Lymph Nodes Of Mice Research Articles

Early Stress-Response Gene REDD1 Controls Oxazolone-Induced Allergic Contact Dermatitis.

REDD1 is an energy sensor and stress-induced mTOR inhibitor. Recently, its novel role in linking metabolism and inflammation/immune responses has emerged. In this study, we assessed the role of REDD1 in murine oxazolone-induced allergic contact dermatitis (ACD), a T cell-dependent model with features of human ACD. A variety of immune indices, including edema, cellular infiltration, inflammatory gene expression, and glucocorticoid response, were compared in Redd1 knockout (KO) and isogenic (C57BL/6 × 129)F1 wild-type mice after sensitization and subsequent ear challenge with oxazolone. Despite relatively normal thymic profiles and similar T cell populations in the lymph nodes of naive Redd1 KO mice, early T cell expansion and cytokine production were profoundly impaired after sensitization. Surprisingly, higher steady-state populations of CD4+ and CD8+ T cells, as well as macrophages (CD45+/Ly-6G-/CD11b+), dendritic cells (CD45+/Ly-6G-/CD11c+), neutrophils (CD45+/Ly-6G+/CD11b+), and innate lymphoid cells (CD45+/Lineage-/IL-7Ra+/ST2+/c-Kit+), were observed in the ears of naive Redd1 KO mice. Upon challenge, ear edema, T cell, macrophage, neutrophil, and dendritic cell infiltration into the ear was significantly reduced in Redd1 KO animals. Accordingly, we observed significantly lower induction of IFN-γ, IL-4, and other cytokines as well as proinflammatory factors, including TSLP, IL-33, IL-1β, IL-6, and TNF-α, in challenged ears of Redd1 KO mice. The response to glucocorticoid treatment was also diminished. Taken together, these data establish REDD1 as an essential immune modulator that influences both the initiation of ACD disease, by driving naive T cell activation, and the effector phase, by promoting immune cell trafficking in T cell-mediated skin inflammation.

Interferon-λ Improves the Efficacy of Intranasally or Rectally Administered Influenza Subunit Vaccines by a Thymic Stromal Lymphopoietin-Dependent Mechanism.

Previous work showed that interferon-λ (IFN-λ) can trigger the synthesis of thymic stromal lymphopoietin (TSLP) by specialized epithelial cells in the upper airways of mice, thereby improving the performance of intranasally administered influenza vaccines. Here we demonstrate that protein-only influenza vaccines containing either IFN-λ or TSLP boosted antigen-specific IgG1 and IgA responses and enhanced the resistance of mice to influenza virus challenge, irrespective of whether the vaccines were applied via the intranasal or the rectal route. TSLP receptor deficiency negatively influenced vaccine-induced antiviral immunity by impairing the migration of dendritic cells from the airways to the draining lymph nodes of immunized mice, thereby restraining follicular helper T cell and germinal center B cell responses. As previously observed during intranasal vaccination, the adjuvant effect of IFN-λ on a rectally administered influenza vaccine was no longer observed when TSLP receptor-deficient mice were used for immunization, highlighting the central role of the IFN-λ/TSLP axis for vaccine-induced antiviral immunity in the mucosa.

Ex vivo Demonstration of Functional Deficiencies in Popliteal Lymphatic Vessels From TNF-Transgenic Mice With Inflammatory Arthritis.

