Key Points
Only a small subset of flagellin-induced CD40LhiCD4 cells express cytokines.
This CD40LhiCD4 cytokine production is restricted to T effector memory cells.
Abstract
Flagellin is an immunodominant Ag in Crohn disease, with many patients showing anti-flagellin Abs. To study the clonality of flagellin-reactive CD4 cells in Crohn patients, we used a common CD154-based enrichment method following short-term Ag exposure to identify Ag-reactive CD4 cells. CD154 expression and cytokine production following Ag exposure compared with negative control responses (no Ag exposure) revealed that only a small fraction of CD154-enriched cells could be defined by Ag-reactive cytokine responses. This was especially true for low-frequency flagellin-reactive CD4 cells compared with polyclonal stimulation or Candida albicans Ag exposure. Moreover, we found that culture conditions used for the assay contributed to background CD40L (CD154) expression in the CD154-enriched CD4 cells. Using a cut-off rule based on flow cytometry results of the negative control CD154-enriched CD4 cells, we could reliably find the fraction of Ag-reactive cells in the CD154-enriched population. Ag-reactive CD4 cytokine production was restricted to CD4 cells with an effector memory phenotype and the highest levels of induced CD154 expression. This has important implications for identifying Ag-specific T cells of interest for single cell cloning, phenotyping, and transcriptomics.
Footnotes
This work was supported by U.S. Department of Veterans Affairs Grant CX0001530 and Impact funds from University of Alabama at Birmingham.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- CD
- Crohn disease
- IBD
- inflammatory bowel disease
- TEM
- effector memory T cell.
- Received August 6, 2020.
- Accepted November 12, 2020.
- Copyright © 2021 by The American Association of Immunologists, Inc.
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