensitization with BLG plus cholera toxin. The synbiotic abrogated this alarmin response, potentially via increased mucus production. Synbiotic treatment also reduced the proportion and number of T follicular (Tf) helper cells in the ileal-draining lymph node (LN) and increased Tf-regulatory cells in the colon-draining LN, suggesting a role in T/B cell regulation. Ongoing experiments are examining how the synbiotic affects localization and expansion of IgE producing cells in mucosal tissues including Peyer’s patches and mesenteric LNs. These findings will help to uncover the mechanisms by which specific, protective bacteria prevent food allergy and inform the development of effective live biotherapeutics.

Microanatomical lipid niche controls intestinal memory B cell migration and effector function

Abstract Enteric infections are a leading global burden, yet few oral vaccines elicit long-lasting immunity and prevent disease, likely due to our lack of understanding of intestinal immunological memory. Long-lived memory B cells can be rapidly reactivated to differentiate into plasma cells, thus further increasing antibody titers and mediating host protection. Such long-term protection requires memory B cells to migrate and persist in sub-anatomical niches within secondary lymphoid organs. While survival niches have been described for memory B cells in lymph nodes and spleen, no niche have been identified in mucosal lymphoid organs, such as Peyer’s patches (PPs). Here, we show that memory B cells require positioning in the PP subepithelial dome (SED) for maintenance and effector function. In PPs, memory B cells express the migratory receptors EBI2, which binds the oxysterol 7alpha, 25-hydroxycholesterol, and CCR6, which recognizes the chemokine CCL20. Both ligands are present in PPs and drive memory B cell migration in vitro. Conditional removal of EBI2 from memory B cells, either in a constitutive or acute fashion, alters their zonation in PPs, reduces their persistency, and impairs their antigen-specific response. While most memory B cells in PPs are IgA+, IgA B cell receptor expression is dispensable for memory B cell positioning in the SED. Using conditional mice for the enzymes generating the oxysterol gradient and cell specific Cre lines, we are currently mapping the cellular and metabolic requirements for EBI2 dependent memory B cell maintenance and effector function in PPs. Understanding the mechanisms for memory B cell persistence is fundamental to our understanding of the intestinal B cell memory response against pathogens. Previously supported by training grants from NIH (5 T32 AI 132152-2, 5 T32 AI 007349-29)

Macrophages and neutrophils in lymph node granulomas from Mycobacterium tuberculosis-infected macaques have immunoregulatory phenotypes

Abstract Tuberculosis (TB) is caused by infection with the Mycobacterium tuberculosis (Mtb). Lung granulomas are the defining pathologic feature of TB but Mtb can infect other organs, including lymph nodes, and causes granulomas to form in these sites. Lymph node granulomas have received less attention than lung granulomas but granulomas in these organs are sites of bacterial persistence and have implications for TB progression. Macrophage subsets and neutrophils in lymph node granulomas have not been extensively studied but the biology of these cells may yield clues that underlie the pathobiology of lymph node infection. Our objective was to describe expression of functional markers and cytokine expression by macrophage subsets and neutrophils in lymph node granulomas and to compare these myeloid cells in lymph node and lung granulomas. We used thoracic lymph node and lung granulomas from cynomolgus macaques, a nonhuman primate that experiences human-like TB pathology and course of disease, to do this work. We found that lymph node epithelioid macrophages were distinct from lymph node dendritic cells but resembled epithelioid macrophages in lung granulomas suggesting these cells share a common progenitor or exposure to common stimuli leads to a phenotypic convergence in different tissues. Macrophages expressed pro- and anti-inflammatory cytokines including type 1 interferon, a cytokine family associated with exacerbated TB. Neutrophils were present in low numbers in non-granulomatous lymph node regions but were selectively recruited to granulomas. Our data suggest that macrophages and neutrophils in lymph node granulomas have immunoregulatory properties that may contribute to local and systemic outcomes in Mtb infection. This work is supported by NIH grants R21 AI167710, R01AI164970, and the Wellcome Trust DELTA tissue program.

