Chimerism and phenotypic analysis of intraepithelial and lamina propria T cells isolated from human ileal biopsies after intestinal transplantation

Summary

Understanding immune cell dynamics after intestinal transplantation has provided new insights into human lymphocyte biology. However, isolating and characterizing such cells can be challenging. Here, we provide a protocol to isolate intraepithelial and lamina propria lymphocytes from human ileal biopsies. We describe techniques for flow cytometric analysis and determination of multilineage chimerism and T lymphocyte phenotypes. This protocol can be modified to isolate and analyze lymphocytes from other tissues.

For complete details on the use and execution of this protocol, please refer to Fu et al. (2019)¹ and Fu et al. (2021).²

Graphical abstract

Highlights

• •Isolating intraepithelial and lamina propria immune cells from human ileal biopsies
• •Determination of multilineage chimerism using HLA allele-specific antibodies
• •Phenotypic analysis of T cell subsets using multicolor flow cytometry
• •Adaptable to isolate and analyze immune cells from other human tissues

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Understanding immune cell dynamics after intestinal transplantation has provided new insights into human lymphocyte biology. However, isolating and characterizing such cells can be challenging. Here, we provide a protocol to isolate intraepithelial and lamina propria lymphocytes from human ileal biopsies. We describe techniques for flow cytometric analysis and determination of multilineage chimerism and T lymphocyte phenotypes. This protocol can be modified to isolate and analyze lymphocytes from other tissues.

Before you begin

The protocol below describes the specific steps for isolating intraepithelial and lamina propria immune cells from fresh ileal biopsies from intestinal transplant patients. This protocol can also be adapted to isolate and analyze immune cells from other human tissues (e.g., liver, lung, lymph nodes, spleen). The determination of multilineage chimerism using human leukocyte antigen (HLA) allele-specific antibodies, along with the phenotypic analysis of T cell subsets using multicolor flow cytometry, can be applied to other types of human tissues after transplantation.

Institutional permissions

The studies involved in this protocol were approved by the Columbia University Institutional Review Board (IRB nos. AAAJ5056, AAAF2395, and AAAS7927). All subjects or legal guardians provided their written, informed consent and assent when appropriate. Requests to transfer human biospecimens outside of the organization must be submitted for Columbia’s IRB review and approval.

Prepare and prewarm media and buffers

Timing: 0.5 h

• 1.Prepare the necessary reagents before starting the experiment. Recipes and storage condition details are in materials and equipment.
• 2.Prewarm aliquots of dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), and lamina propria lymphocyte (LPL) buffers in a 37°C water bath.

Note: Each patient sample, which contains 1–4 pieces of human ileal biopsies from one patient at one timepoint, will require 20 mL DTT buffer, 20 mL EDTA buffer, and 25 mL LPL buffer.

Prepare individual incubation environments

Timing: 0.25 h

• 3.Prepare an individual water bath for each patient sample. Add 100–150 mL Millipore water to a 1 L beaker and set the beaker on a hot plate stirrer.
• 4.Place a thermometer inside the beaker and adjust the hot plate settings until the water temperature reaches 37°C.
• 5.Label a sterile 125 mL ventilated flask with identifying information for each sample.
• 6.Add a sterile (autoclaved) stir bar to each flask.

Prepare antibody panel

Timing: 0.25–0.5 h

• 7.Prepare the flow cytometry antibody panel (Table 1).

Note: Primary antibodies should be prepared as a master mix immediately before staining samples. This preparation can occur during the LPL buffer incubation. The primary antibody master mix should exclude Fc block, any secondary antibody (Streptavidin BUV737 is the secondary antibody in the Pt25 multilineage panel), and DAPI. Details on staining panel and dilution factors are in materials and equipment.

Note: Antibody panels should be tailored to include donor and/or recipient-specific HLA antibodies and pan-HLA class I (HLA-ABC) antibody for each patient to determine chimerism after transplantation. The 18-color antibody panel is adjusted depending on the patient and which immune cell populations are prioritized. For example, in the Pt25 multilineage panel, we often alternate between recent thymic emigrant (RTE) marker CD31³ and NK cell marker CD56.⁴

Table 1.

Primary surface antibody staining panel: Pt25 multilineage panel

Marker	Fluorophore	Clone
CD45	PE-CF594	HI30
CD19	BUV496	SJ25C1
CD3	PerCP-Cy5.5	SP34-2
CD4	redFluor 710	OKT4
CD8	APC-Cy7	SK1
γδTCR	PE-Cy7	IMMU510
CD45RA	BV510	HI100
CD197 (CCR7)	PE	G043H7
CD56	BV605	HCD56
CD69	BV650	FN50
CD103	BV711	Ber-ACT8
CD49a	BUV395	SR84
CD28	Pacific Blue	CD28.2
CD314 (NKG2D)	APC	1D11
HLA-A2, A28	Biotin	N/A
HLA-B12	FITC	REA138
HLA-ABC	BV786	G46-2.6

