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^1,2. Tfh cells are pivotal for mounting humoral immune responses, particularly in vaccine responses and in the defense against bacterial and viral pathogens. Abnormal Tfh cell responses are implicated in various disorders, including autoimmune diseases, immunodeficiency, and potentially some forms of cancer^3,4,5,6. Understanding their function and regulation is, therefore, important for developing effective vaccine and therapeutic strategies⁷.

In contrast to other CD4⁺ T helper cells like Th1, Treg, and Th17, for which in vitro differentiation systems are well-established⁸, Tfh cells are more complex to study because their development involves dynamic cell-to-cell interactions and specialized anatomical structures⁷. Thus, studies of Tfh cells have largely relied on ex vivo phenotyping of human peripheral circulating Tfh cells (cTfh) and in vivo mouse models. Investigating the regulation of specific molecules on Tfh cells is therefore challenging, particularly for human Tfh cells⁹. Thus, an in-depth ex vivo study of Tfh cells derived from lymphoid tissues is essential for enhancing our understanding of human Tfh cells.

Tonsils, as secondary lymphoid organs, harbor unique cell populations that are not present in peripheral blood, such as pre-Tfh (CXCR5^intPD-1^int CD4⁺) and GC-Tfh (CXCR5^hiPD-1^hi CD4⁺) cells, both of which are instrumental in forming GCs. Moreover, the palatine tonsils are some of the most commonly removed tissues in pediatric surgeries and are readily accessible, making them an invaluable human tissue source for exploring complex immune mechanisms involving Tfh cells. Because they are situated in the upper airway, a primary site for respiratory viral infections, the tonsils offer an advantageous tissue for investigating immune responses to such viral infections. For example, our lab previously characterized Tfh cells in tonsils and adenoids of children who had convalesced from COVID-19 and compared these tissues to those from uninfected controls¹⁰. Here, we outline our protocol for processing and storing tonsil cells. Using PP2A inhibitor-treated tonsils as an example, we detail how to characterize these cells through high-dimensional spectral flow cytometry and analyze the data using unsupervised analysis.

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 years and older.