Background: Recent studies demonstrated lymphangiogenesis and expansion of draining lymph nodes during chronic inflammatory arthritis, and lymphatic dysfunction associated with collapse of draining lymph nodes in rheumatoid arthritis (RA) patients and TNF-transgenic (TNF-Tg) mice experiencing arthritic flare. As the intrinsic differences between lymphatic vessels afferent to healthy, expanding, and collapsed draining lymph nodes are unknown, we characterized the ex vivo behavior of popliteal lymphatic vessels (PLVs) from WT and TNF-Tg mice. We also interrogated the mechanisms of lymphatic dysfunction through inhibition of nitric oxide synthase (NOS).Methods: Popliteal lymph nodes (PLNs) in TNF-Tg mice were phenotyped as Expanding or Collapsed by in vivo ultrasound and age-matched to WT littermate controls. The PLVs were harvested and cannulated for ex vivo functional analysis over a relatively wide range of hydrostatic pressures (0.5–10 cmH2O) to quantify the end diastolic diameter (EDD), tone, amplitude (AMP), ejection fraction (EF), contraction frequency (FREQ), and fractional pump flow (FPF) with or without NOS inhibitors Data were analyzed using repeated measures two-way ANOVA with Bonferroni’s post hoc test.Results: Real time videos of the cannulated PLVs demonstrated the predicted phenotypes of robust vs. weak contractions of the WT vs. TNF-Tg PLV, respectively. Quantitative analyses confirmed that TNF-Tg PLVs had significantly decreased AMP, EF, and FPF vs. WT (p < 0.05). EF and FPF were recovered by NOS inhibition, while the reduction in AMP was NOS independent. No differences in EDD, tone, or FREQ were observed between WT and TNF-Tg PLVs, nor between Expanding vs. Collapsed PLVs.Conclusion: These findings support the concept that chronic inflammatory arthritis leads to NOS dependent and independent draining lymphatic vessel dysfunction that exacerbates disease, and may trigger arthritic flare due to decreased egress of inflammatory cells and soluble factors from affected joints.

Efficacy of lactic acid bacteria supplementation against Fusarium graminearum growth in vitro and inhibition of Zearalenone causing inflammation and oxidative stress in vivo

Fusarium graminearum invasion and Zearalenone (ZEN)-mycotoxin contamination are considered the most global threat to food and feed. This study investigates the effect Lactobacillus plantarum MON03 viable cells (LPVC) and LP free cells supernatant (LPFCS) against Fusarium graminearum growth and ZEN production in vitro and evaluates if treatment with LP viable cells can counteract the negative effect of ZEN on inflammation and oxidative stress in mesenteric lymph nodes and serum biochemical parameters in mice.For the in vitro study, 7 days of LPVC, LPFCS and F. graminearum co-incubation at different concentrations was done in order to determine the antifungal activity and ZEN- production inhibition. Regarding the in vivo study, Balb/c mice were treated as following: Control, ZEN group, LP group and ZEN + LP group for 30 days.In vitro, LPVC showed an excellent antifungal activity after 7 days of co-incubation (103 CFU/ml). LPVC was succeeded also to inhibit ZEN production by the fungi. In vivo, ZEN has shown an important oxidative damage. As a result of the exposure to ZEN, an increase cytokines, as effectors of an inflammatory response, were observed in the mesenteric lymph nodes (MLN) of intoxicated mice. In parallel, a serum biochemical change was also observed. LPVC induced a reduction of ZEN-induced oxidative stress and counteracts also the biochemical parameters damage and the inflammatory markers increased by ZEN.LPVC can be valorized as an anti-cating agent in the vitro and in the gastro-intestinal tract to decrease ZEN-toxic effects.

Orengedokuto exerts anti-allergic effects via inhibition of effector T cell activation in a murine model of contact hypersensitivity.

Orengedokuto (OGT) is a Kampo prescription that has been used for the treatment of inflammation, hypertension, gastrointestinal disorders, and liver and cerebrovascular diseases. It is also used for the treatment of skin diseases such as urticaria and atopic dermatitis. We previously studied its anti-allergic effects of OGT on the murine model of 2,4,6-trinitrochlorobenzene (TNCB)-induced contact hypersensitivity (CHS) and demonstrated that it significantly suppresses ear swelling in a dose-dependent manner. However, the mechanism underlying this activity remained unknown. Here, we sought to identify the mechanism involved. Using a murine model of TNCB-induced CHS, together with adoptive cell transfer experiments, we found that the anti-allergic effects of OGT may be due to the inhibition of effector T cell activation and not the induction and/or activation of regulatory T cells. Flow cytometry analysis revealed that oral administration of OGT suppressed the increase in CD8+CD44highCD62L+ cell number in draining lymph nodes (dLNs) of mice sensitized with 5% TNCB. Additionally, ex vivo experiments confirmed the suppressive effect of OGT on the activation of effector T cells, as interferon-γ (IFN-γ) production by cultured lymphocytes obtained from 5% TNCB-sensitized mice and stimulated with anti-CD3ε and anti-CD28 monoclonal antibodies was reduced by OGT administration. In conclusion, our finding suggests that OGT exerts anti-allergic effects by regulating the activation of effector T cells involved in inflammatory skin diseases such as atopic dermatitis.