For Pt25, HLA-A2, 28 Biotin and HLA-B12 FITC are used to distinguish recipient and donor cells, respectively. For HLA-A2, A28 Biotin, the secondary antibody Streptavidin BUV737 completes the staining. For other patients, where only a donor or recipient HLA antibody is available and can clearly distinguish donor and recipient cells, FITC or Biotin-Streptavidin-BUV737 channels in this panel may be used to stain for other markers, such as using CD14 FITC to capture monocyte chimerism. Adjustments, such as alternating between CD31 BV605 and CD56 BV605 in the 18-color antibody staining panel for samples, allows for the study of different cell populations.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse monoclonal anti-Human CD3 (clone SK7) APC-Cy7	BD Biosciences	Cat#561800; RRID: AB_396890
Mouse monoclonal anti-Human CD3 (clone SP34-2) PerCP-Cy5.5	BD Biosciences	Cat#552852; RRID: AB_394493
Mouse monoclonal anti-Human CD4 (clone OKT4) AF700	Tonbo	Cat#80-0048; RRID: AB_2621976
Mouse monoclonal anti-Human CD8 (clone SK1) APC-Cy7	BD Biosciences	Cat#561945; RRID: AB_396892
Mouse monoclonal anti-Human CD14 (clone M5E2) APC-Cy7	BioLegend	Cat#301820; RRID: AB_493695
Mouse monoclonal anti-Human CD14 (clone M5E2) FITC	BioLegend	Cat#982502; RRID: AB_2616906
Mouse monoclonal anti-Human CD19 (clone SJ25C1) BUV496	BD Biosciences	Cat#612938; RRID: AB_2870221
Mouse monoclonal anti-Human CD21 (clone Bu32) PerCP-Cy5.5	BioLegend	Cat#354908; RRID: AB_2561544
Mouse monoclonal anti-Human CD24 (clone ML5) BUV395	BD Biosciences	Cat#563818; RRID: AB_2632389
Mouse monoclonal anti-Human CD27 (clone O323) BV711	BioLegend	Cat#302834; RRID: AB_2563809
Mouse monoclonal anti-Human CD28 (clone CD28.2) Pacific Blue	BioLegend	Cat#302928; RRID: AB_10641279
Mouse monoclonal anti-Human CD31 (clone WM-59) BV605	BD Biosciences	Cat#562855; RRID: AB_2737841
Mouse monoclonal anti-Human CD33 (clone P67.6) APC-Cy7	BioLegend	Cat#366614; RRID: AB_2566416
Mouse monoclonal anti-Human CD38 (clone HIT2) PE-Cy7	BioLegend	Cat#303516; RRID: AB_2072782
Mouse monoclonal anti-Human CD45 (clone HI30) PE-CF594	BD Biosciences	Cat#562279; RRID: AB_11154577
Mouse monoclonal anti-Human CD45 (clone HI30) V500	BD Biosciences	Cat#560777; RRID: AB_1937324
Mouse monoclonal anti-Human CD45RA (clone HI100) Brilliant Violet 510	BioLegend	Cat#304142; RRID: AB_2561947
Mouse monoclonal anti-Human CD45RB (clone MEM-55) PE	BioLegend	Cat#310204; RRID: AB_314807
Mouse monoclonal anti-Human CD49a (clone SR84) BUV395	BD Biosciences	Cat#742363; RRID: AB_2740721
Mouse monoclonal anti-Human CD56 (clone HCD56) Brilliant Violet 605	BioLegend	Cat#318334; RRID: AB_2561912
Mouse monoclonal anti-Human CD69 (clone FN50) Brilliant Violet 650	BioLegend	Cat#310934; RRID: AB_256315
Mouse monoclonal anti-Human CD103 (clone Ber-ACT8) Brilliant Violet 711	BioLegend	Cat#350222; RRID: AB_2629651
Mouse monoclonal anti-Human CD138 (clone MI15) redFluor 710	Thermo Fisher Scientific	Cat#46138842; RRID: AB_2815146
Mouse monoclonal anti-Human CD197 (CCR7) (clone G043H7) PE	BioLegend	Cat#353204; RRID: AB_10913813
Mouse monoclonal anti-Human CD314 (NKG2D) (clone 1D11) APC	BioLegend	Cat#320808; RRID: AB_492962
Mouse monoclonal anti-Human CD326 (clone 9C4) APC-Cy7	BioLegend	Cat#324246; RRID: AB_2783194
Mouse monoclonal anti-Human HLA-A2/A28 (clone N/A) Biotin	One Lambda	Cat#BIH0037; RRID: N/A
Mouse monoclonal anti-Human HLA-A2, A28 (clone REA142) FITC	Miltenyi Biotec	Cat#130-099-601; RRID: AB_2652040
Mouse monoclonal anti-Human HLA-A2 (clone BB7.2) FITC	BioLegend	Cat#343304; RRID: AB_1659245
Mouse monoclonal anti-Human HLA-A3 (clone GAP.A3) APC	Thermo Fisher Scientific	Cat#17-5754-42; RRID: AB_2573220
Mouse monoclonal anti-Human HLA-A9 (clone N/A) Biotin	One Lambda	Cat#BIH0964; RRID: N/A
Mouse monoclonal anti-Human HLA-A9 FITC (clone N/A)	One Lambda	Cat#FH0964; RRID: N/A
Mouse monoclonal anti-Human HLA-A29 (clone N/A) Biotin	One Lambda	Cat#BIH0155; RRID: N/A
Mouse monoclonal anti-Human HLA-ABC (clone G56-2.6) APC	BD Biosciences	Cat#555555; RRID: AB_398603
Mouse monoclonal anti-Human HLA-ABC (clone G46-2.6) BV786	BD Biosciences	Cat#740982; RRID: AB_2740606
Mouse monoclonal anti-Human HLA-B8 (clone N/A) FITC	One Lambda	Cat#FH0536A; RRID: N/A
Mouse monoclonal anti-Human HLA-B12 (clone REA138) FITC	Miltenyi Biotec	Cat#130-099-860; RRID: AB_2652098
Mouse monoclonal anti-Human HLA-Bw6 (clone REA143) APC	Miltenyi Biotec	Cat#130-099-845; RRID: AB_2652026
Mouse monoclonal anti-Human IgA (clone IS11-8310) APC	Miltenyi Biotec	Cat#130113472; RRID: AB_1036150
Mouse monoclonal anti-Human IgD (clone IA6-2)	BD Biosciences	Cat#563313; RRID: AB_2738134
Mouse monoclonal anti-Human IgG (clone G18-145) V450	BD Biosciences	Cat#561299; RRID: AB_10611575
Mouse monoclonal anti-Human IgM (clone G20-127) PE-CF594	BD Biosciences	Cat#562539; RRID: AB_2737641
Streptavidin, Alexa Fluor 594 Conjugate	Thermo Fisher Scientific	Cat#S32356; RRID: N/A
Streptavidin BUV737	BD Biosciences	Cat#564293; RRID: AB_2869560
Mouse monoclonal anti-Human γδTCR (clone IMMU510) PE-Cy7	Beckman Coulter	Cat#B10247; RRID: N/A
Biological samples
Patient intestinal biopsies	N/A	N/A
Buffycoats	New York Blood Center	N/A
Chemicals, peptides, and recombinant proteins
AIM V Medium	Thermo Fisher Scientific	Cat#12055-091
Bovine Serum Albumin, Heat Shock Treated	Thermo Fisher Scientific	Cat#BP1600-100
Collagenase D (2.5 G)	Roche Applied Science	Cat#11088882001
4′6-diamidino-2-phenylindole (DAPI)	Sigma-Aldrich	Cat#D9542-10MG
Dimethyl sulfoxide (DMSO)	Millipore Sigma	Cat#D2438-5X10ML
DL-Dithiothreitol solution, ∼1 M in H₂O	Sigma-Aldrich	Cat#43816
0.5 M EDTA, pH 8.0	Thermo Fisher Scientific	Cat#15575020
Hanks’ Buffered Saline Solution (HBSS) (1×), without phenol red, calcium or magnesium	Lonza	Cat#10-547F
HBSS (10×), no calcium, no magnesium, no phenol red	Thermo Fisher Scientific	Cat#14185052
HEPES, 1 M	Thermo Fisher Scientific	Cat#15630080
Human Serum AB	GeminiBio	Cat#100512
2-Mercaptoethanol, 14.3 M	Sigma-Aldrich	Cat#M3148
Phosphate Buffered Saline (PBS) (1×)	Thermo Fisher Scientific	Cat#SH3025602
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	Cat#15140122
RPMI 1640, 1× without L-glutamine	Thermo Fisher Scientific	Cat#MT15040CV
Sodium azide	Sigma-Aldrich	Cat#S2002
Sodium hydroxide, 1.0 N Standardized Solution	Thermo Fisher Scientific	Cat#AA35629K2
Trypan Blue Solution, 0.4%	Fisher Scientific	Cat#15250061
Software and algorithms
FlowJo	BD Biosciences	www.flowjo.com (v9.9.6 and v10.8.0)
Other
BD LSR II Flow Cytometer	BD Biosciences
Cell Strainers, 40 μm	Thermo Fisher Scientific	Cat#08-771-1
125 mL Erlenmeyer Flask (Flat Base)	TriForest Labware	Cat#FPC0125S
Magnetic Stir Bars	Thermo Fisher Scientific	Cat#14-513-60SIX
Sterile Disposable Filter Units with PES Membranes	Thermo Fisher Scientific	Cat#09-741-02

