Project description:NOD mice (8- to 13-wk-old) were bred and maintained at the La Trobe University Central Animal House (Bundoora, Melbourne, Australia). All experiments were conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes (National Health and Medical Research Council 1997), after approval by the La Trobe University Animal Ethics committee (Melbourne, Australia). A total of 124 female mice were divided into three groups. The immunized group comprised 91 mice injected s.c. into the lower flanks with 200 micrograms of MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep) emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis (Difco). An i.v. injection of 350 ng of Bordetella pertussis toxin was administered both immediately thereafter and 48 h later. The 26 mice in the control group were injected with adjuvant and pertussis toxin only. The seven animals used for the baseline group (time, day 0) were naive, not injected mice. Except for the seven naive animals from the baseline group, nine animals (seven from the EAE and two from the control group) were sacrificed (at the same time of the day) at each of 13 time points after immunization, which are detailed as days 3, 5, 7, 9, 11, 12, 13, 14, 15, 16, 17, 18, and 19 postimmunization. Lymph nodes from the remaining four animals of the immunized and from the two control groups were dissected, immersed in RNAlater (Ambion), and frozen at 20C. Lymph nodes were removed from RNAlater and homogenized in TRIzol (Invitrogen Life Technologies) using an electric homogenizer. After resuspending the final RNA pellet in water, samples were repurified using the RNeasy kit (Qiagen). cDNA was synthesized from 15 micrograms of total RNA using Superscript II RT (Invitrogen Life Technologies) and a modified dNTP mix containing dUTP. Samples were hydrolyzed by adding 10 microliters of 0.1 N NaOH, neutralized with 25 microliters of 1 M HEPES, and precipitated with 3 M sodium acetate and ethanol. Resuspension in 0.05 M sodium bicarbonate was followed by 1 h incubation with either N-hydroxysuccinimide ester Cy3 or Cy5 fluorescent dyes (Amersham Biosciences). Probes were quenched by the addition of 4 M hydroxylamine and neutralized with 100 mM sodium acetate. Final probe cleanup was conducted using the QIA Quick PCR purification kit (Qiagen). We followed a common reference design in which each Cy3-labeled lymph node probe was combined with a Cy5-labeled probe derived from a pool of RNA. Hybridization onto glass slides containing 18,240 spotted 60- to 70-mer oligonucleotides, followed by washing and scanning was performed at the Gladstone microarray core facility at the University of California (San Francisco, CA) Keywords: Time series

Project description:NOD mice (8- to 13-wk-old) were bred and maintained at the La Trobe University Central Animal House (Bundoora, Melbourne, Australia). All experiments were conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes (National Health and Medical Research Council 1997), after approval by the La Trobe University Animal Ethics committee (Melbourne, Australia). A total of 124 female mice were divided into three groups. The immunized group comprised 91 mice injected s.c. into the lower flanks with 200 micrograms of MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep) emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis (Difco). An i.v. injection of 350 ng of Bordetella pertussis toxin was administered both immediately thereafter and 48 h later. The 26 mice in the control group were injected with adjuvant and pertussis toxin only. The seven animals used for the baseline group (time, day 0) were naive, not injected mice. Except for the seven naive animals from the baseline group, nine animals (seven from the EAE and two from the control group) were sacrificed (at the same time of the day) at each of 13 time points after immunization, which are detailed as days 3, 5, 7, 9, 11, 12, 13, 14, 15, 16, 17, 18, and 19 postimmunization. Lymph nodes from the remaining four animals of the immunized and from the two control groups were dissected, immersed in RNAlater (Ambion), and frozen at 20C. Lymph nodes were removed from RNAlater and homogenized in TRIzol (Invitrogen Life Technologies) using an electric homogenizer. After resuspending the final RNA pellet in water, samples were repurified using the RNeasy kit (Qiagen). cDNA was synthesized from 15 micrograms of total RNA using Superscript II RT (Invitrogen Life Technologies) and a modified dNTP mix containing dUTP. Samples were hydrolyzed by adding 10 microliters of 0.1 N NaOH, neutralized with 25 microliters of 1 M HEPES, and precipitated with 3 M sodium acetate and ethanol. Resuspension in 0.05 M sodium bicarbonate was followed by 1 h incubation with either N-hydroxysuccinimide ester Cy3 or Cy5 fluorescent dyes (Amersham Biosciences). Probes were quenched by the addition of 4 M hydroxylamine and neutralized with 100 mM sodium acetate. Final probe cleanup was conducted using the QIA Quick PCR purification kit (Qiagen). We followed a common reference design in which each Cy3-labeled lymph node probe was combined with a Cy5-labeled probe derived from a pool of RNA. Hybridization onto glass slides containing 18,240 spotted 60- to 70-mer oligonucleotides, followed by washing and scanning was performed at the Gladstone microarray core facility at the University of California (San Francisco, CA) Keywords: Time series NOD mice (8- to 13-wk-old) were injected s.c. into the lower flanks with 200 micrograms of MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep) emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis (Difco). An i.v. injection of 350 ng of Bordetella pertussis toxin was administered both immediately thereafter and 48 h later. The mice in the control group were injected with adjuvant and pertussis toxin only. Disease and control mice were sacrificed at 13 identical timepoints and microarrays were processed for each timepoint.

