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Isolation of Mouse Small Intestinal Intraepithelial Lymphocytes, Peyer's Patch, and Lamina Propria Cells

Leo Lefrançois¹, Nils Lycke²

¹University of Connecticut Health Center, Farmington, Connecticut
²University of Göteborg, Göteborg, Sweden, Sweden

Publication Name:

Current Protocols in Immunology

Unit Number:

Unit 3.19

DOI:

10.1002/0471142735.im0319s17

Online Posting Date:

May, 2001

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Abstract

The intestinal mucosal immune system is composed of three major lymphoid areas: the lamina propria (LP), which lies just underneath the basement membrane in the intestinal villi; (2) the intraepithelial compartment, which contains the intraepithelial lymphocytes (IEL) and is located just above the basement membrane, between the columnar epithelial cells; and (3) Peyer's patches (PP), lymphoid nodules (akin to lymph nodes) embedded in the gut wall, separated from the LP and IEL. The LP, PP, and IEL lymphoid populations form a complex, interconnected network that responds to immunological insults in the intestine. Therefore, these lymphocyte populations should be analyzed when studying the immunological status of the intestine, for example in oral immunization or in intestinal disease (including infectious disease and tumors). This unit details techniques for isolation of IEL, PP cells, and LP cells from the small intestine of the mouse.

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Unit Introduction
Basic Protocol 1: Isolation of IEL
Basic Protocol 2: Isolation of PP Lymphocytes
Basic Protocol 3: Isolation of Lymphocytes from Intestinal LP
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Isolation of IEL

Materials

Mice: e.g., strain C57BL/6, 10 weeks old (Table A.1C.1)
CMF solution (see recipe), 4°C
CMF/FBS/DTE solution (see recipe), 37°C
RPMI 1640, 4°C
Fetal bovine serum (FBS; Life Technologies), heat-inactivated (appendix 2A)
44% and 67% Percoll solutions (see recipe)

Iris scissors (straight) and forceps
20-ml syringe with 18- or 20-G needle
50-ml conical centrifuge tubes (Becton Dickinson)
50-ml Erlenmeyer flasks, silanized (see recipe or appendix 3K)
Stir plate (e.g., Bellco 4-position)
Refrigerated centrifuge (Sorvall RC-3B with HL-8 rotor or equivalent)
Prewashed nylon wool (Type 200L, Du Pont NEN)
10-ml syringe
17 × 100–mm round-bottom polystyrene centrifuge tubes (Falcon)

Additional reagents and equipment for euthanasia (unit 1.8), trypan blue exclusion (appendix 3B), panning (unit 3.5A), and cell sorting (unit 5.4)

Basic Protocol 2: Isolation of PP Lymphocytes

Materials

Mice: e.g., strain C57BL/6, 8 to 10 weeks old (Table A.1C.1)
CMF/HEPES solution (see recipe), 4°C
CMF/FBS/Dispase/DNase solution (see recipe), 37°C
2- to 3-mm-diameter glass rod
Sharp straight scissors
Small forceps, sharp, thin, and curved at the tip
Nylon screen (e.g., Pes 250/37 polyester precision filters, SAATI) or 250 µm-gauge steel mesh
Magnetic stir plate with a minimum of 4 to 6 positions and stir-bars
Thermostat-controlled incubator large enough to contain the stir plate, 37°C

Basic Protocol 3: Isolation of Lymphocytes from Intestinal LP

Materials

Mice: e.g., strain C57BL/6, 8 to 10 weeks old (Table A.1C.1)
CMF/HEPES solution (see recipe), 4°C
PBS (appendix 2A)
CMF/FBS/EDTA solution (see recipe), 37°C
Complete RPMI-10 medium (without sodium bicarbonate; see recipe)
Complete RPMI-10/collagenase (see recipe), 37°C
100% and 40% Percoll solutions (see recipe), ice cold
Curved forceps
Straight iris scissors with a ball tip on one point/blunt end
Scalpel, scissors, or razor blades
Nylon screen (e.g., Pes 250/37 polyester precisions filters, SAATI) or 250-µm-gauge steel mesh
50-ml Erlenmeyer flask, silanized (see recipe or appendix 3K)
Magnetic stir plate with a minimum of 4 to 6 positions and stir-bars
Thermostat-controlled incubator large enough to contain the stir plate, 37°C
2.5-ml plastic transfer pipets (Sarstedt)
50-ml conical centrifuge tubes
10- to 15-ml conical centrifuge tubes

Additional reagents and equipment for ELISPOT assay (unit 7.14), panning (positive selection; unit 3.5A), and cell sorting (negative selection; unit 5.4)

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Figures

Figure 3.19.1

Flow cytometer light-scatter profiles for IEL preparations before (A) and after (B) panning. The Percoll fraction or panned IEL were analyzed on a FACScan (Becton Dickinson) for forward-light scatter (linear scale) and side scatter (logarithmic scale).

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Figure 3.19.2

Flow chart for the isolation of lamina propria lymphocytes by enzymatic digestion of intestinal mucosa.

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Figure 3.19.3

Longitudinal section of a small intestinal villus.