Materials and equipment

Reconstituted Collagenase D

Reagent	Final concentration	Amount
Hanks’ Buffered Saline Solution (HBSS) (1×), without phenol red, calcium or magnesium	N/A	49 mL
Collagenase D (2.5 G)	50 mg/mL	2.5 g
Total	N/A	50 mL

Note: Reconstitute Collagenase D in HBSS (1X) and filter through a 0.22 μm sterile vacuum filter before use. Although the manufacturer (Roche Diagnostics, Mannheim, Germany) recommends the reconstitution of only the amount of lyophilizate needed for immediate use, our practice has shown that reconstituted Collagenase D aliquotted in 1.5 mL microcentrifuge tubes (0.5 mL/tube and 1 mL/tube) can be stored at –20°C for at least 2 months. Thaw new aliquots each time for LPL buffer preparation. Avoid repeated freezing and thawing.

Prepared 2-Mercaptoethanol (2-ME)

Reagent	Final concentration	Amount
HBSS (1×), without phenol red, calcium or magnesium	N/A	28.45 mL
2-ME, 14.3 M	25 mM	0.05 mL
Total	N/A	28.5 mL

Note: Reconstitute the entire vial of 2-Mercaptoethanol in HBSS (1X) and aliquot into 1.5 mL microcentrifuge tubes (1 mL/tube). Prepared 2-ME can be stored at –20°C for at least 2 months. Thaw fresh aliquots each time for MLR media preparation.

CRITICAL: 2-ME is combustible as a liquid or vapor, with a very low odor threshold (0.12–0.64 ppm), and can be toxic if ingested and fatal if inhaled or absorbed through the skin. 2-ME preparation should be performed in the chemical fume hood.

DTT Buffer

Reagent	Final concentration	Amount
HBSS (10×), no calcium, no magnesium, no phenol red	1×	5 mL
Penicillin-Streptomycin (10,000 U/mL)	100 U/mL	0.5 mL
0.5 M EDTA, pH 8.0	5 mM	0.05 mL
DL-Dithiothreitol solution, ∼1 M in H2O	0.2 mM	0.1 mL
Sterile dH2O	N/A	44.35 mL
Total	N/A	50 mL

Note: Autoclave Millipore water to obtain sterile dH₂O. Filter buffer through a 0.22 μm sterile vacuum filter. DTT buffer can be prepared in advance in a 500 mL volume and stored at 4°C for at least 2 months. Aliquot the necessary amount of buffer and prewarm at 37°C before use.

EDTA Buffer

Reagent	Final concentration	Amount
HBSS 10× (no calcium, no magnesium, no phenol red)	1×	5 mL
Penicillin-Streptomycin (10,000 U/mL)	100 U/mL	0.5 mL
0.5 M EDTA, pH 8.0	5 mM	0.05 mL
Sterile dH2O	N/A	44.45 mL
Total	N/A	50 mL

Note: Autoclave Millipore water to obtain sterile dH₂O. Filter buffer through a 0.22 μm sterile vacuum filter. EDTA buffer can be prepared in advance at a 500 mL volume and stored at 4°C for at least 2 months. Aliquot the necessary amount of buffer and prewarm at 37°C before use.