Clonal dynamics of SARS-CoV-2 specific CD4 T cells in lymph nodes after vaccination

Abstract The ongoing COVID-19 pandemic highlight the importance of vaccination. Past studies have surveyed SARS-CoV-2 specific B cell response in the lymph node (LN) of vaccinated patient, but the quality, the dynamics, and the specificity of the T helper response following mRNA vaccination remains incompletely understood. We address this question in healthy individuals receiving mRNA vaccine (BNT162b or mRNA-1273). We collected axillary draining LN using fine needle aspiration following vaccination and analyzed CD4+ T cells by single cell transcriptome, proteome analysis and TCR sequencing. We observed an accumulation T cell having characteristic signature of activated T follicular helper cell (Tfh), through the course of the 6 months, as well as an increase expansion of Tfh clones. Using TCR sequences to track progenies of same T cell over time, we observed that some Tfh cells in the 6 months’ time point were already detectable after the first vaccine dose. We then generated a library of SARS-CoV-2 specific TCR sequences by performing TCR sequencing on cells that proliferated in response to a spike peptide pool. The majority of these TCR sequences mapped to cells with a strong Tfh signature on the transcriptomic level and accumulated after 6 months. In conclusion, we observed an expansion of SARS-CoV-2 specific Tfh clones after the first dose of vaccine, some of which persisted after 6 months. We hypothesize that, in a protective SARS-CoV-2 response, short-lived clones are progressively replaced by long-lasting SARS-CoV-2 specific Tfh clones. We plan to test this hypothesis by analyzing clonal dynamics at other time points and by using aggregate TCR sequences from the publicly available sequences from individuals with mild or severe COVID-19. NIH R01AO66358

Intracellular pH controls death and proliferation of lymphocytes in immune response and tumor cells

Abstract Cancer and highly proliferating normal cells have high demand for energy but paradoxically switch energy production from the highly efficient mitochondrial respiration to the low efficient cytosolic glycolysis. This phenomenon is known as the Warburg effect, but the reason for which is not fully understood. This study found that low intracellular pH (pHi) induced apoptosis, whereas high pHi enhanced proliferation, of normal lymphocytes. In tumor cells, low pHi also induced apoptosis whereas high pHi maintained high rates of proliferation. In the draining mediastinal lymph nodes (MLNs) of mice sensitized and challenged with ovalbumin, lymphocytes had dramatically higher pHi and became more heterogeneous than lymphocytes in the non-draining lymph nodes. The pHi of the lymphocytes was inversely related to mitochondrial energetic activities, and the rise of the lymphocyte pHi in the MLNs was accompanied by the reduction of mitochondrial energetic activities. Furthermore, high lymphocyte proliferation in the MLNs powered by strong mitochondrial energy led to low pHi thereby predestined the lymphocytes to death by apoptosis. These findings support a proposed explanation for the Warburg effect as a substitution for mitochondrial respiration that allows the highly proliferating cells to avoid low pHi-induced apoptosis. None

Chemokine CCL19 promotes type 2 helper T cell differentiation and allergic airway inflammation

Abstract Allergic asthma is largely driven by the actions of allergen-specific CD4 +type 2 T helper (Th2) cells. Following allergen inhalation, these cells are induced by the interaction of naïve CD4 +T cells with antigen-bearing dendritic cells (DCs) in lung-draining lymph nodes (LNs). The molecular mechanisms underlying the induction of T helper cells have been well studied and include the migration of allergen-bearing DCs from the lung to regional LNs in a manner dependent on the chemokine receptor, CCR7. Previous studies have demonstrated that CCL21, a CCR7 ligand, is required for the accumulation of naïve T cells and activated DCs in the LNs, but the function of another ligand, CCL19, has been unknown. In the present study, we investigated the role of CCL19 in allergic airway inflammation using CCL19-deficient mice. In a mouse model of asthma, eosinophils and lymphocytes were significantly reduced in airways of CCL19-deficient mice compared with those of wildtype (WT) animals, and production of IL-4 and IL-13 in the lung was significantly diminished, suggesting that CCL19 is required for type 2 immune responses. Co-cultures of naïve WT CD4 +T cells with CCL19-deficient DCs produced less type 2 cytokines than did T cells co-cultured with WT DCs. Further, fibroblastic reticular cells (FRCs) from CCL19-deficient mouse LNs produced less Th2 cytokine production compared with WT FRCs. Addition of recombinant CCL19 to the culture of naïve CD4 +T cells increased phosphorylation of STAT5 and gene expression of Il2, Il2ra, Il2rb, Il2rg, Sta5a, Stat5b and Stat6. Together, our findings suggest that CCL19 produced by DCs and FRCs promotes Th2 differentiation through the activation of STAT5-signaling, and that this promotes allergic airway inflammation. This work was supported by Intramural Research Program of NIEHS, NIH (ZIA ES102025-09).

