T Cell Metabolism Is Dependent on Anatomical Location within the Lung

Roberts, Lydia M.; Evans, Thomas C.; Bosio, Catharine M.

doi:10.4049/immunohorizons.1900063

Cited by 2 publications

(6 citation statements)

References 17 publications

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“…However, in comparison to bead sorted cells, basal ECAR levels were significantly reduced among Tyto sorted CD4 + T cells ( figure 3B). Moreover, the basal ECAR observed among Tyto sorted cells was similar to that observed in immune populations in our previous publication [5]. These data highlight the importance of having a highly purified cellular population for downstream assays and confirm our hypothesis that contaminating cells in our bead purified CD4 + T cell samples contribute significantly to basal ECAR.…”

Section: Tyto Sorted Cells Are Highly Pure and Superior To Bead Purifsupporting

confidence: 89%

“…We previously established that anatomical location within the pulmonary compartment influences a CD4 + effector T cell’s (T eff ) glycolytic capacity [ 5 ]. As an internal control for this study, we included total pulmonary CD4 + T cells isolated from naïve mice in each metabolic flux experiment.…”

Section: Resultsmentioning

confidence: 99%

“…We have implemented bead-based purification techniques for less complicated studies. However, when purifying cells from the pulmonary compartment these techniques result in approximately 85-90% pure cell populations, at best [5]. In some instances, this degree of purity is sufficient but in other experimental conditions, contaminating cells affect downstream analyses.…”

Section: Discussionmentioning

confidence: 99%

“…All T cell activation assays to determine changes in metabolic flux were performed as previously described [ 5 ]. Briefly, 2×10 5 purified T cells were seeded per well in a poly-D-lysine (100 μg/ml) coated Seahorse 96 well plate and centrifuged for 5 minutes at 300xg.…”

Section: Methodsmentioning

confidence: 99%

“…Post-purification flow analysis revealed total naïve CD4 + T cells consistently had 10-15% of contaminating lung cells. Therefore, we attributed the elevated level of basal ECAR in our naïve CD4 + T cell samples to these contaminating cells [5].…”

Section: Tyto Sorted Cells Are Highly Pure and Superior To Bead Purifmentioning

confidence: 99%

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Validation and Application of a Bench Top Cell Sorter in a BSL-3 Containment Setting

Roberts

Anderson

Carmody

et al. 2020

Preprint

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Rigorous assessment of the cellular and molecular changes during infection typically requires isolation of specific immune cell subsets for downstream application. While there are numerous options for enrichment/isolation of cells from tissues, fluorescent activated cell sorting (FACS) is accepted as a method that results in superior purification of a wide variety of cell types. Flow cytometry requires extensive fluidics and aerosol droplets can be generated during collection of target cells. Pathogens such as Francisella tularensis, Mycobacterium tuberculosis, Yersinia pestis, and SARS-CoV-2 require manipulation at biosafety level-3 (BSL-3). Due to the concern of potential aerosolization of these pathogens, use of flow cytometric-based cell sorting in these laboratory settings requires placement of the equipment in dedicated biosafety cabinets within the BSL-3. For many researchers, this is often not possible due to expense, space, or expertise available. Here we describe the safety validation and utility of a completely closed cell sorter that results in gentle, rapid, high purity, and safe sorting of cells on the benchtop at BSL-3. We also provide data demonstrating the need for cell sorting versus bead purification and the applicability of this technology for BSL-3 and potentially BSL-4 related infectious disease projects. Adoption of this technology will significantly expand our ability to uncover important features of the most dangerous infectious diseases leading to faster development of novel vaccines and therapeutics.

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Section: Tyto Sorted Cells Are Highly Pure and Superior To Bead Purifsupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Tyto Sorted Cells Are Highly Pure and Superior To Bead Purifmentioning

confidence: 99%

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Validation and Application of a Bench Top Cell Sorter in a BSL-3 Containment Setting

Roberts

Anderson

Carmody

et al. 2020

Preprint

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Dynamics of pulmonary mucosal cytotoxic CD8 T-cells in people living with HIV under suppressive antiretroviral therapy

Alexandrova,

Yero,

Olivenstein

et al. 2024

Respir Res

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Background Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) suffer from a high burden of pulmonary diseases, even after accounting for their smoking status. Cytotoxic CD8 T-cells are likely implicated in this phenomenon and may act as a double-edged sword. While being essential in viral infection control, their hyperactivation can also contribute to lung mucosal tissue damage. The effects of HIV and smoking on pulmonary mucosal CD8 T-cell dynamics has been a neglected area of research, which we address herein. Methods Bronchoalveolar lavage (BAL) fluid were obtained from ART-treated PLWH (median duration of supressed viral load: 9 years; smokers: n = 14; non-smokers: n = 21) and HIV-uninfected controls (smokers: n = 11; non-smokers: n = 20) without any respiratory symptoms or active infection. Lymphocytes were isolated and CD8 T-cell subsets and homing markers were characterized by multiparametric flow cytometry. Results Both smoking and HIV infection were independently associated with a significant increase in frequencies of total pulmonary mucosal CD8 T-cell. BAL CD8 T-cells were primarily CD69 + expressing CD103 and/or CD49a, at least one of the two granzymes (GzmA/GzmB), and little Perforin. Higher expression levels of CD103, CD69, and GzmB were observed in smokers versus non-smokers. The ex vivo phenotype of GzmA + and GzmB + cells revealed increased expression of CD103 and CXCR6 in smokers, while PLWH displayed elevated levels of CX3CR1 compared to controls. Conclusion Smoking and HIV could promote cytotoxic CD8 T-cell retention in small airways through different mechanisms. Smoking likely increases recruitment and retention of GzmB + CD8 Trm via CXCR6 and CD103. Heightened CX3CR1 expression could be associated with CD8 non-Trm recruitment from the periphery in PLWH.

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T Cell Metabolism Is Dependent on Anatomical Location within the Lung

Cited by 2 publications

References 17 publications

Validation and Application of a Bench Top Cell Sorter in a BSL-3 Containment Setting

Validation and Application of a Bench Top Cell Sorter in a BSL-3 Containment Setting

Dynamics of pulmonary mucosal cytotoxic CD8 T-cells in people living with HIV under suppressive antiretroviral therapy

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