LPL Buffer

Reagent	Final concentration	Amount
RPMI 1640, 1× without L-glutamine	N/A	48.5 mL
Penicillin-Streptomycin (10,000 U/mL)	100 U/mL	0.5 mL
Reconstituted Collagenase D (50 mg/mL)	1 mg/mL	1 mL
Total	N/A	50 mL

Note: Collagenase D reconstitution details are above. Filter buffer through a 0.22 μm sterile vacuum filter. LPL buffer base (consisting of Penicillin-Streptomycin and RMPI 1640, 1× without L-glutamine) can be prepared in advance at a 490 mL volume and stored at 4°C for at least 2 months. Add a freshly thawed aliquot of reconstituted Collagenase D to an aliquot of the necessary amount of LPL buffer base needed each time and prewarm at 37°C before use.

Mixed Lymphocyte Reaction (MLR) Media

Reagent	Final concentration	Amount
AIM-V Medium	N/A	470 mL
Human Serum AB	5%	25 mL
HEPES, 1 M	10 mM	5 mL
Prepared 2-ME (25 mM)	50 μM	1 mL
Total	N/A	500 mL

Note: Heat inactivate the human serum before use. 2-ME needs to be prepared as detailed above. Filter media through a 0.22 μm sterile vacuum filter before use. MLR media can be prepared in advance and stored at 4°C for at least 2 months. Aliquot the necessary amount of buffer and prewarm at 37°C before use.

Fluorescence-Activated Cell Sorting (FACS) Buffer

Reagent	Final concentration	Amount
HBSS 10× (no calcium, no magnesium, no phenol red)	1×	100 mL
Millipore H2O	N/A	900 mL
Sodium hydroxide (NaOH), 1.0N Standardized Solution	210 μN	0.210 mL
Sodium azide (NaN3)	1 mg/mL	1 g
Bovine Serum Albumin, Heat Shock Treated	1 mg/mL	1 g
Total	N/A	1 L

Note: Add HBSS 10× and Millipore H₂O first before adding the remaining reagents. NaOH is used to adjust the pH to 7.0–7.2. NaN₃ is added as a preservative. FACS buffer can be prepared in advance and stored at 4°C for at least 4 months.

Trypan Blue Live/Dead Viability Dye

Reagent	Final concentration	Amount
Trypan Blue Solution, 0.4%	0.2%	10 mL
Phosphate Buffered Solution (1×), no calcium, no magnesium	N/A	10 mL
Total	N/A	20 mL

Note: Trypan Blue viability dye can be prepared in advance and stored at 21°C for at least 2 months.

DAPI Stock Solution

Reagent	Final concentration	Amount
DAPI powder	5 mg/mL	10 mg
Dimethyl sulfoxide (DMSO)	N/A	2 mL
Total	N/A	2 mL

Note: Add 2 mL DMSO to the DAPI vial to create a 14.3 mM (5 mg/mL) DAPI stock solution, making sure the DAPI is thoroughly dissolved. DAPI stock solution can be prepared in advance and stored in the dark at −20°C for at least 6 months.

DAPI Staining Solution

Reagent	Final concentration	Amount
DAPI Stock Solution (5 mg/mL)	2 μg/mL	2 μL
FACS Buffer	N/A	5 mL
Total	N/A	5 mL

Note: FACS Buffer should be prepared as detailed above. Add 2 μL of the DAPI stock solution (details above) to 5 mL FACS Buffer. Vortex and then filter through a 0.22 μm sterile vacuum filter before use. DAPI staining solution can be prepared in advance and stored in the dark at 4°C for at least 6 months.

Surface antibody staining panel: Pt25 multilineage panel (enough for up to 1.5 × 10⁶ live cells, which includes up to 0.5 × 10⁶ intraepithelial lymphocytes (IELs) and up to 0.5 × 10⁶ LPLs from a one-time ileoscope sample that includes 1–4 biopsy pieces from the same patient, and 0.5 × 10⁶ healthy control peripheral blood mononuclear cells (PBMCs).

Reagent	Final concentration	Amount
CD45 (PE-CF594)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:64 titer
CD19 (BUV496)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:16 titer
CD3 (PerCP-Cy5.5)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:16 titer
CD4 (AF700)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:8 titer
CD8 (APC-Cy7)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:16 titer
γδTCR (PE-Cy7)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:4 titer
CD45RA (BV510)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:64 titer
CD197/CCR7	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:16 titer
CD56 (BV605)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:4 titer
CD69 (BV650)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:4 titer
CD103 (BV711)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:16 titer
CD49a (BUV395)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:4 titer
CD28 (Pacific Blue)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:16 titer
CD314/NKG2D (APC)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:4 titer
HLA-A2, A28 (Biotin)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:4 titer
HLA-B12 (FITC)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:2 titer
HLA-ABC (BV786)	Additional 27.5× dilution of designated titer	15 μL from diluted stock at 1:16 titer
Streptavidin (BUV737)	Additional 11× dilution of designated titer	15 μL from diluted stock at 1:32 titer
DAPI staining solution	Approximately at 0.1 μg/mL	7.5 μL from the 2 μg/mL stock
Total	N/A	255 μL for 17 colors without secondary antibody and DAPI

Note: Freshly prepare the antibody panel each time. For HLA-A2, A28 Biotin, the secondary antibody Streptavidin BUV737 is used. For each antibody with a new manufacturer lot number, an antibody titration should be performed to determine an appropriate titer for that specific lot. Then, the stock antibody can be diluted with FACS buffer to create aliquots at the designated titer for staining and sorting purposes.

Step-by-step method details

IELs and LPLs were separated either from intestinal graft biopsy specimens or surgically obtained intestinal graft specimens at the time of stoma closure/revision, according to a protocol adapted from previous reports⁵ and used in our previous studies.^1,2,6

Isolating intraepithelial lymphocytes

Timing: 1 h

These steps isolate IELs from patient samples. The epithelial integrity of ileal biopsy is disrupted by DTT, a reducing agent, and EDTA, a calcium chelator that targets calcium-dependent junctions and promotes the detachment of intestinal epithelial cells (IECs). The DTT and EDTA incubation steps liberate IELs and IECs into the medium without altering the basement membrane.⁷ Supernatant collected from both steps are combined to enrich IELs.

