Differential Modulation of Phenotypic Composition of Hiv-Infected and -Uninfected PBMCS During Cryopreservation

Shete, Ashwini; Jayawant, Priyanka; Thakar, Madhuri; Kurle, Swarali; Singh, Dharmesh P.; Paranjape, Ramesh

doi:10.1080/15321819.2012.741087

Cited by 7 publications

(8 citation statements)

References 23 publications

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“…Percentages of total T regs , measured by the phenotype CD4 + CD25 + CD127 low/− , were not affected by cryopreservation of PBMC, which agreed with previous reports (Van Hemelen et al, 2010,Venet et al, 2010,Shete et al, 2013). These percentages were also well correlated with those measured by the phenotype CD4 + CD25 + FoxP3 + , regardless of types of the samples and HIV status.…”

Section: Discussionsupporting

confidence: 93%

“…Several studies have examined percentages of total T regs before and after cryopreservation, with some showing lower percentages after cryopreservation (Elkord, 2009,Sattui et al, 2012) and others finding no difference (Van Hemelen et al, 2010,Venet et al, 2010,Shete et al, 2013). To our knowledge no study has evaluated the effect of cryopreservation on different subsets of T regs .…”

Section: Introductionmentioning

confidence: 99%

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The effect of cellular isolation and cryopreservation on the expression of markers identifying subsets of regulatory T cells

Zhang

Nilles

Johnson

et al. 2016

Journal of Immunological Methods

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Background The role of CD4+ regulatory T cells (Tregs) and their subsets during HIV infection is controversial. Cryopreserved peripheral blood mononuclear cells (PBMC) are an important source for assessing number and function of Tregs. However, it is unknown if PBMC isolation and cryopreservation affect the expression of CD120b and CD39, markers that identify specific subsets of Tregs. Methods HIV-uninfected (HIV−) and -infected (HIV+) men were randomly selected from the Multicenter AIDS Cohort Study (MACS). Percentages of CD120b+ and CD39+ Tregs measured by flow cytometry in whole blood and in corresponding fresh and cryopreserved PBMC were compared. Results Percentages of CD120b+ Tregs were significantly lower in a) fresh PBMC relative to whole blood, and b) freshly thawed frozen PBMC relative to fresh PBMC when the recovery of viable cryopreserved cells was low. When present, low expression of CD120b in frozen PBMC was reversible by 4 hours of in vitro culture. In contrast, expression of CD39 on Tregs was not affected by isolation and/or cryopreservation of PBMC, or by relative recovery of cryopreserved PBMC. These findings were unaffected by the HIV status of the donor. Conclusion The data suggest that percentages of CD120b+ Tregs and CD39+ Tregs can be validly measured in either whole blood or PBMC (fresh and frozen) in HIV− and HIV+ men. However, for measurement of CD120b+ Tregs one type of sample should be used consistently within a given study, and thawed frozen cells may require in vitro culture if recovery of viable cells is low.

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Section: Discussionsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

The effect of cellular isolation and cryopreservation on the expression of markers identifying subsets of regulatory T cells

Zhang

Nilles

Johnson

et al. 2016

Journal of Immunological Methods

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“…Some published studies have reported that cryopreservation and subsequent thawing did not alter the proportion of T cell subsets, but levels of CD62L expression of CD4 + T cells were reduced on thawed cells but restored if these cells were cultured overnight after thawing [19][20][21]. Shete et al reported differential modulation of phenotypic composition of HIV-infected and HIVuninfected peripheral blood mononuclear cells during cryopreservation, and they reported a significant decrease in CD62L-expressing CD8 + cells [22]. These reports indicated the lability of CD62L in response to cryopreservation [22].…”

Section: Discussionmentioning

confidence: 99%

“…Shete et al reported differential modulation of phenotypic composition of HIV-infected and HIVuninfected peripheral blood mononuclear cells during cryopreservation, and they reported a significant decrease in CD62L-expressing CD8 + cells [22]. These reports indicated the lability of CD62L in response to cryopreservation [22]. Additionally, several studies have indicated that in vitro generation and maintenance of T memory-like cells require antigenic restimulation and supplementation of cytokines such as Interleukin (IL)-2, IL-7 and IL-15 during cell culture [23][24][25][26].…”

