Effect of cryopreservation of peripheral blood mononuclear cells (PBMCs) on the variability of an antigen-specific memory B cell ELISpot

Trück, Johannes; Mitchell, Ruth M.; Thompson, Amber; Morales-Aza, Begonia; Clutterbuck, Elizabeth A.; Kelly, Dominic F.; Finn, Adam; Pollard, Andrew J.

doi:10.4161/hv.29318

Cited by 21 publications

(23 citation statements)

References 36 publications

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“…These results are in contrast to those in existing publications, where no alterations regarding the enumeration of ASC following cryopreservation of PBMCs were found (Kyu et al, 2009;Trück et al, 2014).…”

Section: Cryopreservation Induces Changes In the Percentages Of Plasmcontrasting

confidence: 86%

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Comparative phenotypical analysis of B cells in fresh and cryopreserved mononuclear cells from blood and tissue of rhesus macaques

Klippert

Neumann

Stahl–Hennig∥

2016

Journal of Immunological Methods

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Section: Cryopreservation Induces Changes In the Percentages Of Plasmcontrasting

confidence: 86%

“…Two other publications mentioned a good comparability between fresh and frozen PBMCs regarding the enumeration of antigen-specific memory B cells (Kyu et al, 2009;Trück et al, 2014).…”

Section: Introductionmentioning

confidence: 98%

Comparative phenotypical analysis of B cells in fresh and cryopreserved mononuclear cells from blood and tissue of rhesus macaques

Klippert

Neumann

Stahl–Hennig∥

2016

Journal of Immunological Methods

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“…In healthy individuals, the performance of functional tests that assess T and B memory cells by methods such as enzyme-linked immunosorbent spot assay (ELISPOT), after polyclonal stimulation, is usually adequate (40), but there is still controversy as to whether these findings are similar in PBMCs of the sick (41). As has been previously noted, the cryopreservation did not affect the functionality of cells from healthy children (Fig.…”

Section: Discussionmentioning

confidence: 55%

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

Perdomo‐Celis

Salgado

Castañeda

et al. 2016

Clin Vaccine Immunol

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bCryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosisinducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-␥]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r ‫؍‬ 0.74; P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P < 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3 ؉ CD8 ؉ amine-positive cells was found before cryopreservation (P ‫؍‬ 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-␥-producing CD3؉ cells after stimulation compared with healthy controls and convalescent-phase patients (P < 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho ‫؍‬ ؊0.56; P ‫؍‬ 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs. Cryopreservation is the maintenance of cells and biological tissues at low temperatures and is based on the use of various media or solutions that form hydrogen bonds with water molecules, preventing cellular damage. The low temperatures allow the cells to enter a quiescent state in which cellular functions are suspended without affecting their intrinsic characteristics (1). Peripheral blood mononuclear cells (PBMCs) are frequently cryopreserved for use in transplants or immunological studies (2, 3). However, the cryopreservation process may affect viability, phenotype, and cellular functionality due to factors such as inadequate temperatures, the freezing protocol used, the expertise of the personnel, and freezing time (4, 5). The disease of the individual from whom the PBMCs come also affects cryopreservation. For example, PBMCs from subjects infected with human immunodeficiency virus (HIV) presented reduced viability after cryopreservation, possibly due to the increased numbers of apoptotic cells circulating during the course of the disease (6). Similar findings have been found in the acute phases of diseases, such as visceral leishmaniasis (7). Particularly for HIV, great efforts have been undertaken to optimize the evaluation and comparability of immune tests...

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“…Enumeration of memory B cells by ELISpot was carried out according to the original method of Crotty et al, [ 29 ] with minor modifications including the use of cryopreserved PBMC [ 36 ]. PBMC (1x10 6 per mL) were rested in culture medium (RPMI 1640 with 10% fetal calf serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 2mM L-Glutamine, 10mM Hepes and 1mM Sodium Pyruvate) at 37°C for one hour before being stimulated with 1μg/mL R848 (resimiquimod; MABTECH, Cincinnati, OH) and 10ng/mL recombinant human IL-2 (interleukin-2; MABTECH, Cincinnati, OH) for 5 days.…”

Section: Methodsmentioning

confidence: 99%

Relationship between Humoral Immune Responses against HPV16, HPV18, HPV31 and HPV45 in 12-15 Year Old Girls Receiving Cervarix® or Gardasil® Vaccine

et al. 2015

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BackgroundHuman papillomavirus (HPV) vaccines confer protection against the oncogenic genotypes HPV16 and HPV18 through the generation of type-specific neutralizing antibodies raised against virus-like particles (VLP) representing these genotypes. The vaccines also confer a degree of cross-protection against HPV31 and HPV45, which are genetically-related to the vaccine types HPV16 and HPV18, respectively, although the mechanism is less certain. There are a number of humoral immune measures that have been examined in relation to the HPV vaccines, including VLP binding, pseudovirus neutralization and the enumeration of memory B cells. While the specificity of responses generated against the vaccine genotypes are fairly well studied, the relationship between these measures in relation to non-vaccine genotypes is less certain.MethodsWe carried out a comparative study of these immune measures against vaccine and non-vaccine genotypes using samples collected from 12–15 year old girls following immunization with three doses of either Cervarix® or Gardasil® HPV vaccine.ResultsThe relationship between neutralizing and binding antibody titers and HPV-specific memory B cell levels for the vaccine genotypes, HPV16 and HPV18, were very good. The proportion of responders approached 100% for both vaccines while the magnitude of these responses induced by Cervarix® were generally higher than those following Gardasil® immunization. A similar pattern was found for the non-vaccine genotype HPV31, albeit at a lower magnitude compared to its genetically-related vaccine genotype, HPV16. However, both the enumeration of memory B cells and VLP binding responses against HPV45 were poorly related to its neutralizing antibody responses. Purified IgG derived from memory B cells demonstrated specificities similar to those found in the serum, including the capacity to neutralize HPV pseudoviruses.ConclusionsThese data suggest that pseudovirus neutralization should be used as the preferred humoral immune measure for studying HPV vaccine responses, particularly for non-vaccine genotypes.

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Effect of cryopreservation of peripheral blood mononuclear cells (PBMCs) on the variability of an antigen-specific memory B cell ELISpot

Cited by 21 publications

References 36 publications

Comparative phenotypical analysis of B cells in fresh and cryopreserved mononuclear cells from blood and tissue of rhesus macaques

Comparative phenotypical analysis of B cells in fresh and cryopreserved mononuclear cells from blood and tissue of rhesus macaques

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

Relationship between Humoral Immune Responses against HPV16, HPV18, HPV31 and HPV45 in 12-15 Year Old Girls Receiving Cervarix® or Gardasil® Vaccine

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