• 1.Collect research ileoscope samples from consented patients during clinical endoscopy. Each patient sample consists of 1–4 ileal biopsy pieces, ideally around 4–6 mm³ per piece, from one patient at one timepoint (Figure 1A) (troubleshooting 1).

CRITICAL: Ileal biopsies should be processed the same day they are collected, and no later than the next day. If a specimen will not be immediately processed, it should be stored in a sterile vial with saline solution or MLR media at 4°C.

• 2.For each patient sample, add 20 mL of prewarmed DTT buffer to a sterile 125 mL flask.
• 3.Transfer the ileal biopsy piece(s) into the 125 mL flask, ensuring each piece is floating in the DTT buffer (Figure 1B).
• 4.Place the flask into the prewarmed water bath beaker set on the hot plate stirrer.

Note: The water level in the beaker should be above the buffer level in the flask but below the flask cap (Figure 1C). If there is a large volume of buffer being used, such as when processing larger pieces of tissue, this is not necessary. The flask should sit still in the beaker. If it is floating, consider adding a weight on top of the flask.

Note: A 37°C incubator with agitation could be used for each incubation, but the beaker with hot plate stirrer may be optimal: because a stir bar is better at ensuring the small biopsy pieces move around in the buffer for efficient cell isolation. Additionally, each hotplate stirrer functions independently. When processing different tissue samples from multiple patients at once, it is useful to be able to adjust each incubation setting on its own.

• 5.Incubate biopsy pieces in DTT buffer with constant stirring, 125–150 rpm, for 20 min at 37°C.

Note: If processing larger pieces of tissue, the stirring rate can be increased to 150–250 rpm.

• 6.Allow pieces to settle to the bottom of the flask and filter the DTT cell suspension through a 40 μm strainer into a 50 mL conical tube (Figure 1D).
• 7.Centrifuge the DTT cell suspension at 465 × g for 5 min at 4°C. Pour off the supernatant to retain the cell pellet.
• 8.Add 5–10 mL MLR media to the cell pellet in the conical tube and keep it on ice.
• 9.Add 20 mL prewarmed EDTA buffer to the flask (Figure 1E).
• 10.Incubate biopsy pieces in EDTA buffer with constant stirring, 150–200 rpm, for 30 min at 37°C.

CRITICAL: Between incubation steps, biopsy pieces can adhere to the flask wall while the cell suspension from the previous incubation is poured through the cell strainer. If the tissue pieces stay in this position during subsequent incubations, they will dry up and cell viability and yield will be negatively impacted. Thus, it is important to ensure each biopsy piece is re-submerged in the new buffer before proceeding with the next incubation.

• 11.Allow pieces to settle to bottom of the flask and filter the EDTA cell suspension through 40 μm strainer into the 50 mL conical tube that already has the DTT cell suspension (Figure 1F).
• 12.Centrifuge the DTT and EDTA cell suspension at 465 × g for 5 min at 4°C.
• 13.Pour off the supernatant to retain the cell pellet.
• 14.Resuspend the cell pellet in 1 mL MLR media, transfer to a 5 mL flow cytometry tube with a cap, and keep on ice or in a refrigerator at 4°C.

Figure 1.

Isolating intraepithelial lymphocytes

(A) Collect research samples, which consist of 1–4 ileal biopsy pieces from consented patient during clinical endoscopy.

(B) Place the sample into the DTT buffer in the labeled 125 mL ventilated flask a with sterile stir bar.

(C) Place the flask into the prewarmed water bath beaker at 37°C, ensuring the water level in the beaker is above the buffer level in the flask.

(D) Filter DTT cell suspension through 40 μm single-cell strainer into 50 mL conical tube. Centrifuge the cell suspension, pouring off the supernatant after and resuspending the pellet in 5 mL MLR media.

(E) Add 20 mL prewarmed EDTA buffer to the flask. Ensure the sample is floating in the buffer.

(F) Filter EDTA cell suspension through 40 μm single-cell strainer into the 50 mL conical tube from the previous DTT step with MLR media.

Isolating lamina propria lymphocytes

Timing: 1.25 h

These steps subsequently isolate LPLs from the patient samples. The lamina propria regions of ileal biopsies are disrupted by the collagenase D treatment.⁷

• 15.Add 25 mL of prewarmed LPL buffer to the 125 mL flask, ensuring the biopsies are submerged in the LPL buffer (Figure 2A).
• 16.Place the flask back into the beaker and incubate with constant stirring, 150–200 rpm, at 37°C for 1–1.5 h.

CRITICAL: During longer incubation periods, it is especially important to periodically monitor the water bath temperature to ensure it does not exceed 37°C, as incubating the tissue at higher temperatures may reduce cell viability and yield. Adjust the hotplate settings if the temperature begins to climb.

• 17.Pour the LPL cell suspension through a 40 μm strainer into a new 50 mL conical tube (Figure 2B).
• 18.Wash the sides of the 125 mL flask with 25 mL MLR media and pour through a 40 μm strainer into the 50 mL conical tube that already has the LPL cell suspension (Figure 2C).
• 19.Centrifuge at 465 × g for 5 min at 4°C.
• 20.Pour off supernatant to retain cell pellet.
• 21.Resuspend the cell pellet in 1 mL MLR media, transfer to a new 5 mL flow cytometry tube with a cap, and keep on ice or in a refrigerator at 4°C.

Note: Collagenase D function is calcium-dependent, and its activity is inhibited by low temperature and reagents such as EDTA, DTT, and 2-ME.

Pause point: The experiment can be paused at this point, ideally for no longer than 4 h, before proceeding to flow cytometry staining. However, proceeding with the antibody staining steps sooner is suggested to ensure better viability of cells.

Figure 2.

Isolating lamina propria lymphocytes

(A) Add 25 mL prewarmed LPL buffer to the flask, ensuring the sample is in the buffer.

(B) Filter LPL cell suspension through 40 μm single-cell strainer into new 50 mL conical tube.

