Mucosal Immunology (2016) 9, 68–82; doi:10.1038/mi.2015.36; published online 22 April 2015

High-dimensional immune profiling of total and rotavirus VP6-specific intestinal and circulating B cells by mass cytometry

N Nair1,2, E W Newell2,3, C Vollmers4, S R Quake4, J M Morton5, M M Davis2,6,7, X S He1,2 and H B Greenberg1,2

  1. 1Department of Medicine, Stanford University, Stanford, California, USA
  2. 2Department of Microbiology and Immunology, Stanford University, Stanford, California, USA
  3. 3Agency for Science, Technology and Research (A*STAR), Singapore Immunology Network (SIgN), Singapore, Singapore
  4. 4Department of Bioengineering, Stanford University, Stanford, California, USA
  5. 5Department of Surgery, Stanford University School of Medicine, Stanford, California, USA
  6. 6The Howard Hughes Medical Institute, Stanford, California, USA
  7. 7Institute for Immunity, Transplantation and Infection, Stanford University, Stanford, California, USA

Correspondence: HB Greenberg, (

Received 10 September 2014; Accepted 7 March 2015
Advance online publication 22 April 2015



In-depth phenotyping of human intestinal antibody secreting cells (ASCs) and their precursors is important for developing improved mucosal vaccines. We used single-cell mass cytometry to simultaneously analyze 34 differentiation and trafficking markers on intestinal and circulating B cells. In addition, we labeled rotavirus (RV) double-layered particles with a metal isotope and characterized B cells specific to the RV VP6 major structural protein. We describe the heterogeneity of the intestinal B-cell compartment, dominated by ASCs with some phenotypic and transcriptional characteristics of long-lived plasma cells. Using principal component analysis, we visualized the phenotypic relationships between major B-cell subsets in the intestine and blood, and revealed that IgM+ memory B cells (MBCs) and naive B cells were phenotypically related as were CD27 MBCs and switched MBCs. ASCs in the intestine and blood were highly clonally related, but associated with distinct trajectories of phenotypic development. VP6-specific B cells were present among diverse B-cell subsets in immune donors, including naive B cells, with phenotypes representative of the overall B-cell pool. These data provide a high dimensional view of intestinal B cells and the determinants regulating humoral memory to a ubiquitous, mucosal pathogen at steady-state.