Efficient Tonsillar T Follicular Helper Cell Processing and Functional Analysis through High-dimensional Flow Cytometry

Summary

Here, we present a protocol for collecting and processing tonsil samples, phenotyping using high-dimensional spectral flow cytometry, and conducting unsupervised analysis.

Abstract

T follicular helper cells (Tfh) are a subset of CD4⁺ T helper cells that aid in B cell isotype switching, germinal center (GC) formation, somatic hypermutation, and affinity maturation, thereby helping to orchestrate adaptive humoral immune responses in secondary lymphoid tissues during infection, autoimmunity, and vaccination. Understanding the development and function of Tfh cells is crucial for designing effective vaccines and developing targeted treatment strategies for diseases involving this population. Human tonsil cells are accessible mucosal lymphoid organs. They offer a unique opportunity to profile distinct immune populations like Tfh and GC cells, interrogate cell-to-cell interactions, evaluate dynamic cellular functions, and uncover their regulatory mechanisms at the organ level. In addition, in vitro cultures of tonsil cells are straightforward to process and enable the assessment of Tfh cell biology under various conditions. Here, we introduce a protocol for collection, isolation, preservation, and culture of human tonsil cells. We also describe two optimized spectral flow cytometry panels designed for in-depth characterization of Tfh cells. Last, using a phosphatase protein 2A (PP2A) inhibitor treatment as an example, we demonstrate the efficiency and power of unsupervised analyses of high-dimensional flow cytometry data, effectively uncovering treatment-induced differences on a high-dimensional scale.

Introduction

T follicular helper cells (Tfh) are a distinct subset of CD4⁺ helper T cells that are essential for the formation and function of germinal centers (GCs) and B cell antibody production. Tfh cells originate from naïve CD4⁺ T cells through interactions with antigen-presenting cells and the integration of intrinsic and extrinsic factors, including T cell receptor signaling, costimulatory interactions with B cells, cytokines, and chemokines¹. Tfh cells express CXCR5 and PD-1, which guide their position within or near the GC. BCL-6 is the master transcription factor critical for Tfh development, maintenance, and function

Protocol

The Code of Ethics of the World Medical Association (Declaration of Helsinki) was followed in all the human research in this study. Human tonsil specimens were obtained from patients with adenotonsillar hypertrophy causing sleep disordered breathing or obstructive sleep apnea, sourced from the Division of Pediatric Otolaryngology, Children's National Hospital, Washington, DC, USA. This study received approval from the Institutional Review Board (IRB) at the Children's National Hospital (IRB protocol number 00009806). Informed consent was signed by the parents or guardians of all enrolled participants, and assent was obtained from minor participants aged 7 year....

Results

To compare overall cell viability, we cut the tonsil into three similar pieces and processed them using our protocol under three conditions: (1) same-day processing, (2) overnight storage at 4 °C, and (3) overnight storage on ice. The viability among these three conditions was quite similar without any significant difference (Figure 1A). We also assessed the impact of the thawing process on the viability of cells. The dropwise thawing method we used resulted in significantly higher cell viab.......