Schistosoma japonicum peptide SJMHE1 inhibits acute and chronic colitis induced by dextran sulfate sodium in mice

BackgroundHarnessing helminth-based immunoregulation is a novel therapeutic strategy for many immune dysfunction disorders, including inflammatory bowel diseases (IBDs). We previously identified a small molecule peptide from Schistosoma japonicum and named it SJMHE1. SJMHE1 can suppress delayed-type hypersensitivity, collagen-induced arthritis and asthma in mice. In this study, we assessed the effects of SJMHE1 on dextran sulfate sodium (DSS)-induced acute and chronic colitis.MethodsAcute and chronic colitis were induced in C57BL/6 mice by DSS, following which the mice were injected with an emulsifier SJMHE1 or phosphate-buffered saline. The mice were then examined for body weight loss, disease activity index, colon length, histopathological changes, cytokine expression and helper T (Th) cell subset distribution.ResultsSJMHE1 treatment significantly suppressed DSS-induced acute and chronic colitis, improved disease activity and pathological damage to the colon and modulated the expression of pro-inflammatory and anti-inflammatory cytokines in splenocytes and the colon. In addition, SJMHE1 treatment reduced the percentage of Th1 and Th17 cells and increased the percentage of Th2 and regulatory T (Treg) cells in the splenocytes and mesenteric lymph nodes of mice with acute colitis. Similarly, SJMHE1 treatment upregulated the expression of interleukin-10 (IL-10) mRNA, downregulated the expression of IL-17 mRNA and modulated the Th cell balance in mice with chronic colitis.ConclusionsOur data show that SJMHE1 provided protection against acute and chronic colitis by restoring the immune balance. As a small molecule, SJMHE1 might be a novel agent for the treatment of IBDs without immunogenicity concerns.Graphical abstract

Abstract MP229: Signaling Mechanisms Leading To CD4+ T-lymphocyte Activation During Ischemic Heart Failure

CD4 + T-cells turn pathological during chronic heart failure (HF). Phenotypic changes that mediate this transition are unknown. Thus, at 8 weeks post-infarction we conducted limited cell RNA-sequencing on 150 CD4 + T-cells isolated from rodent failing hearts and using ingenuity pathway analysis (z score&gt;2) compared them either with CD4 + T-cells isolated from the mediastinal lymph nodes (mLNs) of the same mice or cardiac CD4 + T-cells from sham mice. T-cells isolated from the mLNs of HF mice showed enhanced TCR signaling (p&lt;2X10 -4 ) with 3-6 fold increase in several downstream mediators such as ITK, MAPK, LCK and Zap70. We also observed heightened leukocyte extravasation signaling (p&lt;2X10 -5 ) with significant increase in PKCθ, and Rho GTPase activating and Ras association domain proteins. These are consistent with enhanced antigen-presentation, and TCR activation during HF. Moreover, we observed significant upregulation of oxidative phosphorylation (p&lt;3X10 -15 ) in cardiac CD4 + T-cells as compared to mLN T-cells with 2-3 fold increase in the gene expression of ATP synthase subunits and cytochrome C indicative of their increased metabolic activity upon infiltration into the failing hearts. Compared to CD4 + T-cells from sham hearts, significant upregulation of inflammatory genes (p&lt;3X10 -5 ), chemotactic factors (p&lt;8X10 -4 ), and several pro-inflammatory cytokine-mediated pathways such as IL-6 (p&lt;2X10 -4 ), IL-1a (p&lt;2X10 -3 ) and TNFα (p&lt;2X10 -3 ) was observed in cardiac CD4 + T-cells from HF mice. Interestingly, we also observed a significant upregulation of homing (p&lt;3X10 -4 ) and connective tissue remodeling genes (p&lt;1.8X10 -3 ) with &gt;8-fold increase in genes like LCN2, SLPI, IGF1, FGFR1, LTF, and TIMP2 required for transmigration and homing of CD4 + T-cells into the ischemic hearts. Our studies show enhanced antigenic CD4 + T-cell activation in the mLNs supporting enhanced pro-inflammatory signaling, metabolic activation, and extravasation into the ischemic hearts.