TLR-mediated immune activation is a strong suppressor of the Treg-attracting chemokine CCL22

Abstract The immunoregulatory chemokine CCL22 is mainly secreted by dendritic cells (DC) in lymph nodes and plays a crucial role in maintaining the balance between immune tolerance and efficient immune response by induction of intercellular contacts between DC and regulatory T cells (Treg) expressing the only so far known CCL22 receptor CCR4. In the absence of CCL22, the immune response to vaccination and cancer has been proven to be enhanced. In this work, we aimed to understand how activation of Toll-like receptors (TLR) modulates CCL22 levels mechanistically in vitro and in vivo. We observed that a bacterial infection with salmonella typhimurium and treatment with different TLR agonists such as Poly (I:C), LPS, R848 and CpG led to a potent and long-lasting suppression of systemic CCL22 in vivo. Furthermore, using in vitro co-cultures of DC, B cells and T cells, we identified TLR-mediated CCL22 suppression to be mediated by soluble factors derived from non-DC. Additionally, we pursued to investigate the underlying mechanism and could show in vitro that CCL22 suppression is particularly driven by redundant action of IL-10, IFN-α and IFN-γ. Moreover, inhibition of NF-κB reverted the CCL22-suppressing effect of CpG on splenocytes. In CpG-treated mice, Treg/DC contacts were diminished to the level known from Ccl22 −/−mice, an observation which links TLR-mediated CCL22 suppression to an immunological relevance. Taken together, we demonstrate that TLR signaling results in CCL22 downregulation via different cytokines, a mechanism which is potentially important to enable the induction of a strong immune response and efficient pathogen clearance in the setting of microbial infection.

Signs of Dendritic Cell Activation in the Lungs of Mice Exposed to Aerosols of Flavored Vaping Liquid

Abstract Rationale: Vaping products contain numerous flavor compounds that are known immunological sensitizers. Sensitization to various antigens can be responsible for the onset of serious respiratory diseases, including hypersensivity pneumonitis. However, respiratory tract sensitization and pathological consequences from exposure to aerosols of flavored vaping liquids need to be thoroughly investigated. Methods: Female BALB/c mice were exposed to laboratory made nicotine-free vaping liquid containing a mixture of 1% citral (lemon), 1% cinnamaldehyde (cinnamon), 1% dihydrocoumarin (coconut) and 1% vanillin (vanilla) solubilized in propylene glycol and glycerol (50:50). Mice were exposed 2h/day for 4 days to vaping or room air. Bronchoalveolar lavage was performed to assess total and differential cell counts, and flow cytometry was performed on pulmonary tissue and lung-draining lymph nodes to assess immune cell populations. Results: After 4 days of daily exposure, we found a significant increase in bronchoalveolar lavage immune cells in mice exposed to aerosols from the flavored vaping liquid. We also observed a significant increase in pulmonary conventional dendritic cells (cDCs) as well as their expression of the activation markers CD40, CD86 and MHC-II. This activation is mainly observed on type 2 cDCs. In the lung-draining lymph nodes, flavored vaping liquid lead to a significant increase in total immune cells (CD45+ cells). Conclusions: Pulmonary conventional dendritic cells activation and the increase in total immune cells in lung-draining lymph nodes caused by exposure to aerosols from flavored vaping liquid suggest the early initiation of immunological sensitization processes in the lungs. Fonds de recherche du Québec – Santé, Ministère de la Santé et des Services sociaux, Fondation de l’Institut de cardiologie et de pneumologie de Québec, Réseau de recherche en santé respiratoire du Québec.

Loss of testosterone leads to impaired T cell function via glucocorticoids in mouse glioblastoma models

