The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cells

Law, Jacqueline; Hirschkorn, Dale F.; Owen, Rachel E.; Biswas, Hope H.; Norris, Philip J.; Lanteri, Marion C.

doi:10.1002/cyto.a.20843

Cited by 26 publications

(42 citation statements)

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“…Tumors were then homogenized and filtered through a nylon filter (70 mm), and the lymphocytes were isolated in RF-10. Tumorinfiltrating lymphocytes (TILs) were studied for (1) IFN-g, tumor necrosis factor (TNF)-a, and interleukin (IL)-4 responses; (2) perforin expression; and (3) naïve memory effector differentiation and FoxP3 staining, 19 as detailed in the supplemental Methods.…”

Section: Tumor-infiltrating Lymphocytesmentioning

confidence: 99%

Cord blood T cells mediate enhanced antitumor effects compared with adult peripheral blood T cells

Hiwarkar

Qasim

Ricciardelli

et al. 2015

Blood

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Key Points• CB T cells mediate enhanced antitumor responses compared with PB T cells in a murine model of B-cell lymphoma.• The antitumor activity correlates with increased tumor-homing of CCR7 high CB CD8 1 T cells and rapid gain of cytotoxic and Th1 function. CD81 T cells and prompt induction of cytotoxic CD8 1 and CD4 1 T-helper (Th1) T cells in the tumor microenvironment. In contrast, in the PB group, this antilymphoma effect is impaired because of delayed tumoral infiltration of PB T cells and a relative bias toward suppressive Th2 and T-regulatory cells. Our data suggest that, despite being naturally programmed toward tolerance, reconstituting T cells after unrelated T-replete CBT may provide superior Tc1-Th1 antitumor effects against high-risk hematologic malignancies. (Blood. 2015;126(26):2882-2891

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Section: Tumor-infiltrating Lymphocytesmentioning

confidence: 99%

Cord blood T cells mediate enhanced antitumor effects compared with adult peripheral blood T cells

Hiwarkar

Qasim

Ricciardelli

et al. 2015

Blood

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“…The best flow cytometry staining panels to identify human Treg cells has been published by Law et al (2009), but no study has determined the best combination of antibody and fixation/permeabilization buffer for the staining of Treg cells in PBMCs and spleen lymphocytes in other mammal species. Therefore, we chose the mouse as a model, and compared two clones of anti-mouse Foxp3 antibodies and two Foxp3 staining buffer sets, which were the most frequently used in published studies in various species (Tsang et al, 2006;Izcue et al, 2008;Bolzer et al, 2009;Prochazkova et al, 2009;Gerner et al, 2010;Janikashvili et al, 2011;Lei et al, 2011;Pinheiro et al, 2011;Robbin et al, 2011;Rocchi et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

“…Several publications noted concerns about the influence of different clones of Foxp3 antibodies coupled to different fluorochromes and paired with different fixation/permeabilization buffers on the staining of human Treg cells (Tran et al, 2007;Pillai and Karandikar, 2008;Grant et al, 2009;Law et al, 2009;Presicce et al, 2010). However, the variables influencing the staining of Treg cells in other mammal species have not been reported.…”

Section: Cd4mentioning

confidence: 99%

“…+ and Foxp3 + T cells CD25 and Foxp3 are thought to be the key markers of mouse CD4 + Treg cells, which are different from the markers of human CD4 + Treg cells (Law et al, 2009). To determine whether there was a correlation between CD25 + T cells and Foxp3 + T cells, we compared their numbers and percentages in CD4 + T cells of murine PBMCs, respectively.…”

Section: Correlation Analysis Between Cd25mentioning

confidence: 99%

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Application and effects of mouse Foxp3 antibody and fixation/permeabilization buffer on the detection of CD4+ regulatory T cells in various mammal species

Sun

Lin²,

Zhang³

et al. 2013

Genet. Mol. Res.

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ABSTRACT. CD4+ regulatory T lymphocytes (Treg cells) play a crucial role in maintaining the normal immune homeostasis. Foxp3, as a key marker for Treg cells, is widely used to identify Treg cells, not only in humans but also in other species, like mouse, porcine, ovine, and bovine. To detect reproducible Treg cells is important for evaluating the state of the immune system, and thus, it is necessary to optimize Foxp3 staining. Here, we present a comparative study of MF23 and FJK-16s clones of anti-mouse Foxp3 antibodies, used in combination with two different fixation/permeabilization buffers. For Foxp3 staining, the fixation/permeabilization buffer and Foxp3 antibody FJK-16s clone from eBioscience were better than those from BD Pharmingen, with the best fluorochrome PE. Moreover, when using the best combination, there was a highly significant positive correlation between CD25 + T cells + T cells. Therefore, the CD25 marker can be used as an alternative to the Foxp3 antibody. As FJK-16s is also applicable for detecting bovine, porcine, canine, ovine, and equine Foxp3 antibodies, these results will be helpful not only in quantifying the frequencies of mouse Treg cells, but also in accurately detecting Treg cells of the other species mentioned above by multicolor flow cytometry.

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“…þ have been reported from 0.44 to 2.8% of total lymphocytes depending on the method of fixation and antibody combination used (Law et al 2009), underscoring the benefit of using a standardized method for Treg detection.…”

Section: Qualification Of Foxp3 Ip Panel For Quantification Of Circulmentioning

confidence: 99%

Determination of Absolute Counts of Circulating Regulatory T Cells in Cynomolgus Macaques Using an Optimized Flow Cytometric Method

2011

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Regulatory T cells (Tregs) are a rare subset of lymphocytes that inhibit the activation and effector functions of T cells and are important regulators of immune responses. Although Tregs are well characterized in humans and rodents, little is known about their immunophenotyping (IP) profile in cynomolgus macaques (Macaca fascicularis), which is an important species for pharmacological and toxicological evaluation of potential immune modulators because of their similar physiologic, genetic, and metabolic response patterns to humans. The authors have developed an immunophenotyping panel using a high-throughput 96-well microtiter plate-based assay to detect circulating Tregs (and have determined the normal range for the number of Tregs in naive healthy cynomolgus macaques to be 56.4 to 179.7 cells/mL (mean + SEM ¼ 113.6 + 5.1 cells/mL; n ¼ 25). Furthermore, the authors compared the resulting FoxP3 þ Treg profiles with a CD127 lo cell-surface panel (CD3) and found a close correlation between the absolute numbers of CD3hi CD127 lo cells (mean + SD ¼ 120 + 8.0 cells/mL). Quantification of circulating Tregs in cynomolgus macaques in this high-throughput assay may help to identify drug candidates that affect this rare, but critical, immunoregulatory cell population.

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The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4⁺CD25⁺Foxp3⁺ regulatory T cells

Cited by 26 publications

References 0 publications

Cord blood T cells mediate enhanced antitumor effects compared with adult peripheral blood T cells

Cord blood T cells mediate enhanced antitumor effects compared with adult peripheral blood T cells

Application and effects of mouse Foxp3 antibody and fixation/permeabilization buffer on the detection of CD4+ regulatory T cells in various mammal species

Determination of Absolute Counts of Circulating Regulatory T Cells in Cynomolgus Macaques Using an Optimized Flow Cytometric Method

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