Mouse parabiosis model promotes recovery of lymphocytes in irradiated mice

Purpose Total body irradiation (TBI) -induced hematopoietic system injury is mainly due to the failure of self-renewal and to the differentiation ability of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) after radiation exposure. The mouse parabiosis model is mainly used in the field of aging research to explore whether circulating factors in peripheral blood can improve the functions of aged tissues and organs. In this study, we generated a mouse model to verify whether non-irradiated peripheral circulation can improve the circulatory environment in irradiated mice and ameliorate TBI-induced hematopoietic system injury. Materials and methods Six- to eight-week-old male C57BL/6 mice were adjoined by a surgical operation. Four weeks later, one mouse in the pair was exposed to 8 Gy or 6 Gy X-ray, and B and T cells in the peripheral blood, bone marrow, spleen, mesenteric lymph nodes and thymus were then detected by flow cytometry. Hematopoietic stem/progenitor cells in bone marrow cells and their levels of ROS and apoptosis were also detected in this study. Results The results showed decreased percentages of B and T lymphocytes in the peripheral blood, bone marrow (BM), spleen and mesenteric lymph nodes (MLNs) in the isotype irradiated mice. The proportions of CD4-positive, CD8-positive, and CD4 and CD8 double-negative cells were also increased, while the proportion of CD4 and CD8 double-positive cells in the irradiated thymus was decreased. Thus, all of the above lymphocyte injuries in the parabiosis model were improved to nearly the levels of the control. We further detected radiation-induced HSC and HPC injury; however, the reduced HSC and HPC numbers, ROS levels and apoptosis percentages were not ameliorated in the parabiotic irradiated mice. Conclusions Above all, our results showed that non-irradiated peripheral circulation can promote the recovery of TBI-induced lymphocyte injury, further indicating that the recovery of immune cells may play a very important role in the repair of TBI-induced damage.

Influenza virus infection selectively triggers the accumulation and persistence of more potent Helios-expressing Foxp3+ regulatory T cells in the lungs.

Foxp3+ regulatory T cells (Tregs) represent a special lineage of CD4+ T cells. Analysis of Treg response during primary and secondary influenza virus infection clearly demonstrates a robust accumulation of Tregs into the infected lungs and the existence of a population of long-lived antigen-specific memory Tregs in the same tissues after resolution of the infection. However, it remains unknown whether these Tregs co-express Helios, a member of the Ikaros transcription factor family. In this study, Foxp3+ Helios+ and Foxp3+ Helios- Tregs in the lungs, mLNs and spleens of influenza virus-infected and uninfected control mice were tracked. The data show that while there is a co-existence of Foxp3+ Helios+ and Foxp3+ Helios- Tregs in the tissues, the accumulated Tregs in the lungs and lung-draining mediastinal lymph nodes (mLNs) of the infected mice are highly enriched for Foxp3+ Helios+ cells. It was further demonstrated that, after the clearance of primary infection, Foxp3+ Helios+ cells have the ability to persist in the tissues over their Helios- counterparts. More importantly, Foxp3+ Helios+ Tregs accumulated in an accelerated kinetics during recall response to reinfection. In vitro analysis of Treg suppressive function revealed that Foxp3+ Helios+ Tregs are more capable of suppressing influenza virus-specific CD8+ T cell activation, cytokine production and proliferation. Together, the data provide new insights into Treg responses during primary and secondary influenza virus infection and suggest that Foxp3+ Helios+ Tregs predominantly drive the Treg responses.

Discovery, Synthesis, and Evaluation of Highly Selective Vascular Endothelial Growth Factor Receptor 3 (VEGFR3) Inhibitor for the Potential Treatment of Metastatic Triple-Negative Breast Cancer.

