Deep phenomics of human PBMCs using a 31-marker spectral flow cytometry panel
- 1Precision Vaccines Program, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA
Abstract
Functional cytokines orchestrate leukocyte-mediated innate and adaptive immune responses within the host following infection or immunization. In contrast, lymphocyte-mediated activation-induced markers (AIMs) are predominantly associated with adaptive immune responses. Regulatory T cells (Tregs) primarily function to balance the immune response by preventing excessive inflammation and maintaining self-tolerance. Currently, conventional intracellular cytokine tracking or cytokine-independent AIMs methodology is available for studying antigen-specific lymphocyte or T cell responses in humans. However, this approach faces challenges due to the difficulty in simultaneously deep immunophenotyping the key functional cytokine signatures, AIMs, and nuclear signature of FoxP3+ Tregs. Using spectral flow cytometry, we developed a 31-parameter panel to profile simultaneously major immune cell subsets (B cells, NK cells, DCs, monocytes) along with T cells, memory phenotypes, functional cytokines by tracking established Th1 markers IFN{gamma}, TNF and IL-2; Th2 markers IL-4/5/13 and the Th17 marker, IL-17, AIMs and FoxP3+ Tregs in human PBMCs in steady state as well as in mitogen stimulated conditions. For AIMs, CD69, CD134 (OX40) and CD137 (4-1BB) are included and validated. We also re-validated our panel to profile antigen specific Th1/2/17 cells or AIMs distribution after peptide stimulation. This optimized multicolor spectral flow panel allows a detailed immune profiling of functional cytokines, AIMs and FoxP3+ Tregs in the T cell compartment from human and might be useful for exploratory analysis of how these functional cytokines or AIMs or Tregs shape host immunity after infection or vaccination. Our panel is highly customizable, allowing researchers to tailor it to their specific requirements.
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BibTeX
@misc{barman2026deep,
title = {{Deep phenomics of human PBMCs using a 31-marker spectral flow cytometry panel}},
author = {S. Barman and A. Patel and A. Kelly and B. Janoschek and R. Hingorani and D. J. Dowling},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.31.763346},
url = {https://www.biorxiv.org/content/10.64898/2026.10.31.763346v1},
note = {CC BY-ND},
}
RIS
TY - UNPB
TI - Deep phenomics of human PBMCs using a 31-marker spectral flow cytometry panel
AU - S. Barman
AU - A. Patel
AU - A. Kelly
AU - B. Janoschek
AU - R. Hingorani
AU - D. J. Dowling
PY - 2026
DA - 2026/10/31
PB - bioRxiv
DO - 10.64898/2026.10.31.763346
UR - https://www.biorxiv.org/content/10.64898/2026.10.31.763346v1
AB - Functional cytokines orchestrate leukocyte-mediated innate and adaptive immune responses within the host following infection or immunization. In contrast, lymphocyte-mediated activation-induced markers (AIMs) are predominantly associated with adaptive immune responses. Regulatory T cells (Tregs) primarily function to balance the immune response by preventing excessive inflammation and maintaining self-tolerance. Currently, conventional intracellular cytokine tracking or cytokine-independent AIMs methodology is available for studying antigen-specific lymphocyte or T cell responses in humans. However, this approach faces challenges due to the difficulty in simultaneously deep immunophenotyping the key functional cytokine signatures, AIMs, and nuclear signature of FoxP3+ Tregs. Using spectral flow cytometry, we developed a 31-parameter panel to profile simultaneously major immune cell subsets (B cells, NK cells, DCs, monocytes) along with T cells, memory phenotypes, functional cytokines by tracking established Th1 markers IFN{gamma}, TNF and IL-2; Th2 markers IL-4/5/13 and the Th17 marker, IL-17, AIMs and FoxP3+ Tregs in human PBMCs in steady state as well as in mitogen stimulated conditions. For AIMs, CD69, CD134 (OX40) and CD137 (4-1BB) are included and validated. We also re-validated our panel to profile antigen specific Th1/2/17 cells or AIMs distribution after peptide stimulation. This optimized multicolor spectral flow panel allows a detailed immune profiling of functional cytokines, AIMs and FoxP3+ Tregs in the T cell compartment from human and might be useful for exploratory analysis of how these functional cytokines or AIMs or Tregs shape host immunity after infection or vaccination. Our panel is highly customizable, allowing researchers to tailor it to their specific requirements.
ER -