(C) Wash the sides of the 125 mL flask with 25 mL MLR media and filter contents through 40 μm single-cell strainer into the same 50 mL conical tube with the LPL cell suspension.

Antibody staining for flow cytometric analysis

Timing: 1.25 h

These steps stain the IELs and LPLs isolated from patient samples with various antibodies in an 18-color panel (troubleshooting 2).

Prior to antibody staining and acquisition of patient samples, fluorescence compensation of the antibody panel must be performed on the flow cytometer that will be used for cell acquisition (in this case, the BD LSR II). This is critical because different fluorophores can have partially overlapping emission spectra, and it is necessary to correct for this fluorescence spillover.

To perform fluorescence compensation, stain single color controls (0.5 × 10⁶ PBMCs isolated from a healthy human donor per control) for each fluorophore (general ratio for each Ab is 10 μL diluted Ab at pre-determined titer per 1 × 10⁶ cells, where the cells are already resuspended at a ratio of 10 × 10⁶ cells/mL in FACS buffer), in addition to an unstained control (troubleshooting 3). When acquiring each single-color control on the flow cytometer, adjust voltage settings such that the target fluorophore displays the highest mean fluorescence intensity (MFI) in comparison to the spillover seen in the other fluorophores to minimize ambiguity on the origin of fluorescence and compensate the full panel.

• 22.Add 1 mL FACS buffer to each flow cytometry tube.
• 23.Centrifuge samples at 465 × g for 5 min at 4°C.
• 24.Pour off the supernatant and, keeping the tube inverted, quickly touch tube rim with a clean Kim Wipe to absorb away any last drops of supernatant.
• 25.Resuspend cells at 10 × 10⁶ cells/mL in FACS buffer.

Note: For samples around the ideal size, it can be assumed that there are 0.5 × 10⁶ cells in each IEL and LPL flow cytometry tube. This cell count for antibody staining is an overestimation of the targeted CD45⁺ cell population based on our previous flow cytometry data from patient biopsies, where around 2,000–100,000 CD45⁺ cells were obtained from up to 4 ileal biopsy pieces around the ideal size described earlier. Given that the target cell number from these biopsies is low, we omit the regular counting step to save more cells for chimerism and phenotypic analysis.

Note: When larger tissue pieces are processed and stained (for example, from a stoma revision/closure, or graft explant), perform cell counting and adjust the amount of each antibody needed for staining.

• 26.Stain each cell sample with Fc block, pipetting up and down to mix.

Note: The general ratio for Fc block staining is 5 μL Fc block per 1 × 10⁶ cells, where the cells are already resuspended at a ratio of 10 × 10⁶ cells/mL in FACS buffer.

• 27.Incubate at room temperature for 5 min.
• 28.Stain each sample with the prepared primary antibody master mix.

Note: The general ratio for antibody staining is 10 μL diluted of each Ab at a pre-determined titer per 1 × 10⁶ cells, where the cells are already resuspended at a ratio of 10 × 10⁶ cells/mL in FACS buffer. In the situation of a 18-color panel staining (including DAPI) where the Ab cocktail volume (5 μL × 17 = 85 μL) is greater than the volume of FACS buffer (50 μL) used to reconstitute 0.5 × 10⁶ cells at 10 × 10⁶/mL, at least a 2-fold lower dilution should be used of each Ab (e.g.: 1:4) rather than the titer selected at the time of Ab titration of a single color panel (e.g., 1:8).

• 29.Incubate for 30 min at 4°C in the dark, ideally in a fridge at 4°C. However, placing the cell samples in a bucket of ice with a lid should also work.
• 30.Add 1 mL FACS buffer to each flow cytometry tube.
• 31.Centrifuge samples at 465 × g for 5 min at 4°C.
• 32.Pour off the supernatant and, keeping the tube inverted, quickly touch tube rim with a clean Kim Wipe to absorb away any last drops of supernatant.
• 33.Reconstitute cells at 10 × 10⁶/mL in FACS buffer (add 50 μL FACS buffer to 0.5 × 10⁶ cells per tube).
• 34.Stain each sample with the secondary antibody (Streptavidin BUV737 is the secondary antibody in the Pt25 multilineage panel) and incubate for 15 min at 4°C in the dark.

Note: The general ratio for antibody staining is 10 μL diluted Ab at a pre-determined titer per 1 × 10⁶ cells, where the cells are already resuspended at a ratio of 10 × 10⁶ cells/mL in FACS buffer.

• 35.Add 1 mL FACS buffer to each flow cytometry tube.
• 36.Centrifuge samples at 465 × g for 5 min at 4°C.
• 37.Pour off the supernatant. Keeping the tube inverted, quickly touch tube rim with a clean Kim Wipe to absorb away any last drops of supernatant.
• 38.Reconstitute cells at 10 × 10⁶/mL in FACS buffer (add 50 μL FACS buffer to 0.5 × 10⁶ cells per tube).
• 39.Stain each sample with DAPI, pipetting up and down to mix.

Note: The general ratio for DAPI staining is 5 μL of DAPI staining solution (2μg/mL stock) per 1 × 10⁶ cells, where the cells are already resuspended at a ratio of 10 × 10⁶ cells/mL in FACS buffer.

• 40.Keep cell samples at 4°C in the dark before proceeding to next steps.

Pause point: The experiment can be paused for up to 12 h at this point before proceeding. Samples should be kept at 4°C in the dark during this period.

Data collection

Timing: 1 h

These steps acquire flow cytometry data from the prepared patient samples for downstream analysis.

• 41.Collect data on the flow cytometer (LSR II, BD Biosciences). Prior to acquiring patient samples, fluorescence compensation must be performed to adjust settings on the flow cytometer. Analyze data with FlowJo v9.9.6 and v10.8.0 software (FlowJo, LLC).

CRITICAL: Prior to acquiring patient samples, HLA quality control (QC) should be performed on the flow cytometer using pre-transplant donor and recipient PBMCs or lymphoid tissue cells with known HLA typing. This is to determine appropriate commercially available HLA antibodies that work best for chimerism tracking on post-transplant specimens.

