| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||
,,,¶,¶
* Division of Nephrology,
Department of Biostatistics and Computational Biology,
Center for Vaccine Biology and Immunology,
Center for Biodefense Immune Modeling, and
¶ Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642
During the recall response by CD27+ IgG class-switched human memory B cells, total IgG secreted is a function of the following: 1) the number of IgG-secreting cells (IgG-SC), and 2) the secretion rate of each cell. In this study, we report the quantitative ELISPOT method for simultaneous estimation of single-cell IgG secretion rates and secreting cell frequencies in human B cell populations. We found that CD27+ IgM– memory B cells activated with CpG and cytokines had considerable heterogeneity in the IgG secretion rates, with two major secretion rate subpopulations. BCR cross-linking reduced the frequency of cells with high per-cell IgG secretion rates, with a parallel decrease in CD27high B cell blasts. Increased cell death may account for the BCR-stimulated reduction in high-rate IgG-SC CD27high B cell blasts. In contrast, the addition of IL-21 to CD40L plus IL-4-activated human memory B cells induced a high-rate IgG-SC population in B cells with otherwise low per-cell IgG secretion rates. The profiles of human B cell IgG secretion rates followed the same biphasic distribution and range irrespective of division class. This, along with the presence of non-IgG-producing, dividing B cells in CpG plus cytokine-activated B memory B cell populations, is suggestive of an on/off switch regulating IgG secretion. Finally, these data support a mixture model of IgG secretion in which IgG secreted over time is modulated by the frequency of IgG-SC and the distribution of their IgG secretion rates.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grants N01-AI-50020 (to A.D.H., O.H., J.R., T.M., and M.S.Z.), N01-AI-50029 (to T.M. and M.S.Z.), and R01 AI069351 (to M.S.Z., O.H., and M.N.C.).
A.D.H. designed the bead standards, performed experiments, analyzed data, and wrote the paper. J.R., T.M., and M.A.B. designed the image analysis system. M.N.C., A.J.M., and T.P. performed experiments, analyzed data, and made the figures for the manuscript. O.H. performed statistical analyses. M.S.Z. directed the experimental design and implementation, analyzed data, and wrote the paper.
2 Address correspondence and reprint requests to Dr. Martin S. Zand, University of Rochester Medical Center, 601 Elmwood Avenue, Box 675, Rochester, NY 14642. E-mail address: Martin_Zand{at}urmc.rochester.edu
3 Abbreviations used in this paper: PBlive, total number of living cells; 
ASC, fraction of cells secreting IgG; 
IgG, rate at which IgG is secreted by each cell; ASC, Ab-secreting cell; BAFF, B cell activating factor; BCR-x, BCR cross-linking Ab; CpG plus CK, CpG and cytokines; DSB, desthiobiotin; IgG-SC, IgG-secreting cell; Int-U, intensity unit; qELISPOT, quantitative ELISPOT; rhuIL, human rIL; tinc, incubation time.
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
