Changes in chemokine levels in plasma in acute SHIVsf162p3 (filled circles) and SIVmac251-infected RMs (open squares).

<p>Means±SEM are shown. *Asterisks indicate significant differences between groups (P<0.05).</p

Dynamics of activated T cells in blood as determined by HLA-DR expression on T cell subsets.

<p>A) Representative gating strategy showing HLA-DR expression on CD3+ T cells prior to infection and 21 days after infection. B) Mean percentages of HLA-DR expression on total T cells (CD3+), and on CD4+ and CD8+ T cell subsets in SHIVsf162p3 (filled circles) and SIVmac251-infected (open squares) RMs in early and chronic (3 months) infection. Means±SEM are shown. Asterisks indicate significant differences (P<0.05) between groups.</p

Plasma viremia in early SHIVsf162p3 (n = 11; filled circles) and SIVmac251 (n = 16; open squares) infected rhesus macaques.

<p>Means ±SEM are shown.</p

Comparison of central memory CD4+ T cells in SIV (open squares) and SHIVsf162p3 (filled circles) infected macaques.

<p>A) Representative FACS dot plot of central memory CD4+T cells (CD28+CD95 gated CD4+ T cells) in blood at 28 days after SHIVsf162p3/SIVmac251 infection. B, Dynamic of central memory CD4+ T cells in peripheral blood of SIVmac251 (open squares) versus SHIVsf162P3 (filled circles) infected macaques. Although percentages are almost identical in early infection, significantly higher percentages of central memory CD4+ T cells recover in SHIVsf162p3-infected macaques by 28 days of infection. Means ± SEM are shown. *Indicates significant differences between groups (P<0.05).</p

T-cell proliferation during acute SIV infection.

<p>A) Representative gating strategy showing Ki-67 expression on CD3+ T cells prior to and 21 days after infection. B) Longitudinal analysis of early T-cell proliferation in blood from SHIVsf162p3 (filled circles) and SIVmac251-infected RMs (open squares). Means±SEM are shown. Significant differences (P<0.05) between groups are indicated by asterisks.</p

Reduced Expression of CD27 by Collagenase Treatment: Implications for Interpreting B Cell Data in Tissues

<div><p>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.</p></div

Identification and localization of CD27+ cell subsets in intestinal tissues.

<p>Immunohistochemistry staining intestinal B cells (CD20+) (A), CD27+ cells (B), and T cells (CD3+) (C) on the serial adjacent sections. Three-color confocal microscopy images show that CD27+ B cells are mainly located in lymphoid aggregates in the deeper lamina propria of the gut (D), whereas CD27+ cells in the intestinal villi are all T cells (E). Flow cytometry of intestinal cell suspensions confirms most CD27+ cells are CD3+ T cells, with fewer percentages of CD20+ B cells co-expressing CD27 (F).</p

Expression of CD27 on lymphocytes isolated from various tissues of rhesus macaques detected by flow cytometry.

<p>(A) Representative plots generated from whole blood samples showing gating strategy for analysis of CD27+ lymphocytes isolated from all tissues. Specifically, singlets were gated first followed by lymphocytes, and then live lymphocytes (DLneg) were selected for further analysis of CD3+, CD20+, CD27+ as shown as in A; (B) Comparison of percentages of various cells including CD3+, CD20+ and CD27+ after different processing of the same tissues and gating through live lymphocytes. Note that significantly decreased CD27 expression is only detected on lymphocytes subjected to collagenase digestion procedures when compared to no digestion. In contrast, there are no changes in expression of CD3 or CD20 on lymphocytes indicating this is selective for CD27. Bars represent mean percentages ± SEM in each group (n = 7), P values ≤ 0.05 were considered statistically significant.</p

CD27 expression recovers on lymphocytes following treatment with collagenase.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116667#pone.0116667.g001" target="_blank">Fig. 1</a>. (B) Rapid recovery of CD27 expression on collagenase treated cells occurs in all tissues including intestinal tissues. Histograms are representative of four independent experiments.</p

Comparison of CD27 expression in lymph node tissue sections before and after collagenase treatment.

<p>Note marked loss of CD27 signal is detected following collagenase treatment of adjacent frozen sections of the same lymph node (a and b). In contrast, collagenase treatment has no effect on CD3 expression (c).</p