• 42.PBMCs isolated from a healthy human donor can serve as a staining control. They are stained with the same antibodies and then acquired on the flow cytometer alongside patient sample(s) (troubleshooting 4).

Note: The control helps determine chimerism gating as well as resting stage phenotypic markers on circulating T cell subsets during flow cytometry analysis.

• 43.When analyzing the flow cytometry data, gate on the control sample first, and then apply those gates to the patient samples. Lineage markers and chimerism gates can then be adjusted as needed, based on the contour plot distribution (troubleshooting 5).

Expected outcomes

This protocol outlines the isolation of live immune cells, especially IELs and LPLs, from human ileal biopsies for analysis of multilineage chimerism and phenotypes of T cells. With this protocol, donor and/or recipient-derived immune cells can be defined using HLA-allele-specific antibodies versus pan-HLA-ABC. This includes T cells (CD3⁺), B cells (CD19⁺), NK cells (CD56⁺) and myeloid cells (CD14⁺). Different T cell subsets such as CD4⁺ and CD8⁺ αβ T cells, γδTCR⁺ T cells, and their functional phenotypes, naïve and memory T cells (CD45RA vs CCR7 to categorize naïve, central memory, effector memory and terminally-differentiated effect memory T cells), tissue-resident memory T cells (TRM: CD69⁺ CD103^+/− CD49a^+/−), effector T cells (CD28⁺ NKG2D^+/−), and recent thymic emigrants (RTEs: CD3⁺ CD45RA⁺ CD31⁺), can be determined as previously described,^1,2,6,8,9 and as illustrated in an gating example of ileal IEL of Pt25 POD241 (Figure 3).

Figure 3.

Gating strategy for multilineage chimerism, as well as donor and recipient T cell phenotypes, in post-transplant ileal biopsies, using ITx Pt25 POD241 IEL as an example

(A) Representative contour plots depicting gating strategy of HLA-ABC⁺ CD45⁺ cells, CD3⁺ T cells, CD3⁺ γδTCR⁺ T cells, CD3⁺ γδTCR⁻ CD4⁺ T cells, CD3⁺ γδTCR⁻ CD8⁺ T cells, CD19⁺ B cells, and CD3^-CD56⁺ NK cells.

(B) Percentages of donor (“D”) and recipient (“R”) multilineage chimerism in Pt25 POD241 IELs. Pt25’s donor is HLA-B12⁺. Pt25, the recipient, is HLA-A68⁺, which has serological cross-reactivity with HLA-A28.

(C) Gating strategy for donor (“D”) and recipient (“R”) CD4⁺ and CD8⁺ T cells in Pt25 POD241 IELs. CD45RA vs. CCR7 to categorize naïve T cells (CD45RA⁺ CCR7⁺), central memory T cells (TCMs: CD45RA⁻ CCR7⁺), effector memory T cells (TEMs: CD45RA⁻ CCR7⁻), and terminally-differentiated effector memory T cells (TEMRAs: CD45RA⁺ CCR7^-), tissue-resident memory T cells (TRMs: CD69⁺ CD103^+/− CD49a^+/−), effector T cells (CD28⁺ NKG2D^+/−).

The wide range of detectable cell types and phenotypes allows for the detailed study of donor- and recipient-derived immune cell dynamics within the intestinal graft mucosa after intestinal transplantation in conjunction with clinical observations.

This protocol can be modified to study the immune cell composition of other tissue types (such as bone marrow, liver, lung, lymph nodes, peripheral blood, and spleen) as well as the dynamics of mixed chimerism and T cell phenotypes in other types of transplantation in humans over time. Although beyond the scope of this protocol, it is worth mentioning that when disassociating other tissue such as spleen, lymph nodes, and lung, IEL and LPL isolation steps are not performed, as it is irrelevant for these tissues. However, enzymes such as Collagenase D and DNAse are used in incubation steps under the same condition as described in this protocol’s LPL isolation to aid in tissue disaggregation and prevent cell clumping. Depending on the tissue type and tissue size being processed, subsequent gentleMACS disassociation, sterile filtration, and Ficoll or Percoll gradients are also needed to isolate the immune cells for downstream experimental steps and analysis.

Limitations

Clinical biopsy scopes are not always consistently performed in depth to capture enough intestinal mucosa layers for lymphocyte isolation. This can affect the cellularity obtained from IEL and LPL isolation, and subsequently affect the targeted immune cell yield (DAPI^- HLA-ABC⁺ CD45⁺) for flow cytometric analysis.

While chimerism gating in flow cytometric analysis ideally stays consistent across all samples for one patient, sample-specific adjustments may be needed (troubleshooting 5). This is likely due to the enzyme digestion effect of Collagenase D on MHC-I expression,¹⁰ which can impact the binding and staining intensity of HLA antibodies in our settings.

While the antibody panel detailed in this protocol can capture many populations and be tailored to different patient HLA backgrounds, it is not sufficient to analyze all lymphocyte populations at once. To analyze other immune cell populations in depth, certain markers will need to be swapped out for others, or different flow panels would have to be applied to additional clinical specimens (as described in troubleshooting 3). Alternatively, expanded flow cytometry panels that include more than 30 markers will need to be developed to obtain more information from limited clinical specimens.

Troubleshooting

Problem 1

A much larger tissue piece is obtained from the patient following a relevant clinical procedure, such as a stoma revision/closure or graft explant procedure (step 1).

Potential solution

Before isolating immune cells from larger intestinal tissue pieces, the intestinal mucosa must first be separated from other layers that can interfere with lymphocyte isolation, such as the intestinal submucosa, muscular layers, and serosa. In a sterile Petri dish, use sterile forceps and scissors to trim the mucosa away from the other tissue layers. Use sterile PBS (1×) as needed to wash away any debris during this process. Then, cut the mucosa into small pieces, approximately 6–10 mm³ per piece, and transfer to a sterile 125 mL ventilated flask to begin incubation steps.

