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Reduced expression of CD27 by collagenase treatment: implications for interpreting b cell data in tissues.

Shen C, Xu H, Alvarez X, Lackner AA, Veazey RS, Wang X - PLoS ONE (2015)

Bottom Line: Further investigation showed the marked loss of CD27 expression on processed intestinal cells was due to collagenase digestion of intestinal tissues, yet CD27 expression was recoverable within hours of cell isolation.By combining confocal microscopy, we confirmed that only a fraction of B cells express CD27, in contrast to expression on all T cells from tissues examined including the gut.Taken together, our results suggest that CD27 may be a memory marker for B cells, but not for T cells, since essentially all CD3 T cells expressed CD27.

View Article: PubMed Central - PubMed

Affiliation: Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, United States of America.

ABSTRACT
Surface markers have been used to identify distinct cell subpopulations and to delineate various stages of maturation or activation of lymphocytes. In particular CD27 is used for delineation of naïve and memory B cell populations, and is readily detected by flow cytometry. We here used flow cytometry to examine the expression of CD27 on lymphocytes isolated from various tissues of rhesus macaques, and found its expression was consistently low to absent on intestinal cell suspensions. However, immunohistochemistry revealed abundant CD27+ cells in intestinal tissue sections. Further investigation showed the marked loss of CD27 expression on processed intestinal cells was due to collagenase digestion of intestinal tissues, yet CD27 expression was recoverable within hours of cell isolation. By combining confocal microscopy, we confirmed that only a fraction of B cells express CD27, in contrast to expression on all T cells from tissues examined including the gut. Taken together, our results suggest that CD27 may be a memory marker for B cells, but not for T cells, since essentially all CD3 T cells expressed CD27. In summary, it is important to consider the influence of isolation procedures on cell surface expression of phenotypic markers, especially when examining tissue-resident lymphocytes by flow cytometry.

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CD27 expression recovers on lymphocytes following treatment with collagenase.(A) Dot plots show collagenase treated PBMCs lose surface CD27 expression, but expression is almost entirely recovered after 4, and entirely recovered after 24 hrs rest / incubation. Numbers in each quadrant indicate the percentage of total live lymphocytes as indicated in Fig. 1. (B) Rapid recovery of CD27 expression on collagenase treated cells occurs in all tissues including intestinal tissues. Histograms are representative of four independent experiments.
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pone.0116667.g003: CD27 expression recovers on lymphocytes following treatment with collagenase.(A) Dot plots show collagenase treated PBMCs lose surface CD27 expression, but expression is almost entirely recovered after 4, and entirely recovered after 24 hrs rest / incubation. Numbers in each quadrant indicate the percentage of total live lymphocytes as indicated in Fig. 1. (B) Rapid recovery of CD27 expression on collagenase treated cells occurs in all tissues including intestinal tissues. Histograms are representative of four independent experiments.

Mentions: To determine if CD27 expression would recover after collagenase treatment, collagenase treated lymphocytes were incubated with C10 medium at 37°C in CO2 incubator for 4, 8, 12, 16 and 24 hrs, and found a rapid recovery of CD27 expression. As shown as in Fig. 3, after 4 hours incubation, CD27 expression on collagenase treated lymphocytes were comparable to untreated lymphocytes. In fact, CD27 expression was apparently completely recovered by 8 hrs after treatment, and was maintained at pre-treatment levels through 24 hrs of incubation in all tissues examined.


Reduced expression of CD27 by collagenase treatment: implications for interpreting b cell data in tissues.

Shen C, Xu H, Alvarez X, Lackner AA, Veazey RS, Wang X - PLoS ONE (2015)

CD27 expression recovers on lymphocytes following treatment with collagenase.(A) Dot plots show collagenase treated PBMCs lose surface CD27 expression, but expression is almost entirely recovered after 4, and entirely recovered after 24 hrs rest / incubation. Numbers in each quadrant indicate the percentage of total live lymphocytes as indicated in Fig. 1. (B) Rapid recovery of CD27 expression on collagenase treated cells occurs in all tissues including intestinal tissues. Histograms are representative of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4355594&req=5

pone.0116667.g003: CD27 expression recovers on lymphocytes following treatment with collagenase.(A) Dot plots show collagenase treated PBMCs lose surface CD27 expression, but expression is almost entirely recovered after 4, and entirely recovered after 24 hrs rest / incubation. Numbers in each quadrant indicate the percentage of total live lymphocytes as indicated in Fig. 1. (B) Rapid recovery of CD27 expression on collagenase treated cells occurs in all tissues including intestinal tissues. Histograms are representative of four independent experiments.
Mentions: To determine if CD27 expression would recover after collagenase treatment, collagenase treated lymphocytes were incubated with C10 medium at 37°C in CO2 incubator for 4, 8, 12, 16 and 24 hrs, and found a rapid recovery of CD27 expression. As shown as in Fig. 3, after 4 hours incubation, CD27 expression on collagenase treated lymphocytes were comparable to untreated lymphocytes. In fact, CD27 expression was apparently completely recovered by 8 hrs after treatment, and was maintained at pre-treatment levels through 24 hrs of incubation in all tissues examined.

Bottom Line: Further investigation showed the marked loss of CD27 expression on processed intestinal cells was due to collagenase digestion of intestinal tissues, yet CD27 expression was recoverable within hours of cell isolation.By combining confocal microscopy, we confirmed that only a fraction of B cells express CD27, in contrast to expression on all T cells from tissues examined including the gut.Taken together, our results suggest that CD27 may be a memory marker for B cells, but not for T cells, since essentially all CD3 T cells expressed CD27.

View Article: PubMed Central - PubMed

Affiliation: Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, United States of America.

ABSTRACT
Surface markers have been used to identify distinct cell subpopulations and to delineate various stages of maturation or activation of lymphocytes. In particular CD27 is used for delineation of naïve and memory B cell populations, and is readily detected by flow cytometry. We here used flow cytometry to examine the expression of CD27 on lymphocytes isolated from various tissues of rhesus macaques, and found its expression was consistently low to absent on intestinal cell suspensions. However, immunohistochemistry revealed abundant CD27+ cells in intestinal tissue sections. Further investigation showed the marked loss of CD27 expression on processed intestinal cells was due to collagenase digestion of intestinal tissues, yet CD27 expression was recoverable within hours of cell isolation. By combining confocal microscopy, we confirmed that only a fraction of B cells express CD27, in contrast to expression on all T cells from tissues examined including the gut. Taken together, our results suggest that CD27 may be a memory marker for B cells, but not for T cells, since essentially all CD3 T cells expressed CD27. In summary, it is important to consider the influence of isolation procedures on cell surface expression of phenotypic markers, especially when examining tissue-resident lymphocytes by flow cytometry.

Show MeSH