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CD3-positive B cells: a storage-dependent phenomenon.

Nagel A, Möbs C, Raifer H, Wiendl H, Hertl M, Eming R - PLoS ONE (2014)

Bottom Line: Since cellular subsets are often classified by phenotypic analyses, our results indicate that ex vivo cellular classification in peripheral blood might result in misleading interpretations.Therefore, in order to obtain results reflecting the in vivo situation, it is suggested to minimize times of ex vivo blood storage after isolation of PBMC.Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology and Allergology, Philipps University Marburg, Marburg, Germany; Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

ABSTRACT
The majority of clinical studies requires extensive management of human specimen including e.g. overnight shipping of blood samples in order to convey the samples in a central laboratory or to simultaneously analyze large numbers of patients. Storage of blood samples for periods of time before in vitro/ex vivo testing is known to influence the antigen expression on the surface of lymphocytes. In this context, the present results show for the first time that the T cell antigen CD3 can be substantially detected on the surface of human B cells after ex vivo storage and that the degree of this phenomenon critically depends on temperature and duration after blood withdrawal. The appearance of CD3 on the B cell surface seems to be a result of contact-dependent antigen exchange between T and B lymphocytes and is not attributed to endogenous production by B cells. Since cellular subsets are often classified by phenotypic analyses, our results indicate that ex vivo cellular classification in peripheral blood might result in misleading interpretations. Therefore, in order to obtain results reflecting the in vivo situation, it is suggested to minimize times of ex vivo blood storage after isolation of PBMC. Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.

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Transfer of CD3 from T to B cells requires cell-cell contact and is impaired by monensin treatment.(A) Shown are numbers of CD3lowCD20+ B cells in one patient after co-culture of MACS-purified CD4+ T cells and CD20+ B cells overnight (oN) at 4°C. While CD3lowCD20+ B cells were found to be elevated with increasing T-B cell ratios, fixation of antigens on the T cell surface using 1% PFA before co-culture with B cells oN at 4°C did prevent CD3 acquisition by CD20+ B cells. Moreover, co-culture of CD4+ T cells and CD20+ B cells using transwell (Tw) chambers (pore size of 0.4 µm) prevented the appearance of CD3lowCD20+ B cells, excluding the possibility that membrane vesicles like exosomes transfer T cell surface markers from T to B cells. (B) The occurrence of CD3lowCD20+ B cells after oN storage of blood samples at 4°C was impaired by the addition of monensin (applied by the protein transport inhibitor BD GolgiStop). Shown are the results of four independent experiments.
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pone-0110138-g005: Transfer of CD3 from T to B cells requires cell-cell contact and is impaired by monensin treatment.(A) Shown are numbers of CD3lowCD20+ B cells in one patient after co-culture of MACS-purified CD4+ T cells and CD20+ B cells overnight (oN) at 4°C. While CD3lowCD20+ B cells were found to be elevated with increasing T-B cell ratios, fixation of antigens on the T cell surface using 1% PFA before co-culture with B cells oN at 4°C did prevent CD3 acquisition by CD20+ B cells. Moreover, co-culture of CD4+ T cells and CD20+ B cells using transwell (Tw) chambers (pore size of 0.4 µm) prevented the appearance of CD3lowCD20+ B cells, excluding the possibility that membrane vesicles like exosomes transfer T cell surface markers from T to B cells. (B) The occurrence of CD3lowCD20+ B cells after oN storage of blood samples at 4°C was impaired by the addition of monensin (applied by the protein transport inhibitor BD GolgiStop). Shown are the results of four independent experiments.

Mentions: The pronounced increase of CD3lowCD20+ B cell numbers was confirmed after oN/4°C co-culture of both MACS-purified fresh CD4+ T cells and CD20+ B cells (ratio 4∶1 versus 9∶1) compared to CD20+ B cells alone (Figure 5A). In contrast, same conditions but fixation of antigens on the T cell surface using 1% PFA before co-culture with CD20+ B cells prevented the occurrence of CD3lowCD20+ B cells (Figure 5A). Additionally, separation of CD4+ T cells and CD20+ B cells using transwell chambers did not result in elevated CD3lowCD20+ B cell numbers compared to CD20+ B cells alone (Figure 5A). Therefore, transfer of CD3 from T cells to B cells might not depend on small membrane vesicles such as frequently described exosomes, since these nanovesicles – with a diameter of 30–100 nm [17], [18] – are able to pass the transwell membrane (pore size: 400 nm) [19], [20].