Discussion

In contrast to previous reports that fresh tonsils should be processed within 3 h after surgery¹⁹, we found that fresh tonsil samples could retain their viability when stored at 4 °C and processed within 24 h. To optimize cell viability from tonsil tissue upon thawing from liquid nitrogen storage, we adopted the dropwise thawing method recommended in single-cell sequencing, where high cell viability is greatly valued¹². This approach allowed for an optimized recovery o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Machines and instruments
3 mL Plastic Syringe	BD	309657
96 Well U Bottom Plate	Thermo Scientific	163320
1.2 mL Cryogenic Vials	Corning	430487
Cell strainer 70 µm Nylon	Falcon	352350
Cell Culture Dish (60 mm)	VWR	10062-890
Centrifuge	Thermo Scientific	Sorvall Legend XTR
Benchmark B2000-2 MyBath 2L Digital Water Bath	Benchmark	B2000-2
Fine Scissors - Sharp	F.S.T	14060-11
Plastic Instrument Cases	F.S.T	20830-05
Spectral flow cytometer, Aurora	Cytek		5L 16UV-16V-14B-10YG-8R
Standard Pattern Forceps	F.S.T	11000-13
Standard Pattern Forceps	F.S.T	11000-20
Vi-cell Blu cell viability analyzer	Beckman Coulter	C19201
Reagents
0.5 M EDTA pH 8.0	Quality Biology	351-027-101
2-Mercaptoethanol	Gibco	21985-023
ACK lysing buffer	Gibco	A10492-01
Antibiotic-antimycotic mix (Penicillin-Streptomycin-Amphotericin B Suspension, 100x)	Gibco	15240-062
Brilliant Stain Buffer Plus (RUO)	BD	566385
Cantharidin	Sigma	C7632-25MG
Cytofix Fixation Buffer	BD	554655
Dimethyl sulphoxide (DMSO)	Sigma	D8418-250ml
DNAase I	Roche	10104159001
Fetal Bovine Serum (FBS)	VWR	97068-085
FoxP3/Transcription Factor Staining Buffer Set	eBioscience	00-5523-00
Gentamicin (50 mg/mL)	Gibco	15750-060
Golgiplug	BD	555029
GolgiSTOP	BD	554724
HEPES (1 M)	Gibco	15630-080
Ionomycin	Sigma	I0634-1mg
L-Glutamine (200 mM)	Gibco	25030-081
MEM NEAA (100x)	Gibco	11140-050
Paraformaldehyde 16% solution, EM grade (PFA)	Electron Microscopy  Science	15710	Dilute with PBS
PBS, pH 7.4	Gibco	10010072
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140-122
Phorbol 12-Myristate 13-Acetate (PMA)	Sigma	P8139-1mg
Rate-controlled freezing containers	Millipore	Corning CoolCell FTS30
RPMI (+L-Glutamine)	Gibco	11875-085
Sodium Pyruvate (100 mM)	Gibco	11360-070
True-Stain Monocyte Blocker	BioLegend	426103
Buffers
Culture medium			10% heat-inactivated FBS (VWR), 2 nM glutamine, 0.055 mM 2-mercaptoethanol, 1% penicillin/streptomycin, 1 mM sodium pyruvate, 10 mM HEPES, 1% non-essential amino acids in RPMI (+L-Glutamine)
FACS buffer			2 mM EDTA and 2% heat inactivated FBS in PBS
Freezing medium			90% heat-inactivated FBS and 10% DMSO
Thaw buffer			Wash buffer + 0.1 mg/mL DNaseI
Tonsil medium			RPMI supplemented with 5% heat-inactivated FBS, 10 mM glutamine, 0.05 mg/mL gentamicin, 1% antibiotic-antimycotic mix  (penicillin, streptomycin, and amphotericin B).
Wash buffer			RPMI (+L-Glutamine) supplemented with 10%  heat-inactivated FBS and 10 mM HEPES
Antibodies & other regents for cytokine panel
Biotin anti-human CD107a (LAMP-1) Antibody	BioLegend	328604	2 μL in 200 μL complete culture medium per well. This antibody was added with cells during PMA/ionomycin stimulation.
Live Dead staining	100 μL mix per well
LIVE DEAD Blue	Thermo	L23105	1 : 800, 0.125 μL in 100 μL PBS per well.
Monocyte block	50 μL mix per well
Monocyte blocker buffer	BioLegend	426103	5 μL True Stain monocyte blocker and 45 μL FACS buffer per well
Surface antibodies for cytokine panel
Chemokine receptors mix			μL/well
Anti-human CCR6-BV711	BioLegend	353436	1
Anti-human CCR7-BV421	BioLegend	353208	1
Anti-human CXCR3-PE-Cy5	BD	551128	5
Anti-human CXCR5-BV750	BD	747111	1
Brilliant Stain Buffer Plus (RUO)	BD	566385	10
	18 μL mix per well for chemokine receptors; Add directed to cells with the monocyte block buffer
Other surface antibody mix			μL/well
Anti-human CD103-BUV661	BD	749993	2.5
Anti-human CD14-Spark Blue 550	BioLegend	367148	2.5
Anti-human CD19-Spark NIR 685	BioLegend	302270	2.5
Anti-human CD25-BB515	BD	564467	10
Anti-human CD27- Super Bright 436	Thermo	62-0279-42	5
