Naïve and memory B cells in the rhesus macaque can be differentiated by surface expression of CD27 and have differential responses to CD40 ligation

Abstract

The rhesus macaque (RM) model has the potential to be an invaluable tool for studying B cell populations during pathogenic infections, however, to date, there has been no definitive delineation of naïve and memory B cell populations in the RM. This has precluded a rigorous analysis of the generation, persistence and resolution of a pathogen-specific memory B cell response. The present study utilized multiple analyses to demonstrate that CD27 expression on B cells is consistent with a memory phenotype. Compared to CD20+CD27- B cells, CD20+CD27+ B cells were larger in size, and preferentially accumulated at effector sites. Direct sequence analysis revealed that CD20+CD27+ B cells had an increased frequency of point mutations that were consistent with somatic hypermutation and at a functional level, CD40 ligation improved CD20+CD27- but not CD20+CD27+ B cell survival in vitro. These data provide definitive evidence that the naïve and memory B cell populations of the RM can be differentiated using surface expression of CD27.

Fig. 1

Surface expression of CD27 and activation markers on RM B cells. (a.) Representative flow cytometry analysis of CD20+ peripheral blood B cells based on CD27 expression. The gate for positive CD27 levels was set against background staining of myeloid cells which are not known to express this marker. The CD27+ and CD27− subsets were subsequently examined for expression of CD95, CD86 and CD40. Averaged results for percent of cells expressing CD95 (b.), CD86 (c.) and CD40 (d.). n=6 independent animals. Means were compared using paired Student’s T test.

Fig. 2

CD20+ CD27+ cells are larger than CD20+ CD27− peripheral blood cells. (a.) Representative flow cytometry analysis of relative sizes of CD20+ CD27− (black line) and CD20+ CD27+ subsets (gray line). (b.) Averaged geometric mean values of FSC for 6 independent animals. Means were compared using a paired Student’s T test.

Fig. 3

Surface expression of CD27 and activation markers on B cells from tissue compartments. (a.) Representative flow cytometry analysis of CD27 expression on CD20+ cells in peripheral blood, axillary lymph node, mediastinal lymph node, small intestine and bronchial alveolar lavage samples. (b.) Plotted values for each replicate, the line represents the average value for all samples (n=3 independent animals).

Fig. 4

CD20+ B cells from umbilical cord blood do not express CD27. (a.) Analysis of CD27 expression on CD20+ B cells from 3 umbilical cord blood samples. (b.) Averaged CD27+ B cell expression from adult peripheral blood (n=6 independent animals) and cord blood samples (n=3 independent animals). Means were compared using a T test with unequal variance.

Fig. 5

CD20+CD27+ cells but not CD20+CD27− cells demonstrate somatic hypermutations at Ig variable regions. (a) Representative plots for cell sorting parameters and post-sort purity analysis of sorted populations. (b) Representative sequence samples from IGHV1p family genes, shaded areas denote complementarity determining regions. (c) Percent homology to germline sequences for IGHV1 family genes isolated from CD20+CD27− and CD20+CD27+ B cells. Results are representative of 40 unique sequences obtained from 5 individual animals. Means were compared using a T test with unequal variance.

Fig. 6

Binding of CD40 on CD20+CD27− but not CD20+CD27+ B cells results in protection from spontaneous cell losses in a 24 hour culture system. PBMC were cultured in the presence or absence of 200 ng/ml of anti-CD40 antibody for 24 h. Cultures were harvested and then analyzed for CD20 and CD27 expression in order to differentiate the percentages and numbers of B cell populations. (a) Percentage of CD20+CD27− B cells and (b) CD20+CD27+ B cells. (c) Absolute number of CD20+CD27− B cells and (d) CD20+CD27+ B cells. Results are representative of 6 independent experiments run in duplicate. Error bars represent the standard error of the mean. Mean values for both culture conditions and freshly isolated cells were compared using 1 way ANOVA with repeated measures and Bonferroni’s multiple comparison test.

Fig. 7

The percentage of CD20+CD27− B cells expressing CD95 increases and the percentage of CD20+CD27− B cells that bind Annexin V decreases after anti-CD40 antibody treatment. PBMC were cultured in the presence or absence of 200 ng/ml of anti-CD40 in triplicate for 24 h. Cultures were harvested and then analyzed for CD95 expression within (a) CD20+CD27− or (b) CD20+CD27+ B cells. Representative flow cytometry plots of the CD20+CD27− B cell population following 24 hour culture (c) without anti-CD40 antibody treatment and (d) with anti-CD40 antibody treatment. The average percentage of Annexin V staining for all 6 animals within the (e) CD20+CD27− or (f) CD20+CD27+ B cell populations. Mean values for both culture conditions and freshly isolated cells were compared using 1 way ANOVA with repeated measures and Bonferroni’s multiple comparison test.