1. Human tonsil collection and single-cell isolation and storage

1. Isolation of tonsillar single cells
   1. Surgical specimen collection
      1. Obtain tonsils from patients undergoing tonsillectomy and collect the fresh tissue pieces in the operating room in a 50 mL centrifuge tube filled with 25-35 mL of ice-cold sterile tonsil medium: RPMI supplemented with 5% heat inactivated fetal bovine serum (FBS), 0.05 mg/mL gentamicin, 10 mM glutamine, and antibiotic-antimycotic mix (100 units/mL streptomycin, 100 units/mL penicillin, and 0.25 µg/mL amphotericin B).
         NOTE: Tonsils in the above medium can be stored at 4 °C overnight and processed the following day. Minimal loss of cell viability was noted with overnight storage versus same-day processing (Figure 1A). Tonsils can also be shipped overnight on ice.
   2. Tonsil processing
      NOTE: All media, buffers, and surgical tools must be sterile, and the tonsil cell isolation should be performed on ice in a biosafety hood.
      1. Place the tonsils on a 60 mm plastic cell culture dish containing 5 mL of chilled tonsil medium. Work on ice. Using sterile tweezers and scissors or a scalpel, carefully remove any visible blood clots, fat, and connective tissue (Figure 1B,C).
      2. Move the tonsils to a new 60 mm cell culture dish with 5 mL of ice-cold tonsil medium. Slice the tonsil pieces into 3-5 mm fragments.
      3. Prepare a new 60 mm cell culture dish with 10 mL of cold tonsil medium and place a 70 μm cell strainer in the dish.
      4. Move the tonsil fragments to the strainer and smash the tonsil fragments by using the plunger end of a sterile 3 mL syringe through the strainer.
      5. Collect the medium containing cells outside the strainer in the dish and transfer to a 50 mL conical tube.
      6. Add an additional 10 mL of tonsil medium to the dish with the strainer, smash the tonsil fragments with the plunger, and collect the medium in the conical tube. For large tonsils, repeat this step 2-3x more until only connective tissue fragments are left in the strainer. Put the remaining tissue and used plates in a biohazard bag and in the medical waste.
      7. Fill the 50 mL conical tube to the top with tonsil medium.
      8. Centrifuge at 400 × g for 5 min at 4 °C.
         NOTE: Extending the centrifugation to 20 min may increase the yield.
      9. Remove the supernatant by carefully suctioning or pipetting out and resuspend the pellet in 5 mL of ammonium-chloride-potassium (ACK) lysing buffer at room temperature (RT) to lyse red blood cells. Incubate for 5 min at RT, then add 35 mL of chilled PBS to prevent further lysis. Centrifuge at 400 × g for 5 min at 4 °C.
      10. Wash once with 50 mL of chilled PBS and centrifuge. Resuspend with 10 mL of PBS and strain cells through a 70 μm plastic strainer. Count cells.
2. Cryopreservation of tonsillar cells
   1. Prepare freezing medium (90% heat-inactivated FBS and 10% dimethyl sulfoxide (DMSO)).
      ​NOTE: This may be stored at 4 °C for up to 1 month.
   2. Centrifuge the cell suspension (step 1.1.2.10) at 400 × g for 5 min at 4 °C. Discard the supernatant and resuspend the cell pellet with freezing medium.
      NOTE: We advise freezing the cells at a density of 10 × 10⁶ to 50 × 10⁶ cells/mL of freezing medium¹¹.
   3. Aliquot 0.5-1 mL of the cell suspension into sterile cryovials. Freeze the vials in rate-controlled freezing containers in a -80 °C freezer. Transfer the vials into a storage tank containing liquid nitrogen within one week for long-term preservation.
3. Thawing frozen cells
   NOTE: A sequential-dilution thawing approach referenced from a sample preparation protocol for single-cell RNA sequencing is outlined below¹². Other thawing methods may also work, but we found greater cell viability with this approach compared with immediately putting cells into warm thaw buffer (Figure 1D). The wash buffer can be prepared ahead of time. Thaw buffer is comprised of wash buffer plus 0.1 mg/mL DNase I (DNase I should be added just prior to starting the thaw). Increasing centrifugation time to 1 min/mL (for 15 mL tubes) is optional to increase the cell yield.
   1. Warm a water bath to 37 °C. Prewarm the thaw and wash buffer in the 37 °C water bath.
      NOTE: All cell washes are performed at RT.
   2. Thaw the cryovials in the 37 °C water bath for 2-3 min immediately after removing from storage. Avoid fully submerging the vial in the water bath. Remove the vial from the water bath while a small ice crystal remains visible.
   3. Gently transfer thawed cells to a 15 mL conical tube using a wide-bore pipette tip in a biosafety hood. Rinse the cryovial with 0.5 mL of prewarmed thawing buffer, and add the rinse drop by drop (1 drop every 5 s) to the 15 mL conical tube, gently shaking the tube to mix.
   4. Dilute cells sequentially in the 15 mL conical tube by incrementally adding 2 mL and then 4 mL of medium with ~1 min waiting time between additions. Add medium at a rate of 1 mL every 3-5 s to the tube, gently swirling to mix.
   5. Centrifuge at 400 × g for 5 min at RT.
   6. Remove the supernatant; resuspend the cell pellet in 300 μL of thawing buffer; and incubate at RT for 5 min.
   7. Add 10 mL of wash buffer (no DNase I). Centrifuge again at 400 × g for 5 min at RT.
   8. Wash one more time: remove the supernatant and resuspend the cell pellet in 10 mL of wash buffer. Centrifuge at 400 × g for 5 min at RT.
   9. Filter the cells through a 70 μm strainer. Centrifuge again for 5 min at 400 × g at RT and count the cells. Resuspend in culture medium.

2. Measuring expression of intracellular cytokines and transcription factors after ex vivo treatment

NOTE: Here, we detail our method for phenotypic characterization of tonsillar Tfh cells using two high-dimensional flow cytometry panels. Given that PP2A activity is essential for optimal BCL-6 protein expression and Tfh differentiation⁴, we illustrate our evaluation of Tfh cell maintenance and functionality through the examination of tonsil cells treated with the PP2A inhibitor cantharidin (CAN).