Abstract While sex differences in glioblastoma (GBM) are well established, the contribution of sex hormones on these differences has not been fully explored. Recently, the immunoregulatory effect of testosterone in other type of cancers has been reported. To evaluate the effect of testosterone on GBM, we performed castration or sham surgery on mice, followed by intracranial implantation of syngeneic glioma cells. Surprisingly, the castrated B6 mice had more aggressive tumors compared to sham surgery controls and the castration induced aggressive tumor growth could be rescued by exogenous testosterone administration. This was further confirmed by chemical blockade of androgen receptor signaling using enzalutamide. In contrast, castration in Rag1KO mouse models did not show a survival difference compared to controls, suggesting a role for lymphocytes. Interestingly, upregulated levels of circulating corticosterone in castrated mice was detected by mass spectrometry, and glucocorticoid receptor blocker reversed the survival of castrated mice, suggesting glucocorticoid-mediated systemic immunosuppression upon castration. Flow analysis revealed decreased cytokine production in CD4+ and CD8+ T cells from tumors and lymph nodes in castrated mice. We also found that tumor growth was delayed in castrated mice when tumor cells were subcutaneously implanted in the flank while decreased cytokine production in T cells in tumor and lymph nodes from flank tumor model as well. These data suggest brain-specific effect of steroid hormone on tumor control. Taken together, our findings demonstrate that testosterone regulates T cell function via glucocorticoids, and ultimately affects GBM tumor growth in a site-specific manner. Supported by grants from NIH (R35 NS127083, P01 CA245705).

Examining methotrexate’s safety and efficacy in combination with immune checkpoint inhibitors to control immune related inflammatory arthritis in cancer

Abstract Immune checkpoint inhibitors (ICIs) are associated with high rates of toxic side effects known as immune related adverse events such as inflammatory arthritis (ir-RA). ir-RA is empirically treated with drugs for rheumatoid arthritis such as methotrexate (MTX) and hydroxychloroquine which was recently shown to reduce the anti-tumor benefit of ICIs in mouse models of cancer. We have set out to determine the safety of MTX use in combination with ICIs, its effect on the anti-tumoral immune response and its efficacy in treating ir-RA in a novel arthritis-B16 PD-L1 tumor mouse model. We observed that MTX did not diminish the anti-tumor benefit of ICIs in our melanoma model and significantly altered the intra-tumoral CD8 +T cell population by promoting the formation of central memory cells and diminishing PD1 expression. We have identified that MTX acts on neuropilin-1 (NRP1), a receptor identified as a CD8 +T cell exhaustion marker promoting terminal exhaustion and limiting memory cell formation in tumors. We have shown that MTX treatment in vitro reduces the expression of NRP1 on stimulated CD8 +T cells. We have also advanced in the development of a novel arthritis-tumor model showing that MTX can control and diminish inflammation in the delayed type hypersensitivity arthritis (DTHA) and reduced the frequency of Th1 and Th17 cells in the popliteal lymph nodes of mice, with Th17 cells known to have a crucial role in ir-RA. Together these results show that MTX is safe for use in combination with ICIs in our tumor model and is effective in our DTHA model that mimics the immune profile of ir-RA. With the development of our combined arthritis-tumor model we will be able to assess MTX’s efficacy in controlling arthritis in ICIs treated tumor bearing mice.

Regulatory maturation of lung cDC1 is required for resolution of Influenza infection

Abstract Pulmonary infection with Influenza A virus (IAV) is responsible for 20,000–30,000 deaths in the US yearly. During the acute stage of infection, pattern recognition receptors (PRRs)-dependent recognition of the virus leads to DC maturation and subsequent expansion of virus-specific effector T cells in the lung-draining lymph node. Recruitment of cytotoxic T cells into the lungs is crucial for the control of IAV infection, however unchecked effector T cell activation often leads to lung immunopathology even after the virus has been cleared. Reducing the availability of IL-2 by CD25+ regulatory T cells is thought to be the principal mechanism limiting the expansion and differentiation of effector CD8 T cells. Recently, efferocytotic maturation of cDC1 (mregDC) has been implicated in impairing CD8 T cells responses, however mregDC has not been studied in the context of IAV infection. Here we described expression of maturation markers, CCR7, PD-L1, and CD40 of lung cDC subsets. PD-L1+ CCR7+ CD40+ cDC1 differentiates in the lungs several days after the virus has been cleared (12–16 days post-infection, dpi). The accumulation of mature cDC1 coincides with increased frequency of Caspase 3+ cells and recovery of disease. At 8dpi, mature cDC1 produce IL-12p70 and induce IFNg+ CD8 T cells, in contrast, at 16dpi mature cDC1 only produce IL-12p40 and fail to induce IFNg+ CD8 T cells. In conclusion, our data suggest that mature DC1 play a role in limiting CD8 T cells mediated immunopathology after IAV infection.