Project description:Tumor-draining lymph nodes (TDLNs) are the first organs where the metastatic spread of different types of cancer, including head and neck cancer (HNC), occurs and have therefore high prognostic relevance. Moreover, first anti-cancer immune responses have been shown to be initiated in such LNs via tumor-educated myeloid cells. Among myeloid cells present in TDLNs, neutrophils represent a valuable population and considerably participate in the activation of effector lymphocytes there. Tumor-supportive or tumor-inhibiting activity of neutrophils strongly depends on the surrounding microenvironment. Thus, type I interferon (IFN) availability has been shown to prime anti-tumor activity of these cells. In accordance, mice deficient in type I IFNs show elevated tumor growth and metastatic spread, accompanied by the pro-tumoral neutrophil bias. To reveal the mechanism responsible for this phenomenon, we have studied here the influence of defective type I IFN signaling on the immunoregulatory activity of neutrophils in TDLNs. Live imaging of such LNs was performed using two-photon microscopy in a transplantable murine HNC model. CatchupIVM-red and Ifnar1-/- (type I IFN receptor- deficient) CatchupIVM-red mice were used to visualize neutrophils and to assess their interaction with T-cells in vivo. We have evaluated spatiotemporal patterns of neutrophil/T-cell interactions in LNs in the context of type I interferon receptor (IFNAR1) availability in tumor-free and tumor-bearing animals. Moreover, phenotypic and functional analyses were performed to further characterize the mechanisms regulating neutrophil immunoregulatory capacity. We demonstrated that inactive IFNAR1 leads to elevated accumulation of neutrophils in TDLNs. However, these neutrophils show significantly impaired capacity to interact with and to stimulate T-cells. As a result, a significant reduction of contacts between neutrophils and T lymphocytes is observed, with further impairment of T-cell proliferation and activation. This possibly contributes to the enhanced tumor growth in Ifnar1-/- mice. In agreement with this, IFNAR1-independent activation of downstream IFN signaling using IFN-λ improved the immunostimulatory capacity of neutrophils in TDLNs and contributed to the suppression of tumor growth. Our results suggest that functional type I IFN signaling is essential for neutrophil immunostimulatory capacity and that stimulation of this signaling may provide a therapeutic opportunity in head and neck cancer patients.

Dataset Information

Adjuvant immunotherapy of experimental autoimmune encephalomyelitis: immature myeloid cells expressing CXCL10 and CXCL16 attract CXCR3+CXCR6+ and myelin-specific T cells to the draining lymph nodes rather than the central nervous system.

Publications

Adjuvant immunotherapy of experimental autoimmune encephalomyelitis: immature myeloid cells expressing CXCL10 and CXCL16 attract CXCR3+CXCR6+ and myelin-specific T cells to the draining lymph nodes rather than the central nervous system.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets