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Reduced expression of CD27 by collagenase treatment: implications for interpreting b cell data in tissues
Chanjuan Shen1, Huanbin Xu1, Xavier Alvarez1
1Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, United States of America.
Insights
Flow cytometry analysis of rhesus macaque lymphocytes revealed that collagenase digestion significantly reduces CD27 expression on intestinal cells. This suggests CD27 is a B cell memory marker, but not for T cells, highlighting the importance of isolation methods.
Area of Science:
- Immunology
- Cell Biology
- Primate Research
Background:
- Surface markers like CD27 are crucial for identifying lymphocyte subpopulations and their activation states.
- CD27 is commonly used to distinguish naive and memory B cells via flow cytometry.
Purpose of the Study:
- To investigate CD27 expression on lymphocytes from various rhesus macaque tissues using flow cytometry.
- To understand the impact of tissue processing on CD27 surface marker detection.
Main Methods:
- Flow cytometry was employed to analyze CD27 expression on lymphocytes from different rhesus macaque tissues.
- Immunohistochemistry and confocal microscopy were used to validate findings and assess cell populations.
- The effect of collagenase digestion on CD27 expression was specifically examined.
Main Results:
- CD27 expression was low to absent on intestinal cell suspensions but abundant in tissue sections.
- Collagenase digestion during tissue processing led to a marked loss of CD27 expression.
- CD27 expression was partially recoverable within hours of cell isolation.
- While a fraction of B cells expressed CD27, nearly all T cells (CD3+) exhibited CD27 expression.
Conclusions:
- CD27 appears to be a reliable memory marker for B cells but not T cells in rhesus macaques.
- Tissue isolation procedures, particularly enzymatic digestion, can significantly affect cell surface marker expression.
- Careful consideration of isolation methods is essential for accurate phenotypic analysis of tissue-resident lymphocytes using flow cytometry.
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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