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Figure 3.19.4

Yield of lamina propria lymphocytes after incubation of intestinal tissue pieces in medium containing 300 U/ml collagenase. The number of viable lymphocytes released into the medium was determined every 30 min. The intestines were handled in pairs. The mean values ± SD of seven experiments are shown.

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Literature Cited

Literature Cited
	Cerf-Bensussan, N., Quaroni, A., Kurnick, J.T., and Bhan, A.K. 1984. Intraepithelial lymphocytes modulate Ia expression by intestinal epithelial cells. J. Immunol. 132:2244-2252.
	Davies, M.D.J. and Parrott, D.M.V. 1981. Preparation and purification of lymphocytes from the epithelium and lamina propria of murine small intestine. Gut. 22:481-488.
	Deusch, K., Luling, F., Reich, K., Classen, M., Wagner, H., and Pfeffer, K. 1991. A major fraction of human intraepithelial lymphocytes expresses the / T cell receptor, the CD8 accessory molecule and preferentially uses the V1 gene segment. Eur. J. Immunol. 21:1053-1059.
	Goodman, T. and Lefrançois, L. 1989. Intraepithelial lymphocytes. Anatomical site, not T cell receptor form, dictates phenotype and function. J. Exp. Med. 170:1569-1581.
	Harriman, G.R., Lycke, N.Y., Elwood, L.J., and Strober, W. 1990. T lymphocytes that express CD4 and the T cell receptor but lack Thy-1. Preferential localization in Peyer's patches. J. Immunol. 145:2406-2414.
	Harriman, G.R., Hörnquist, E., and Lycke, N.Y. 1992. Antigen-specific and polyclonal CD4⁺ lamina propria T cell lines: Phenotypic and functional characterization. Immunology. 75:66-73.
	Hörnquist, E., Lycke, N., Czerkinsky, C., and Holmgren, J. 1993. Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen carrier systems.. In Novel Delivery Systems for Oral Vaccines (D.T. O'Hagan, ed.) pp. 157-173. CRC Press, Boca Raton, Fla.
	Hörnquist, E. and Lycke, N. 1993. Cholera toxin adjuvant promotes antigen priming of T cells. Eur. J. Immunol. 23:2136-2143.
	Jarry, A., Cerf-Bensussan, N., Brousse, N., Selz, F., and Guy-Grand, D. 1990. Subsets of CD3+ (T cell receptor / or ) and CD3^– lymphocytes isolated from normal human gut epithelium display phenotypical features different from their counterparts in peripheral blood. Eur. J. Immunol. 20:1097-1103.
	Lefrançois, L. 1987. Carbohydrate differentiation antigens of murine T cells: Expression on intestinal lymphocytes and intestinal epithelium. J. Immunol. 138:3375-3384.
	Lefrançois, L., Lefrançois, L. 1991a. Intraepithelial lymphocytes of the intestinal mucosa: Curiouser and curiouser. Semin. Immunol. 3:99-108.
	Lefrançois, L. 1991b. Phenotypic complexity of intraepithelial lymphocytes of the small intestine. J. Immunol. 147:1746-1751.
	Lefrançois, L. 1991c. Extrathymic differentiation of intraepithelial lymphocytes: Generation of a separate and unequal T cell repertoire Immunol. Today. 12:436-438.
	Lefrançois, L., LeCorre, R., Mayo, J., Bluestone, J.A., and Goodman, T. 1990. Extrathymic selection of TCR-,d⁺ T cells by class II major histocompatibility molecules. Cell. 63:333-340.
	Lycke, N. 1986. A sensitive method for the detection of specific antibody production in different isotypes from single lamina propria plasma cells. Scand J. Immunol. 24:393-403.
	Nedrud, J.G. and Lamm, M. 1991. Adjuvants and the mucosal immune system.. In Topics in Vaccine Adjuvant Research (D.R. Spriggs and W.C. Koff, eds.) pp. 51-65. CRC Press, Boca Raton, Fla.
	Petit, A., Ernst, P.B., Befus, A.D., Clark, D.A., Rosenthal, K.L., Ishizaka, T., and Bienenstock, J. 1985. Murine intestinal intraepithelial lymphocytes I. Relationship of a novel Thy-1^–, Lyt-1^–, Lyt-2⁺, granulated subpopulation to natural killer cells and mast cells. Eur. J. Immunol. 15:211-215.
	Poussier, P., Edouard, P., Lee, C., Binnie, M., and Julius, M. 1992. Thymus-independent development and negative selection of T cells expressing T cell receptor / in the intestinal epithelium: Evidence for distinct circulation patterns of gut- and thymus-derived T lymphocytes. J. Exp. Med. 176:187-199.
	Spalding, D.M., Koopman, W.J., Eldridge, J.H., McGhee, J.R., and Steinman, R.M. 1983. Accessory cells in murine Peyer's patch. J. Exp. Med. 157:1646-1659.
	Tagliabue, A., Befus, A.D., Clark, D.A., and Bienenstock, J. 1982. Characteristics of natural killer cells in the murine intestinal epithelium and lamina propria. J. Exp. Med. 155:1785-1796.