1. Flow cytometric staining
   1. Intracellular cytokine staining
      NOTE: Incubations are performed in the dark. Reagents, antibodies, and their respective quantities per well used in both panels are listed in the Table of Materials. To assess T cell degranulation, the anti-CD107a antibody is added during the treatment step to minimize false-negative results caused by its internalization¹³.
      1. Plate 2 × 10⁶ tonsil cells with 200 µL of culture medium (1 × 10⁷ cells/mL) in a 96-well U bottom plate well with the following amounts of PP2A inhibitor CAN for 16 h: vehicle control, 3 μM, and 6 μM.
         NOTE: CAN was reconstituted at 0.1 M with 1% DMSO.
      2. Add monensin (0.7 μL/mL), brefeldin (1:1,000), phorbol myristate acetate (50 ng/mL, PMA), ionomycin (1,000 ng/mL), and 2 μL anti-CD107a to each well for 2.5 h in 5% CO₂ incubator at 37 ^oC. Total culture volume is 200 μL per well.
      3. Centrifuge staining antibodies at ~16,000 × g for 3-5 min at 4 °C to remove antibody aggregates. Prepare the surface antibody mix for step 2.1.1.8, which includes all the surface antibodies except the chemokine receptors, Brilliant Stain Buffer Plus, and FACS buffer (volume of each noted in Table of Materials, total 112 μL of mix per reaction).
      4. After completing 2.5 h in culture, wash the cells 2x with 200 μL of ice-cold PBS by centrifuging at 400 × g for 2 min at 4 °C. Between washes, discard the supernatant by inverting the plate quickly. Perform staining in the same plate.
      5. Incubate the cells in 100 μL/well of 1:800 diluted LIVE/DEAD Blue dye in PBS for 15 min at RT. Wash 2x with 200 μL of FACS buffer.
      6. Resuspend the cells with True Stain Monocyte Blocker (5 μL monocyte blocker + 45 μL FACS buffer per well) for 5 min at RT.
      7. Sequentially add chemokine receptor antibodies directly to the staining reaction (first, anti-CXCR3 and anti-CCR7 for 10 min each, followed by a mix of anti-CXCR5 and anti-CCR6 along with 10 μL of Brilliant Stain Buffer Plus for 5 min at RT). Mix well after each addition by pipetting up and down several times. See the Table of Materials for antibody volumes.
      8. Add the surface antibody mix prepared in step 2.1.1.3 directly to the staining reaction and incubate for 30 min at RT. Total staining volume per reaction is 180 μL: 50 μL monocyte blocker in buffer (see step 2.1.1.6) + 18 μL of anti-chemokine receptor antibodies (see step 2.1.1.7) + 112 μL of surface antibody mix (see step 2.1.1.3).
      9. Wash 2x with 200 μL of cold FACS buffer.
      10. Resuspend the cells and incubate with 80 μL/well of paraformaldehyde-based fixation buffer at RT for 20 min.
      11. Add 160 μL of 1x permeabilization buffer to each well, which already contains fixation buffer. Centrifuge the plate at 400 × g for 2 min at RT and wash 1x with 200 μL of permeabilization buffer.
      12. Prepare intracellular cytokine (ICS) antibody mix, which includes anti-cytokine antibodies, Brilliant Stain Buffer Plus, and permeabilization buffer (total volume 50 μL/well, Table of Materials). Incubate the cells with this intracellular cytokine antibody mix at RT for 30 min.
      13. Wash 2x with 200 μL/well permeabilization buffer. Resuspend with 200 μL/well of FACS buffer.
      14. Store the stained cells at 4 °C in the dark and acquire data on a spectral flow cytometer within 24 h to minimize loss of staining intensity¹⁴.
   2. Nuclear transcription factor staining
      1. Culture the cells with CAN as in step 2.1.1.1 (without stimulating the cells).
      2. Add anti-human CD40 antibody (0.5 μg/mL) to the culture medium and culture with cells for 15 min before beginning the staining. If cells are stimulated, add this antibody 15 min before starting the stimulation to prevent the internalization of CD40L upon activation^15,16.
      3. Centrifuge the staining antibodies and prepare the surface antibody mix similar to step 2.1.1.3: surface antibodies in the Table of Materials, 10 μL/well of Brilliant Stain Buffer Plus, and 54.1 μL/well of FACS buffer.
         NOTE: Total staining volume per well is 180 μL: 50 μL of monocyte blocker in buffer + 13 μL of chemokine receptor antibodies + 117 μL of surface antibody mix.
      4. Wash the cells and perform LIVE/DEAD Blue staining and surface staining and washes as in steps 2.1.1.5-2.1.1.8.
         NOTE: The anti-CXCR3 antibody is not included in this panel.
      5. Fix the cells with 80  μL of nuclear transcription factor fixation buffer from FoxP3/Transcription Factor Staining Buffer Set. Incubate for 30 min at RT.
      6. Wash the cells with permeabilization buffer as in step 2.1.1.11.
      7. Prepare the intracellular transcription factor (TF) antibody mix, which includes anti-transcription factor antibodies listed in the Table of Materials, 10 μL of Brilliant Stain Buffer Plus, and 16 μL of permeabilization buffer (total volume 40 μL/well). Incubate the cells with this mix for 1 h at 4 °C.
      8. Wash and acquire on a spectral cytometer as in steps 2.1.1.13-2.1.1.14.