During IEL and LPL isolation steps, scale up the amount of DTT, EDTA, LPL buffer used to 50 mL each for 10–20 mucosa pieces, and extend the incubation time to 1 h for each IEL isolation buffer. The incubation period for LPL isolation can be extended further to 1.5–2 h as well. Both DNAse (10 μg/mL) and Amphotericin B (1 μg/mL) should be added to DTT, EDTA, and LPL buffers when processing larger amounts of intestinal tissue.

Problem 2

The current 18-color antibody panel has a limited number of phenotypic markers for non-T cell populations (antibody staining for flow cytometric analysis).

Potential solution

• •If non-T cell populations, such as B cells and plasma cells, are the priority of study, rebuild the current 18 color panel by replacing T cell phenotypic markers to B cell and plasma cell markers as needed (Table 2).
• •Alternatively, a different flow cytometer can be used, such as the Cytek Aurora. The Cytek Aurora can resolve over 40 colors in combination, allowing for the previously separate T-cell and B-cell panels to be joined into a new panel with more colors. Panel design and template setup will be critical to ensure optimal multicolor detection on precious clinical samples.

Table 2.

Primary surface antibody staining panel: Pt25 B-cell panel

Marker	Fluorophore	Clone
IgM	PE-CF594	G20-127
CD19	BUV496	SJ25C1
CD21	PerCP-Cy5.5	Bu32
CD138	redFluor 710	MI15
CD3, CD14, CD33, and CD326	APC-Cy7	SK7, M5E2, P67.6, and 9C4
CD38	PE-Cy7	HIT2
IgG	V450	G18-145
CD45RB	PE	MEM-55
IgD	BV605	IA6-2
CD69	BV650	FN50
CD27	BV711	O323
CD24	BUV395	ML5
CD45	V500	HI30
IgA	APC	IS11-8310
HLA-A2, A28	Biotin	N/A
HLA-B12	FITC	REA138
HLA-ABC	BV786	G46-2.6
DAPI	DAPI	N/A

For this patient, HLA-A2, 28 Biotin and HLA-B12 FITC antibodies are used to distinguish recipient and donor cells, respectively. Because this is a tailored B-cell panel, CD3, CD14, CD33, and CD326 antibodies used in this panel are conjugated to the same fluorophore to easily gate them out during flow cytometric analysis.

For HLA-A2, A28 Biotin, the secondary antibody Streptavidin BUV737 completes the staining.

Problem 3

The panel that needs to undergo fluorescence compensation contains markers that detect antigens that single-color control cells rarely or do not express (e.g., CD69 and CD103 for healthy human PBMCs), compromising single color staining and panel setup for clear fluorescence detection (antibody staining for flow cytometric analysis).

Potential solution

• •Stain the single-color control cells with antibodies that are conjugated to the same fluorochromes but bind to more common and highly expressed antigens (e.g., CD45 and CD3) instead for fluorescence compensation.
• •Use commercially available antibody-capture beads as compensation controls.

Problem 4

Low cell yield affects the feasibility of flow cytometry analysis (step 42).

Situation I: If the overall immune cell yield (DAPI⁻ HLA-ABC⁺ CD45⁺) is low, it might be due to:

• •Clinical scopes failed to capture enough mucosal tissue.
• •Water bath temperature is unstable and rose too high (over 40°C) during longer incubations (0.5 h–1.5 h) and harmed the cells.
• •Some or all the ileal biopsy pieces in the sample adhered to the flask wall instead of being in the buffers during incubation steps and dried up.

Situation II: If certain cell populations have low cell count in either the isolated IEL or LPL components, it might be due to:

• •Naturally low level of certain cell populations distributed in the IEL or LPL
• •Cell yield was affected by immunosuppressive treatment at different stages post-transplant.

Potential solution

Situation I:

• •Carefully inspect each ileal biopsy piece before processing to ensure enough mucosal tissue has been captured.
• •Closely monitor the water bath temperature to make sure it stays at 37°C, especially during longer incubation periods.
• •Closely monitor the ileal biopsy pieces during each incubation step to ensure they are all floating in the buffers.

Situation II:

• •Research the expected yield of the cell populations of interest in both the isolated IEL and LPL components and adjust the sample amount accordingly.
• •Closely follow the patient’s clinical treatment regimen and note any changes that may have affected the cell populations of interest in the ileal graft. Adjust experimental plan and priorities accordingly to achieve the best utilization of precious limited clinical ileal biopsy specimens.

Problem 5

Cell samples exhibit dim or undistinguishable antibody staining on donor and/or recipient-specific HLA markers, resulting in compromised chimerism determination in flow cytometric analysis (step 43).

Potential solution

• •Performing HLA quality control (HLA-QC) using pre-transplant donor and recipient PBMCs or lymphoid tissue cells with known HLA types is strongly recommended to determine which HLA markers will perform best. FMO (fluorescence minus one) controls for each HLA antibody are critical to be included in HLA-QC steps. Lineage markers including each targeted cell population are also necessary in HLA-QC steps to determine multilineage chimerism.
• •In some cases, donor and/or recipient-specific HLA antibodies may show lower staining intensities in LPL compared to IEL samples collected at the same time point from the same patient. This is likely due to the digestive effect of collagenase D on HLA expression as discussed above. An additional cell washing step with MLR media is recommended after the LPL buffer incubation. Increasing the amount of HLA antibody when staining isolated LPL samples may also help avoid dim staining. When performing flow cytometric analysis of patient chimerism, sample-specific gating could be applied based on the contour plot distribution, in reference to both healthy control PBMCs, and the IELs from the same time point.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to the lead contact, Jianing Fu (jf2977@cumc.columbia.edu).

Materials availability

This study did not generate new unique reagents. Requests to transfer human biospecimens outside of the organization must be submitted for Columbia’s IRB review and approval.

Data and code availability

The flow cytometric data supporting the current study have not been deposited in a public repository because they have not been published yet, but they are available from the corresponding author upon request.