Intracellular cytokine optimization and standard operating procedure

Lamoreaux, Laurie; Koup, Richard A.

doi:10.1038/nprot.2006.268

Cited by 240 publications

(233 citation statements)

References 22 publications

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“…Frozen PBMC were thawed the day before experiments and left in complete media overnight at 371C to recover [56]. Intracellular staining for cytokines and surface expression of CD107a was performed as described elsewhere [56].…”

Section: Functional Assaysmentioning

confidence: 99%

“…Intracellular staining for cytokines and surface expression of CD107a was performed as described elsewhere [56]. In brief, P815 cells, a mouse mastocytoma cell line obtained from the American Type Culture Collection, Manassas, VA, USA, were pre-coated with combinations of antibodies to NKR, 5 mg/mL anti-2B4 (clone C1.7, Beckman Coulter), 5 mg/mL anti-DNAM-1 (DX11, Becton Dickinson), 5 mg/mL anti-NKG2D (1D11, Becton Dickinson), 0.1 or 5 mg/mL anti-CD3 (OKT-3), or 0.1 or 5 mg/mL mouse IgG (MOPC-21, BioLegend, San Diego, CA, USA).…”

Section: Functional Assaysmentioning

confidence: 99%

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Activating NK‐cell receptors co‐stimulate CD4⁺CD28⁻ T cells in patients with rheumatoid arthritis

et al. 2010

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Effector T‐cell responses can be modulated by competing positive or negative signals transduced by NK‐cell receptors (NKR). In the CD4+ T‐cell population, the expression of NKR is primarily found in the CD4+CD28− T‐cell subset, also known as CD28null T cells. These T cells are frequently found in rheumatoid arthritis (RA) and other inflammatory disorders, suggesting that signaling through NKR may play a role in the autoimmune reaction. Here we aimed to dissect the phenotype and function of NKR‐expressing CD4+CD28− T cells in patients with RA. By analyzing a broad array of NKR on CD4+CD28− T cells we found a significant expression of the co‐activating receptors 2B4 (CD244), DNAM‐1 (CD226), and CRACC. Pair‐wise ligations of 2B4 with DNAM‐1 and/or NKG2D lead to increased effector functions of primary CD4+CD28− T cells to suboptimal levels of anti‐CD3 stimulation. Using multi‐parameter flow cytometry, we demonstrate that such co‐ligation led to an increased magnitude in overall responsiveness without changing qualitative aspects of the response. Altogether these results demonstrate a pattern of additive effects in NKR‐mediated functional modulation of CD4+CD28− T cells in RA. This may have consequences for the inflammatory responses imposed by these cells, thus influencing disease manifestations.

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Section: Functional Assaysmentioning

confidence: 99%

Section: Functional Assaysmentioning

confidence: 99%

Activating NK‐cell receptors co‐stimulate CD4⁺CD28⁻ T cells in patients with rheumatoid arthritis

et al. 2010

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“…Some groups have suggested resting PBMC overnight after thawing before testing effector functions (Lamoreaux et al, 2006;Kierstead et al, 2007). It is possible that the resting process would remove cells destined for death and allow "stunned" cells to recover functional ability.…”

