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. 2024 Jun 21;5(2):103119.
doi: 10.1016/j.xpro.2024.103119. Epub 2024 Jun 7.

T-B coculture assay for functional analysis of antigen-specific memory CD4+ T cells

Asgar Ansari  1 , Poonam Coshic  2 , Ashok Sharma  3 , Alessandro Sette  4 , Daniela Weiskopf  4 , Nimesh Gupta  5
Affiliations

T-B coculture assay for functional analysis of antigen-specific memory CD4+ T cells

Asgar Ansari et al. STAR Protoc. .

Abstract

The B cell "help" function of CD4+ T cells is critical in establishing the humoral arm of adaptive immunity. Here, we present a protocol to measure the "help" function of antigen-specific memory T cells using an autologous T-B coculture supplemented with monocytes. We describe steps for cell preparation, human cell sorting, coculture, and a flow cytometry-based assessment of B cell outputs. This protocol demonstrates enhanced sensitivity and proves useful in evaluating T-B collaboration in various contexts of health and disease. For complete details on the use and execution of this protocol, please refer to Ansari et al.1.

Keywords: Cell isolation; Cell-based Assays; Immunology.

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Conflict of interest statement

Declaration of interests N.G. and A.A. are listed as inventor on patent filed by NII that covers the use of described method for vaccine evaluation and adjuvant testing purposes. LJI has filed for patent protection for various aspects of T cell epitope and vaccine design work. D.W. is a consultant for Moderna. A. Sette is a consultant for Gritstone bio, Flow Pharma, Moderna, AstraZeneca, QIAGEN, Fortress, Gilead, Sanofi, Merck, RiverVest, MEDACorp, Turnstone, NA Vaccine Institute, Emervax, Gerson Lehrman Group, and Guggenheim.

Figures

None
Graphical abstract
Figure 1
Figure 1
Sorting strategy and post-sorting purity analysis of memory CD4+ T cells, B cells and monocytes (A) Representative gating strategy for sorting T cells, B cells and Monocytes. PBMCs are selected by plotting FSC-A against SSC-A (Gate-1) followed by doublets exclusion (Gate-2) and gating single live cells by selecting Live Dead eFluor 506 negative cells (Gate-3). CD14+ Monocytes (sorting in Tube-3) and CD20+B cells (sorting in Tube-2) are identified by plotting CD20+ cells against CD14+ cells (Gate-4). Plot CD45RO expression against CD4 expression on cells after excluding monocytes and B cells in Gate-4 and gate memory CD4+ T cells as CD4+CD45RO+ cells (Gate-5) (sorting in Tube-1). (B) Post-sorting purity analysis of CD4+CD45RO+ cells (Tube-1), CD20+ cells (Tube-2) and CD14+ cells (Tube-3).
Figure 2
Figure 2
Gating strategy for the analysis of viable plasmablasts and plasma cells in T-B coculture Representative gating strategy for analyzing the B cell output. Lymphocytes are selected by size (Gate-1) followed by discrimination of single cells (Gate-2) and live cells as Live Dead eFluor 506 negative cells (Gate-3). CD19+ B cells are discriminated from CD3+ T cells as CD19+CD3- cells (Gate-4). Total CD27+ cells are gated to exclude naive B cells (Gate-5) and differentiated plasmablasts and plasma cells are gated as CD20loCD38hi cells (Gate-6). Plasma cells are discriminated from plasmablasts by the expression of CD138 (Gate-7).
Figure 3
Figure 3
Analyzing the plasmablasts differentiation in T-B cocultures Representative FACS plots depicting plasmablast differentiation in T-B cocultures. Plasmablast differentiation was analyzed by flow cytometry after 9 days of coculture stimulated with SARS-CoV-2 spike specific CD4 peptides. No medium was exchanged during incubation. COVID-19 recovered T-B coculture showed plasmablast differentiation, CD20loCD38hi cells within CD27+CD19+ B cells. The coculture of cells from non-infected individuals (pre-pandemic donors) served as negative control of the assay, which showed no detectable plasmablast response.
Figure 4
Figure 4
Measuring the Antibody Secreting Cells (ASCs) in T-B cocultures Representative fluorospot images of IgM, IgG and IgA secreting ASCs in cocultures of pre-pandemic and COVID-19 recovered individuals. Antibody secreting cells (ASCs) were analyzed by Fluorospot assay after 9 days T-B cocultures stimulated with SARS-CoV-2 spike specific CD4 peptides. Appropriately diluted cell suspension was added to anti-IgM, anti-IgG and anti-IgA coated FluoroSpot wells and detected with Fluorochrome conjugated antibodies. The coculture of cells from non-infected individuals (pre-pandemic donors) served as negative control of the assay.
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References

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