We herein report the identification, structural optimization, and structure-activity relationship of thieno[2,3-d]pyrimidine derivatives as a novel kind of selective vascular endothelial growth factor receptor 3 (VEGFR3) inhibitors. N-(4-Chloro-3-(trifluoromethyl)phenyl)-4-(6-(4-(4-methylpiperazin-1-yl)phenyl)thieno[2,3-d]pyrimidin-4-yl)piperazine-1-carboxamide (38k) was the most potent VEGFR3 inhibitor (IC50 = 110.4 nM) among developed compounds. Compared with VEGFR1 and VEGFR2, VEGFR3 was approximately 100 times more selective. Here, compound 38k significantly inhibited proliferation and migration of VEGF-C-induced human dermal lymphatic endothelial cells (HDLEC), MDA-MB-231, and MDA-MB-436 cells by inactivating the VEGFR3 signaling pathway. Additionally, 38k induced cell apoptosis and a prolonged G1/S-phase in MDA-MB-231 and MDA-MB-436 cells. It also presented acceptable pharmacokinetic characteristics in Sprague-Dawley (SD) rats with an oral bioavailability of 30.9%. In the xenograft model in vivo, 38k effectively inhibited breast cancer growth by suppressing the VEGFR3 signaling pathway. 38k pronouncedly resisted the formation of pulmonary metastatic nodules in mice. Collectively, 38k may be a promising therapeutic agent of metastatic breast cancer.

Dendrobine Inhibits γ-Irradiation-Induced Cancer Cell Migration, Invasion and Metastasis in Non-Small Cell Lung Cancer Cells.

The use of ionizing radiation (IR) during radiotherapy can induce malignant effects, such as metastasis, which contribute to poor prognoses in lung cancer patients. Here, we explored the ability of dendrobine, a plant-derived alkaloid from Dendrobium nobile, to improve the efficacy of radiotherapy in non-small cell lung cancer (NSCLC). We employed Western blotting, quantitative real-time (qRT)-PCR, transwell migration assays, and wound-healing assays to determine the effects of dendrobine on the migration and invasion of A549 lung cancer cells in vitro. Dendrobine (5 mm) inhibited γ-irradiation-induced migration and invasion of A549 cells by suppressing sulfatase2 (SULF2) expression, thus inhibiting IR-induced signaling. To investigate the inhibitory effects of dendrobine in vivo, we established a mouse model of IR-induced metastasis by injecting BALB/c nude mice with γ-irradiated A549 cells via the tail vein. As expected, injection with γ-irradiated cells increased the number of pulmonary metastatic nodules in mice (0 Gy/DPBS, 9.8 ± 1.77; 2 Gy/DPBS, 20.87 ± 1.42), which was significantly reduced with dendrobine treatment (2 Gy/Dendrobine, 10.87 ± 0.71), by prevention of IR-induced signaling. Together, these findings demonstrate that dendrobine exerts inhibitory effects against γ-irradiation-induced invasion and metastasis in NSCLC cells in vitro and in vivo at non cytotoxic concentrations. Thus, dendrobine could serve as a therapeutic enhancer to overcome the malignant effects of radiation therapy in patients with NSCLC.

Simultaneous transduction of dendritic cells with A20 and BTLA genes stimulates the development of stable and efficient tolerogenic dendritic cells and induces regulatory T cells