Anti-human CD3-BV510	BioLegend	344828	2.5
Anti-human CD38 APC-Fire810	BioLegend	303550	1
Anti-human CD4 CF YG584	Cytek	R7-20041	2.5
Anti-human CD45RA-BUV395	BD	740315	0.6
Anti-human CD56-BUV737	BD	612766	3.5
Anti-human CD57-FITC	BioLegend	359604	1.2
Anti-human CD69-BUV563	BD	748764	1
Anti-human CD8-BUV805	BD	612889	1.2
Anti-human FAS-BB700	BD	566542	0.6
Anti-human HLA-DR APC-Fire 750	BioLegend	307658	2
Anti-human PD1-BV785	BioLegend	329929	1.2
Anti-human-CD45-PerCP	Thermo	MHCD4531	1.2
Brilliant Stain Buffer Plus (RUO)	BD	566385	10
FACS buffer			61
			112 μL mix per well for other surface antibodies; Add directly to the monocyte block buffer (50 μL) and chemokine receptor mix (18 μL, 50+18+112 = 180 μL in total per well)
Intracelluar cytokine antibodies for cytokine panel			μL/well
Anti-human Granzyme B-PE	BD	561142	1
Anti-human IL10-PE-Dazzle 594	BioLegend	506812	1
Anti-human IL17A-BV605	BioLegend	512326	1
Anti-human IL2-BV650	BioLegend	500334	3
Anti-human IL21- Alexa Fluor 647	BD	560493	10
Anti-human IL4-PerCP Cy5.5	BD	561234	1
Anti-human Perforin-APC	BioLegend	353312	2.5
Anti-human TNFa-PE-Cy7	BioLegend	502930	0.1
Anti-IFNγ-Pacific Blue	BioLegend	502522	1
SAv-BUV615	BD	613013	0.25
Brilliant Stain Buffer Plus (RUO)	BD	566385	10
1x permeabilization buffer	eBioscience	00-5523-00	19.15
	(50 μL mix per well)
Antibodies & other regents for transcription factor panel
CD40 Antibody, anti-human	Miltenyi Biotec	130-094-133	0.5 μg/mL of culture medium for working concentration
Live Dead staining	100 μL mix per well
LIVE DEAD Blue	Thermo	L23105	1 : 800, 0.125 μL in 100 μL PBS per well.
Monocyte blocker buffer	BioLegend	426103	5 μL True Stain monocyte blocker and 45 μL FACS buffer per well
Surface antibodies for transcription factor panel
Chemokine receptors mix			μL/well
Anti-human CCR6-BV711	BioLegend	353436	1
Anti-human CCR7-BV421	BioLegend	353208	1
Anti-human CXCR5-BV750	BD	747111	1
Brilliant Stain Buffer Plus (RUO)	BD	566385	10
	13 μL mix per well
Other surface antibody mix			μL/well
Anti-human 4-1BB-PE-CY7	BioLegend	309818	2.5
Anti-human CD14-Spark Blue 550	BioLegend	367148	2.5
Anti-human CD19-Spark NIR 685	BioLegend	302270	2.5
Anti-human CD200-PerCP-eFluor 710	ThermoFisher	46-9200-42	2.5
Anti-human CD25-PE-Cy5	BioLegend	302608	5
Anti-human CD3-BV510	BioLegend	344828	2.5
Anti-human CD38-APC-Fire810	BioLegend	303550	1
Anti-human CD4-Pacific Blue	BioLegend	317423	1
Anti-human CD40L-PE	BD	557299	15
Anti-human CD45RA-BUV395	BD	740315	0.6
Anti-human CD56-BUV737	BD	612766	3.5
Anti-human CD69-BUV650	BioLegend	310934	2.5
Anti-human CD8-BUV805	BD	612889	1.2
Anti-human HLA-DR APC-Fire 750	BioLegend	307658	2
Anti-human ICOS-BUV563	BD	741421	1.2
Anti-human OX40-APC	BioLegend	350008	5
Anti-human PD1-BV785	BioLegend	329929	1.2
Anti-human-CD45-PerCP	Thermo	MHCD4531	1.2
Brilliant Stain Buffer Plus (RUO)	BD	566385	10
FACS buffer			54.1
			117  μL mix per well for other surface antibodies; Add directly to the monocyte block  buffer (50 μL) and chemokine receptor mix (13 μL, 50+13+117 = 180 μL in total per well)
Transcription factor antibodies for transcription factor panel	μL/well
Anti-human T-bet-BV605	BioLegend	644817	4
Anti-human Bcl6-PE-CF594	BD	562401	4
Anti-human FoxP3-Alexa Fluor 488	BD	560887	1.6
Anti-human GATA3-BB700	BD	566642	2
Anti-human Ki67-Alexa Fluor 700	BD	561277	0.4
Anti-human Rorgt-Alexa Fluor 647	BD	563620	2
Brilliant Stain Buffer Plus (RUO)	BD	566385	10
1 x Permeabilization buffer	eBioscience	00-5523-00	16
			40 μL mix per well
Software and Packages for unsupervised analysis
	Software/package	Version	Source
1	data.table	1.16.2	The Comprehensive R Archive Network
2	FlowJO	10.9.0	Becton, Dickinson and Company
3	pheatmap	1.0.12	The Comprehensive R Archive Network
4	R	4.3.2	The Comprehensive R Archive Network
5	readxl	1.4.3	The Comprehensive R Archive Network
6	Rstudio	2023.12.1+402	Posit PBC
7	Seurat	5.1.0	The Comprehensive R Archive Network
8	SeuratData	0.2.2.9001	The Comprehensive R Archive Network
9	SpectroFlo	3.0	Cytek
10	tidyverse	2.0.0	The Comprehensive R Archive Network
11	viridis	0.6.5	The Comprehensive R Archive Network