CD3-positive B cells: a storage-dependent phenomenon.

Nagel A, Möbs C, Raifer H, Wiendl H, Hertl M, Eming R - PLoS ONE (2014)

Transfer of CD3 from T to B cells requires cell-cell contact and is impaired by monensin treatment.(A) Shown are numbers of CD3lowCD20+ B cells in one patient after co-culture of MACS-purified CD4+ T cells and CD20+ B cells overnight (oN) at 4°C. While CD3lowCD20+ B cells were found to be elevated with increasing T-B cell ratios, fixation of antigens on the T cell surface using 1% PFA before co-culture with B cells oN at 4°C did prevent CD3 acquisition by CD20+ B cells. Moreover, co-culture of CD4+ T cells and CD20+ B cells using transwell (Tw) chambers (pore size of 0.4 µm) prevented the appearance of CD3lowCD20+ B cells, excluding the possibility that membrane vesicles like exosomes transfer T cell surface markers from T to B cells. (B) The occurrence of CD3lowCD20+ B cells after oN storage of blood samples at 4°C was impaired by the addition of monensin (applied by the protein transport inhibitor BD GolgiStop). Shown are the results of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199681&req=5

pone-0110138-g005: Transfer of CD3 from T to B cells requires cell-cell contact and is impaired by monensin treatment.(A) Shown are numbers of CD3lowCD20+ B cells in one patient after co-culture of MACS-purified CD4+ T cells and CD20+ B cells overnight (oN) at 4°C. While CD3lowCD20+ B cells were found to be elevated with increasing T-B cell ratios, fixation of antigens on the T cell surface using 1% PFA before co-culture with B cells oN at 4°C did prevent CD3 acquisition by CD20+ B cells. Moreover, co-culture of CD4+ T cells and CD20+ B cells using transwell (Tw) chambers (pore size of 0.4 µm) prevented the appearance of CD3lowCD20+ B cells, excluding the possibility that membrane vesicles like exosomes transfer T cell surface markers from T to B cells. (B) The occurrence of CD3lowCD20+ B cells after oN storage of blood samples at 4°C was impaired by the addition of monensin (applied by the protein transport inhibitor BD GolgiStop). Shown are the results of four independent experiments.
Mentions: The pronounced increase of CD3lowCD20+ B cell numbers was confirmed after oN/4°C co-culture of both MACS-purified fresh CD4+ T cells and CD20+ B cells (ratio 4∶1 versus 9∶1) compared to CD20+ B cells alone (Figure 5A). In contrast, same conditions but fixation of antigens on the T cell surface using 1% PFA before co-culture with CD20+ B cells prevented the occurrence of CD3lowCD20+ B cells (Figure 5A). Additionally, separation of CD4+ T cells and CD20+ B cells using transwell chambers did not result in elevated CD3lowCD20+ B cell numbers compared to CD20+ B cells alone (Figure 5A). Therefore, transfer of CD3 from T cells to B cells might not depend on small membrane vesicles such as frequently described exosomes, since these nanovesicles – with a diameter of 30–100 nm [17], [18] – are able to pass the transwell membrane (pore size: 400 nm) [19], [20].

Bottom Line: Since cellular subsets are often classified by phenotypic analyses, our results indicate that ex vivo cellular classification in peripheral blood might result in misleading interpretations.Therefore, in order to obtain results reflecting the in vivo situation, it is suggested to minimize times of ex vivo blood storage after isolation of PBMC.Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology and Allergology, Philipps University Marburg, Marburg, Germany; Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

ABSTRACT
The majority of clinical studies requires extensive management of human specimen including e.g. overnight shipping of blood samples in order to convey the samples in a central laboratory or to simultaneously analyze large numbers of patients. Storage of blood samples for periods of time before in vitro/ex vivo testing is known to influence the antigen expression on the surface of lymphocytes. In this context, the present results show for the first time that the T cell antigen CD3 can be substantially detected on the surface of human B cells after ex vivo storage and that the degree of this phenomenon critically depends on temperature and duration after blood withdrawal. The appearance of CD3 on the B cell surface seems to be a result of contact-dependent antigen exchange between T and B lymphocytes and is not attributed to endogenous production by B cells. Since cellular subsets are often classified by phenotypic analyses, our results indicate that ex vivo cellular classification in peripheral blood might result in misleading interpretations. Therefore, in order to obtain results reflecting the in vivo situation, it is suggested to minimize times of ex vivo blood storage after isolation of PBMC. Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.

Show MeSH
Related in: MedlinePlus