Section: Responses Not Restored By Overnight Restingmentioning

confidence: 99%

Loss of T cell responses following long-term cryopreservation

Owen

Sinclair

Emu

et al. 2007

Journal of Immunological Methods

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Although cryopreservation of peripheral blood mononuclear cells (PBMC) is a commonly used technique, the degree to which it affects subsequent functional studies has not been well defined.Here we demonstrate that long-term cryopreservation has detrimental effects on T cell IFN-γ responses in human immunodeficiency virus (HIV) infected individuals. Long-term cryopreservation caused marked decreases in CD4 + T cell responses to whole proteins (HIV p55 and cytomegalovirus (CMV) lysate) and HIV peptides, and more limited decreases in CD8 + T cell responses to whole proteins. These losses were more apparent in cells stored for greater than one year compared to less than six months. CD8 + T cell responses to peptides and peptide pools were well preserved. Loss of both CD4 + and CD8 + T cell responses to CMV peptide pools were minimal in HIV-negative individuals. Addition of exogenous antigen presenting cells (APC) did not restore CD4 + T cell responses to peptide stimulation and partially restored T cell IFN-γ responses to p55 protein.Overnight resting of thawed cells did not restore T cell IFN-γ responses to peptide or whole protein stimulation. A selective loss of phenotypically defined effector cells did not explain the decrement of responses, although cryopreservation did increase CD4 + T cell apoptosis, possibly contributing to the loss of responses. These data suggest that the impact of cryopreservation should be carefully considered in future vaccine and pathogenesis studies. In HIV-infected individuals short-term cryopreservation may be acceptable for measuring CD4 + and CD8 + T cell responses. Long-term cryopreservation, however, may lead to the loss of CD4 + T cell responses and mild skewing of T cell phenotypic marker expression.

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“…This issue was addressed by taking aliquots of highly enriched population of monocytes from humans, RM and SM which were fixed, permeabilized and analyzed for intracellular reactivity with mAbs to the discordant Siglec-1, 5.1, 5.2, 7.1, 7.2 and 9 using standardized techniques for intracellular localization of cell surface molecules [27]. These extensive staining studies, unfortunately did not reveal any difference from the results of the cell surface staining studies except for Siglec-7.…”

Section: Differential Expression Of Siglecs By Cell Lineages From Hummentioning

confidence: 99%

Differences in the constitutive and SIV infection induced expression of Siglecs by hematopoietic cells from non-human primates

Jaroenpool

Rogers

Pattanapanyasat

et al. 2007

Cellular Immunology

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The expression of the Siglec family of molecules by hematopoietic cells from uninfected and SIV infected disease susceptible rhesus macaques (RM) and SIV infected disease resistant sooty mangabeys (SM) and for comparison humans was carried out. The predominant cell lineage in all 3 species expressing Siglec's was monocytes. The major finding by both a cross sectional and a prospective SIV infection study showed that whereas monocytes from RM show marked increase in each Siglec constitutively expressed, monocytes from SM showed marked decreases in Siglec-1 expression. While monocytes from all 3 species constitutively expressed Siglec-3, human monocytes in addition expressed Siglec-5 and 9 and to a lower density 7, monocytes from RM expressed Siglec-7 and those from SM expressed Siglec-1. Monocytes from all 3 species however expressed mRNA for Siglec's-1, 5, 7 and 9. The reasons for the failure to detect these molecules at the protein level and the mechanisms for such distinct effects of SIV infection on Siglec expression are discussed.

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Intracellular cytokine optimization and standard operating procedure

Cited by 240 publications

References 22 publications

Activating NK‐cell receptors co‐stimulate CD4⁺CD28⁻ T cells in patients with rheumatoid arthritis

Activating NK‐cell receptors co‐stimulate CD4⁺CD28⁻ T cells in patients with rheumatoid arthritis

Loss of T cell responses following long-term cryopreservation

Differences in the constitutive and SIV infection induced expression of Siglecs by hematopoietic cells from non-human primates

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Intracellular cytokine optimization and standard operating procedure

Cited by 240 publications

References 22 publications

Activating NK‐cell receptors co‐stimulate CD4+CD28− T cells in patients with rheumatoid arthritis

Activating NK‐cell receptors co‐stimulate CD4+CD28− T cells in patients with rheumatoid arthritis

Loss of T cell responses following long-term cryopreservation

Differences in the constitutive and SIV infection induced expression of Siglecs by hematopoietic cells from non-human primates

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Activating NK‐cell receptors co‐stimulate CD4⁺CD28⁻ T cells in patients with rheumatoid arthritis

Activating NK‐cell receptors co‐stimulate CD4⁺CD28⁻ T cells in patients with rheumatoid arthritis