Analysis of T cells in mouse lymphoid tissue and blood with flow cytometry

Skordos, Ioannis; Demeyer, Annelies; Beyaert, Rudi

doi:10.1016/j.xpro.2021.100351

Cited by 12 publications

(8 citation statements)

References 8 publications

(9 reference statements)

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“…Primary WT lymphocytes were isolated from thymus of 8 weeks of age C57/B6 male mice as described 31 . WT lymphocytes were cultured in RPMI supplemented with 10% FBS, 2 mM L‐glutamine, 0.01 mM 2‐Mercaptoethanol, penicillin/streptomycin and 50 U/mL of Mouse IL‐2.…”

Section: Methodsmentioning

confidence: 99%

Targeting sphingosine kinase 1 in p53KO thymic lymphoma

Velazquez,

Stith,

Zhang

et al. 2023

The FASEB Journal

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Sphingosine kinase 1 (SK1) is a key sphingolipid enzyme that is upregulated in several types of cancer, including lymphoma which is a heterogenous group of malignancies. Treatment for lymphoma has improved significantly by the introduction of new therapies; however, subtypes with tumor protein P53 (p53) mutations or deletion have poor prognosis, making it critical to explore new therapeutic strategies in this context. SK1 has been proposed as a therapeutic target in different types of cancer; however, the effect of targeting SK1 in cancers with p53 deletion has not been evaluated yet. Previous work from our group suggests that loss of SK1 is a key event in mediating the tumor suppressive effect of p53. Employing both genetic and pharmacological approaches to inhibit SK1 function in Trp53KO mice, we show that targeting SK1 decreases tumor growth of established p53KO thymic lymphoma. Inducible deletion of Sphk1 or its pharmacological inhibition drive increased cell death in tumors which is accompanied by selective accumulation of sphingosine levels. These results demonstrate the relevance of SK1 in the growth and maintenance of lymphoma in the absence of p53 function, positioning this enzyme as a potential therapeutic target for the treatment of tumors that lack functional p53.

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Section: Methodsmentioning

confidence: 99%

Targeting sphingosine kinase 1 in p53KO thymic lymphoma

Velazquez,

Stith,

Zhang

et al. 2023

The FASEB Journal

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“…Single‐cell suspensions from the thymus, spleen and cervical lymph nodes (cLN) of 8‐ to 14‐week‐old mice were prepared as described [61].…”

Section: Methodsmentioning

confidence: 99%

“…All incubation steps were done on ice. We investigated CTLA-4 and TNFR-II expression on HOIL-1 KI Tregs by using the same antibodies and protocol described before [61].…”

Section: Flow Cytometrymentioning

confidence: 99%

Normal lymphocyte homeostasis and function in MALT1 protease‐resistant HOIL‐1 knock‐in mice

et al. 2022

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The uniqueness of MALT1 protease activity in controlling several aspects of immunity in humans has made it a very attractive therapeutic target for multiple autoimmune diseases and lymphoid malignancies. Despite several encouraging preclinical studies with MALT1 inhibitors, severe reduction in regulatory T cells and immune-mediated pathology seen in MALT1 protease-dead (MALT1-PD) mice and some, but not all, studies analysing the effect of prolonged pharmacological MALT1 protease inhibition, indicates the need to further unravel the mechanism of MALT1 protease function. Notably, the contribution of individual MALT1 substrates to the immune defects seen in MALT1-PD mice is still unclear. Previous in vitro studies indicated a role for MALT1-mediated cleavage of the E3 ubiquitin ligase HOIL-1 in the modulation of nuclear factor-jB (NF-jB) signalling and inflammatory gene expression in lymphocytes. Here, we addressed the immunological consequences of inhibition of HOIL-1 cleavage by generating and immunophenotyping MALT1 cleavage-resistant HOIL-1 knock-in (KI) mice. HOIL-1 KI mice appear healthy and have no overt phenotype. NF-jB activation in T or B cells, as well as IL-2 production and in vitro T-cell proliferation, is comparable between control and HOIL-1 KI cells. Inhibition of HOIL-1 cleavage in mice has no effect on thymic T-cell development and conventional T-cell homeostasis. Likewise, B-cell development and humoral immune responses are not affected. Together, these data exclude an important role of MALT1-mediated HOIL-1 cleavage in Tand B-cell development and function in mice.

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“…Spleens were removed from each mouse and placed in 5 mL of sterile media (RPMI/HEPES/Pen/Strep/10% FBS). Splenocytes were isolated 33 within 2 hours of collection and used fresh or frozen for later analysis.…”

Section: Methodsmentioning

confidence: 99%

Heteologous saRNA-Prime, DNA Dual-Antigen-Boost SARS-CoV-2 Vaccination Elicits Robust Cellular Immunogenicity and Cross-Variant Neutralizing Antibodies

Rice

Verma

Voigt

et al. 2021

Preprint

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We assessed if immune responses are enhanced in CD-1 mice by heterologous vaccination with two different nucleic acid-based COVID-19 vaccines: a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) and nucleocapsid (N) vaccine (AdS+N) and a self-amplifying and -adjuvanted S RNA vaccine (SASA S) delivered by a nanostructured lipid carrier. The AdS+N vaccine encodes S modified with a fusion motif to increase cell-surface expression. The N antigen is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal compartment to increase the potential for MHC class I and II stimulation. The S sequence in the SASA S vaccine comprises the D614G mutation, two prolines to stabilize S in the prefusion conformation, and 3 glutamines in the furin cleavage region to increase cross-reactivity across variants. CD-1 mice received vaccination by prime > boost homologous and heterologous combinations. Humoral responses to S were the highest with any regimen including the SASA S vaccine, and IgG against wild type S1 and Delta (B.1.617.2) variant S1 was generated at similar levels. An AdS+N boost of an SASA S prime enhanced both CD4+ and CD8+ T-cell responses to both S wild type and S Delta peptides relative to all other vaccine regimens. Sera from mice receiving SASA S homologous or heterologous vaccination were found to be highly neutralizing of all pseudovirus tested: Wuhan, Delta, and Beta strain pseudoviruses. The findings here support the clinical testing of heterologous vaccination by an SASA S > AdS+N regimen to provide increased protection against COVID-19 and SARS-CoV-2 variants.

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Analysis of T cells in mouse lymphoid tissue and blood with flow cytometry

Cited by 12 publications

References 8 publications

Targeting sphingosine kinase 1 in p53KO thymic lymphoma

Targeting sphingosine kinase 1 in p53KO thymic lymphoma

Normal lymphocyte homeostasis and function in MALT1 protease‐resistant HOIL‐1 knock‐in mice

Heteologous saRNA-Prime, DNA Dual-Antigen-Boost SARS-CoV-2 Vaccination Elicits Robust Cellular Immunogenicity and Cross-Variant Neutralizing Antibodies

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