BackgroundThis study investigated the potential of simultaneous overexpression of A20 and B- and T-lymphocyte attenuator (BTLA) genes in dendritic cells (DCs) to develop a tolerogenic phenotype in DCs and investigate their capabilities for induction of immunosuppression. MethodsPlasmid vectors were designed harboring A20, BTLA, and A20 + BTLA genes and were transfected to HEK 293T cells to produce lentiviruses. DCs were transduced by the gene carrying viruses and evaluated for the surface expression of MHCII, CD40, and CD86 molecules by flow-cytometry. The mRNA expression of A20, BTLA, and CCR7 were determined. Mixed-lymphocyte reaction was conducted to evaluate the T cell stimulation potency and ELISA was used to measure the production of IL-10, TGF-β, and TNF-α. The potential of DCs for migration to lymph nodes and Treg induction were assessed by in vivo experiments. ResultsTransduction of DCs resulted in significantly decreased surface expression of CD40 and CD86 co-stimulators and upregulated A20, BTLA, and CCR7 mRNA expression. The IL-10 and TGF-β levels were enhanced significantly in the supernatant of LPS-treated DCs transduced with A20 + BTLA-containing virus group relative to the DCs transduced with pCDH vectors. DCs transduced with A20 + BTLA harboring vectors had higher migratory potential to mouse lymph nodes and caused the development of higher numbers of Treg cells compared with the DCs transduced with pCDH vectors. ConclusionsSimultaneous overexpression of A20 and BTLA genes in DCs caused development of tolerogenic DCs with a promoted potential in induction of Treg cells, accompanied by remarkable stability after inflammatory stimulation. All these offer a promising potential of such DCs in treating autoimmune and inflammatory disorders.

Tbx21 and Foxp3 Are Epigenetically Stabilized in T-Bet+ Tregs That Transiently Accumulate in Influenza A Virus-Infected Lungs.

During influenza A virus (IAV) infections, CD4+ T cell responses within infected lungs mainly involve T helper 1 (Th1) and regulatory T cells (Tregs). Th1-mediated responses favor the co-expression of T-box transcription factor 21 (T-bet) in Foxp3+ Tregs, enabling the efficient Treg control of Th1 responses in infected tissues. So far, the exact accumulation kinetics of T cell subsets in the lungs and lung-draining lymph nodes (dLN) of IAV-infected mice is incompletely understood, and the epigenetic signature of Tregs accumulating in infected lungs has not been investigated. Here, we report that the total T cell and the two-step Treg accumulation in IAV-infected lungs is transient, whereas the change in the ratio of CD4+ to CD8+ T cells is more durable. Within lungs, the frequency of Tregs co-expressing T-bet is steadily, yet transiently, increasing with a peak at Day 7 post-infection. Interestingly, T-bet+ Tregs accumulating in IAV-infected lungs displayed a strongly demethylated Tbx21 locus, similarly as in T-bet+ conventional T cells, and a fully demethylated Treg-specific demethylated region (TSDR) within the Foxp3 locus. In summary, our data suggest that T-bet+ but not T-bet− Tregs are epigenetically stabilized during IAV-induced infection in the lung.

Monitoring Cellular Movement with Photoconvertible Fluorescent Protein and Single-Cell RNA Sequencing Reveals Cutaneous Group 2 Innate Lymphoid Cell Subtypes, Circulating ILC2 and Skin-Resident ILC2

We previously generated a transgenic mouse line expressing skin-specific IL-33 (IL33tg mice) and showed that IL-33 elicits group 2 innate lymphoid cell (ILC2)–dependent atopic dermatitis–like skin inflammation. ILC2s are believed to be tissue-resident cells under steady-state conditions, but the dynamics of ILC2 migration are not fully understood. We sorted ILC2s from the skin and draining lymph nodes of IL33tg mice and analyzed their transcriptomes using the single-cell RNA sequencing technique, which revealed that the skin ILC2s had split into two clusters: circulating ILC2 and skin-resident ILC2. The circulating ILC2s expressed H2-related major histocompatibility complex class II genes. Conversely, the skin-resident ILC2s demonstrated increased mRNA expression of the ICOS, IL-5, and IL-13. Next, we tracked ILC2 migration using IL33tg–Kikume Green-Red mice. Exposing the IL33tg–Kikume Green-Red mice’s inflamed skin to violet light allowed us to label the circulating ILC2s in their skin and track the ILC2 migration from the skin to the draining lymph nodes. Cutaneous local innate responses could transition to systemic type 2 responses by migrating the activated ILC2s from the skin into the draining lymph node. Conversely, the skin-resident ILC2s produced a large number of cytokines. Thus, the skin ILC2s turned out to be a heterogeneous cell population.

Abstract 1916: Lymph node targeting double stranded CpG act effective adjuvant in cancer peptide vaccine

Abstract Background: Adjuvants can enhance immune responses of vaccinated antigen by inducing host innate immune responses. While cancer peptide vaccines were largely studied in recent years, effective cancer peptide vaccines have not been found yet so far because of their poor immunogenicity. Developing an effective vaccine adjuvant suitable for peptide antigen is a promising approach. CpG oligodeoxynucleotides(CpG) are a toll-like receptor 9 agonists and effectively elicit innate immune responses. In order to improve adjuvanticity of CpG, we designed a novel lymph-node targeting CpG, which is composed from a single stranded CpG and a modified complementary strand with amphiphilic chain. Methods: S-540956 is a double stranded oligodeoxynucleotide composed from a single stranded phosphorothioate oligonucleotide and the complementary strand with amphiphilic chain at the 5' end. Accumulating capability to lymph nodes of fluorescent labeled S-540956 was analyzed by flow cytometer, IVIS imaging system, and 2-photon microscopy. Incorporation of S-540956 by antigen-presenting cells in lymph nodes was assessed by Image Stream® imaging cytometer. To test cellular immune responses, three types of MHC class-I epitope peptides were employed as antigenic peptides. Therapeutic effect of S-540956 adjuvanted HPV E7 peptide vaccine was evaluated in TC-1 tumor model. Results:S-540956 exhibited higher accumulating capacity to lymph nodes in mice compared to a typical CpG adjuvant, ODN2006. Systemic proinflammatory cytokine responses were decreased by combining the complementary strand with amphiphilic chain. S-540956 was efficiently incorporated into plasmacytoid DCs and induced the expression of costimulatory molecules. Cellular immune responses induced by S-540956 adjuvanted peptide vaccine were significantly higher than ODN2006 adjuvanted peptide vaccine. And this enhanced adjuvant effect was observed also in protein vaccine. In TC-1 tumor model, therapeutically administered S-540956 adjuvanted HPV-E7 peptide vaccine lead to significant tumor shrinkage compared to ODN2006 adjuvanted peptide vaccine. Conclusion: S-540956 acted as a potent adjuvant for peptide vaccine by inducing cellular immune responses effectively. Accumulating capacity to lymph nodes of S-540956 is a promising adjuvant for not only for peptide but for protein or other biologic antigens. Citation Format: Takayuki Nakagawa, Motoyasu Onishi, Soichi Tofukuji, Shinya Omoto, Kazufumi Katayama, Akira Kugimiya, Morio Nagira, Ken J. Ishii. Lymph node targeting double stranded CpG act effective adjuvant in cancer peptide vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1916.

Metformin generates profound alterations in systemic and tumor immunity with associated antitumor effects

BackgroundMetformin is a commonly used antidiabetic medication which has demonstrated promise as an anticancer agent alone and in combination with conventional treatment regimens. There is increasing evidence that metformin can...

GFAP and desmin expression in lymphatic tissues leads to difficulties in distinguishing between glial and stromal cells

Recently, we found many immune cells including antigen presenting cells neurally hard wired in the T-cell zone of most lymphoid organs like amongst others, lymph nodes in rats, mice and humans. Single immune cells were reached by single neurites and enclosed with a dense neural meshwork. As it is well known that axons are always accompanied by glial cells, we were able to identify Schwann cells in the hilum, medullary and capsule region, like expected. Unexpected was the result, that we found oligodendrocyte-like cells in these regions, myelinating more than one axon. Likewise important was the finding, that one of the standard glial markers used, a polyclonal GFAP antibody equally bound to desmin and therefore marked nearly all stromal cells in cortical, paracortical and medullary cord regions. More detailed analysis showed that these results also appeared in many other non-lymphoid organs. Therefore, polyclonal GFAP antibodies are only conditionally usable for immunohistochemical analysis in peripheral tissues outside the central nervous system. It remains to be elucidated, if the binding of the GFAP antibody to desmin has its reason in a special desmin variant that can give stromal cells glial character.

Lymph-Derived Neutrophils Primarily Locate to the Subcapsular and Medullary Sinuses in Resting and Inflamed Lymph Nodes.