CD4 T cells from mice with alopecia areata express an effector like phenotype and can transfer disease

Abstract Alopecia areata (AA) is an autoimmune disease with a 2% lifetime incidence. AA presents as hair loss and is characterized histologically by the presence of CD4 and CD8 T cells surrounding the hair bulb. Although CD4 T cells comprise a majority of the cellular infiltrate, little is known about the contribution of this population to disease pathogenesis. An emerging animal model of AA involves adoptive transfer of in vitro activated and expanded bulk skin draining lymph node (SDLN) cells from AA mice, resulting in the induction of disease in recipient mice. The objective of this study was to investigate the mechanisms by which those cells transfer disease. Expanded bulk SDLN cells overwhelmingly consisted of CD3+ T cells, including both CD8 and CD4 T cells. To address whether these T cell populations could separately induce AA, we first sorted CD8 T cells from the SDLNs of AA mice and found these cells potently induced disease in recipient mice. Surprisingly, isolated CD4 T cells from AA mice also had a robust capacity to induce disease, and they did so more efficiently than CD4 T cells from unaffected (UA) mice. High dimensional flow cytometric analysis of the SDLNs indicated that an enhanced number of CD4 T cells in AA mice exhibited an effector-like phenotype. Development of AA following transfer of expanded CD4 T cells relied on the presence of endogenous CD8 T cells and host responsiveness to IFN-γ, but, in contrast, loss of IL-17 signaling appeared to have no effect. Together, these data suggest that activated CD4 T-helper type 1 effector cells contribute to the activation of CD8 T cells and the subsequent attack of the hair follicle. Further studies are needed to further define the specific mechanisms by which CD4 T cells contribute to the development of AA. Dept of VA (I01BX004907), NIH/NIAMS (R01AR077194, K08AR069111, T32AI007485)

CE-452779-1 IVABRADINE FOR CONTROLLING HEART RATE IN PERMANENT ATRIAL FIBRILLATION: RESULTS OF THE TRANSLATIONAL CLINICAL TRIAL BRAKE-AF

Ivabradine has been postulated to reduce the ventricular rate in atrial fibrillation. However, the mechanisms responsible for that reduction and its performance as rate-control drug are not known To evaluate the mechanism of inhibition of atrioventricular conduction produced by ivabradine and to determine its efficacy and safety compared with digoxin in permanent atrial fibrillation The effects of ivabradine in atrioventricular nodal and ventricular cells were studied by in-vitro whole-cell patch-clamp experiments . Changes in If, INa, ICaL, ICaT, IKs, IKr, and IK1 conductances were integrated in mathematical simulations of nodal and ventricular action potentials. In parallel, a multicenter, randomized, open-label, phase III clinical trial compared ivabradine (5mg BID increased to 7.5mg BID after one month) versus digoxin (0.10-0.25mg QD adjusted by serum levels) for uncontrolled permanent atrial fibrillation despite beta-blockers or calcium channel blockers. Primary efficacy and safety endpoints were the reduction of the mean daytime heart rate at 3 months and a combination of syncope, clinically significant bradycardia or serious drug-related adverse events, respectively. The primary efficacy endpoint was evaluated by intention to treat using the O'Brien–Fleming method. Safety endpoints were evaluated with χ2 or Fisher’s exact tests Ivabradine 1 μM inhibited If and IKr by 28.9% and 22.8% (P<0.05) respectively. INa and ICaL were also reduced but only at 10 μM. Ivabradine slowed the firing frequency of a modelled human AV nodal action potential by 10.6% and induced a minimal prolongation of the ventricular action potential. The clinical trial randomized 35 patients to ivabradine and 33 to digoxin. Mean daytime heart rate decreased by 11.6 bpm (-11.5%) in the ivabradine arm (p=0.02) Vs 19.6 (-20.6%) in the digoxin arm (p<0.001) however, the non-inferiority margin of efficacy was not met (Z:-1.95; p=0.97). Primary safety endpoint occurred in 3 (8.6%) patients on ivabradine and in 8 (24.2%) on digoxin (p=0.10). Therapeutic adherence was lower in the digoxin arm (78.8% vs. 97.1%; p=0.02) Ivabradine produced a discrete heart rate reduction in patients with permanent atrial fibrillation. Compared with digoxin, ivabradine was less effective, better tolerated and had a similar rate of serious adverse events.The inhibition of If in the AV node seems the main mechanism responsible for the ivabradine-induced heart rate reduction in atrial fibrillation.