3. Unsupervised analysis of high-dimensional flow cytometry data

NOTE: Software, packages, and their versions used for analysis are listed in the Table of Materials. Screen captures from steps 3.2 to 3.4 are in Supplemental File 1. Exemplar scripts from steps 3.5 to 3.7 are in Supplemental File 2 and Supplemental File 3, steps 3.8 to 3.9 are in Supplemental File 4 and Supplemental File 5. ICS raw files and TF raw files for the scripts are provided in Supplemental File 6 and Supplemental File 7, respectively.

1. Open the flow cytometry analysis software workspace where the ".fcs" files are stored and complete gating as shown in Figure 1E.
2. Right-click on the target node (CD4⁺ T cells in this case). Choose Select Equivalent Nodes.
3. Right-click on the target node again. Select Export/Concatenate Populations.
4. Wait for the Populations: Export or Concatenate window to open, choose CSV-channel values, and choose the destination for exported files. Include all the cells in the Include Events box and select All compensated parameters in the Parameters box. Expand the Advanced Options, type a prefix to rename the files, and click export to export the files to the destination folder (export one .csv file for each sample).
5. Read the exported "*.csv" files with R (one for each sample). Assign a sample ID to each cell and combine cells from all samples into a single data frame.
6. Add or merge metadata (additional information about samples) into the data frame.
7. Randomly downsample an equal number of cells per sample for the following analysis. This step is optional.
   NOTE: In this example, 8,000 CD4⁺ T cells were downsampled from the total CD4⁺ T cells of each condition, representing at least 1/7 of the total number of CD4⁺ T cells.
8. Create Seurat object: interpret Channel values for antibody markers as gene expression levels when creating a Seurat object.
   1. Create Seurat object with the data frame generated from step 3.6. Skip the NormalizeData step and save them directly as "data" assay.
   2. Select the surface markers from the flow panel to use as VariableFeatures in the unsupervised clustering.
   3. Run ScaleData, RunPCA, FindNeighbors, FindClusters, RunUMAP as the standard pipeline introduced from Seurat except the NormalizeData step¹⁷.
   4. Adjust the resolution parameter in the FindClusters step to modify the number of clusters, ensuring they more accurately reflect the underlying biological context.
9. Use visualization tools such as DimPlot and FeaturePlot compatible with the Seurat package. For comparing groups that contain multiple samples per group, calculate the percentage of each cluster on a per-subject basis and perform statistical comparisons. Subsequently, use mathematical models for more sophisticated statistical analyses¹⁰.

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