Neutrophils are the first immune cells to be recruited from the blood to the tissue site of an infection or inflammation. It has been suggested that neutrophils are capable of migrating from the infected tissue via lymphatic vessels to the draining lymph nodes. However, it remains elusive as to which areas within the lymph nodes can be reached by such reversely migrating cells. To address this question, we applied a model for adoptive neutrophil transfer into the afferent lymphatic vessel that drains towards the popliteal lymph node in mice. We showed that resting and in vitro-activated neutrophils did not enter the lymph node parenchyma but localized primarily in the subcapsular and medullary sinuses. Within the medulla, neutrophils show random migration and are able to sense laser-induced sterile tissue injury by massively swarming to the damaged tissue site. Co-injected dendritic cells supported the entry of resting neutrophils into the lymph node parenchyma via the subcapsular sinus. In contrast, in vivo-activated adoptively transferred neutrophils were capable of migrating into the interfollicular areas of the lymph node. Collectively, the data presented here give further insights into the functional behavior of neutrophils within the lymph nodes.

Gene Expression Profiling of Lymph Node Sub-Capsular Sinus Macrophages in Cancer.

Lymph nodes are key lymphoid organs collecting lymph fluid and migratory cells from the tissue area they survey. When cancerous cells arise within a tissue, the sentinel lymph node is the first immunological organ to mount an immune response. Sub-capsular sinus macrophages (SSMs) are specialized macrophages residing in the lymph nodes that play important roles as gatekeepers against particulate antigenic material. In the context of cancer, SSMs capture tumor-derived extracellular vesicles (tEVs), a form of particulate antigen released in high amounts by tumor cells. We and others have recently demonstrated that SSMs possess anti-tumor activity because in their absence tumors progress faster. A comprehensive profiling of SSMs represents an important first step to identify the cellular and molecular mechanisms responsible for SSM anti-tumor activity. Unfortunately, the isolation of SSMs for molecular analyses is very challenging. Here, we combined an optimized dissociation protocol, careful marker selection and stringent gating strategies to highly purify SSMs. We provide evidence of decreased T and B cell contamination, which allowed us to reveal the gene expression profile of this elusive macrophage subset. Squamous cell carcinomas induced an increase in the expression of Fc receptors, lysosomal and proteasomal enzymes in SSMs. Imaging of mouse and patient lymph nodes confirmed the presence of the top differentially expressed genes. These results suggest that SSMs respond to tumor formation by upregulating the machinery necessary for presentation of tumor particulate antigens to B cells.

Critical role of the CD44lowCD62Llow CD8+ T cell subset in restoring antitumor immunity in aged mice

CD8+ T cells play a central role in antitumor immune responses that kill cancer cells directly. In aged individuals, CD8+ T cell immunity is strongly suppressed, which is associated with cancer and other age-related diseases. The mechanism underlying this age-related decrease in immune function remains largely unknown. This study investigated the role of T cell function in age-related unresponsiveness to PD-1 blockade cancer therapy. We found inefficient generation of CD44lowCD62Llow CD8+ T cell subset (P4) in draining lymph nodes of tumor-bearing aged mice. In vitro stimulation of naive CD8+ T cells first generated P4 cells, followed by effector/memory T cells. The P4 cells contained a unique set of genes related to enzymes involved in one-carbon (1C) metabolism, which is critical to antigen-specific T cell activation and mitochondrial function. Consistent with this finding, 1C-metabolism-related gene expression and mitochondrial respiration were down-regulated in aged CD8+ T cells compared with young CD8+ T cells. In aged OVA-specific T cell receptor (TCR) transgenic mice, ZAP-70 was not activated, even after inoculation with OVA-expressing tumor cells. The attenuation of TCR signaling appeared to be due to elevated expression of CD45RB phosphatase in aged CD8+ T cells. Surprisingly, strong stimulation by nonself cell injection into aged PD-1-deficient mice restored normal levels of CD45RB and ameliorated the emergence of P4 cells and 1C metabolic enzyme expression in CD8+ T cells, and antitumor activity. These findings indicate that impaired induction of the P4 subset may be responsible for the age-related resistance to PD-1 blockade, which can be rescued by strong TCR stimulation.