Section: Discussionmentioning

confidence: 99%

Effects of enzymatic digestion, cell culture and preservation conditions on surface CD62L expression of primary murine CD3+CD4+ T cells

Liu¹,

Zhao²,

Huang³

et al. 2018

biomedicalresearch

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Surface markers are used to identify distinct cell populations. CD62L, a cell adhesion molecule on leukocytes, is used to identify and segregate central memory T (T CM) and effector memory T (T EM) cells. In the present study, we evaluated the effects of experimental conditions on surface CD62L expression of primary murine CD3 + CD4 + T cells. We found that CD62L expression levels were more resistant to collagenase D treatment. Prolonged culture (at 37°C) and preservation (at 4°C) periods resulted in modulation of surface CD62L expression in vitro. These results provide valuable information for the development and optimization of a reliable method for detecting of CD62L expression in vitro.

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“…With this approach the inherent variability associated with study participants would be removed and the study could focus on differences that might be associated with technical aspects of the procedures. Overnight shipping of fresh mucosal specimens can be confounded by weather-associated delays, and there is also concern about the changes in cell phenotype that might be introduced by cryopreservation [ 13 ]. Further work will be needed to evaluate the impact of cryopreservation on the phenotype of cells isolated from various tissue compartments.…”

Section: Discussionmentioning

confidence: 99%

Exploring the Feasibility of Multi-Site Flow Cytometric Processing of Gut Associated Lymphoid Tissue with Centralized Data Analysis for Multi-Site Clinical Trials

et al. 2015

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The purpose of this study was to determine whether the development of a standardized approach to the collection of intestinal tissue from healthy volunteers, isolation of gut associated lymphoid tissue mucosal mononuclear cells (MMC), and characterization of mucosal T cell phenotypes by flow cytometry was sufficient to minimize differences in the normative ranges of flow parameters generated at two trial sites. Forty healthy male study participants were enrolled in Pittsburgh and Los Angeles. MMC were isolated from rectal biopsies using the same biopsy acquisition and enzymatic digestion protocols. As an additional comparator, peripheral blood mononuclear cells (PBMC) were collected from the study participants. For quality control, cryopreserved PBMC from a single donor were supplied to both sites from a central repository (qPBMC). Using a jointly optimized standard operating procedure, cells were isolated from tissue and blood and stained with monoclonal antibodies targeted to T cell phenotypic markers. Site-specific flow data were analyzed by an independent center which analyzed all data from both sites. Ranges for frequencies for overall CD4+ and CD8+ T cells, derived from the qPBMC samples, were equivalent at both UCLA and MWRI. However, there were significant differences across sites for the majority of T cell activation and memory subsets in qPBMC as well as PBMC and MMC. Standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis, can reduce but not exclude variability in reporting flow data within multi-site studies. Based on these data, centralized processing, flow cytometry, and analysis of samples may provide more robust data across multi-site studies. Centralized processing requires either shipping of fresh samples or cryopreservation and the decision to perform centralized versus site processing needs to take into account the drawbacks and restrictions associated with each method.

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Differential Modulation of Phenotypic Composition of Hiv-Infected and -Uninfected PBMCS During Cryopreservation

Cited by 7 publications

References 23 publications

The effect of cellular isolation and cryopreservation on the expression of markers identifying subsets of regulatory T cells

The effect of cellular isolation and cryopreservation on the expression of markers identifying subsets of regulatory T cells

Effects of enzymatic digestion, cell culture and preservation conditions on surface CD62L expression of primary murine CD3+CD4+ T cells

Exploring the Feasibility of Multi-Site Flow Cytometric Processing of Gut Associated Lymphoid Tissue with Centralized Data Analysis for Multi-Site Clinical Trials

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