Summary

Efficiënte isolatie protocol voor B- en T-lymfocyten van Human Palatine Amandelen

Published: November 16, 2015
doi:

Summary

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Abstract

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term “tonsils” refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Introduction

Tonsils are collections of incompletely encapsulated lymphoid tissues that lie under, and in contact with, the epithelium in the upper aero-digestive tract. Usually the term “tonsils” refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. The paired palatine tonsils together with the nasopharyngeal tonsil (adenoid), paired tubal tonsils and lingual tonsils constitute the so-called “Waldeyer´s ring”. The latter is responsible for the initial contact between inhaled or ingested pathogens and the lymphoid tissues of the aerodigestive tract1,2. Indeed, numerous reports have shown that both bacterial and viral antigens can be detected in palatine tonsil tissue samples2-6.

The palatine tonsils are composed of dense lymphoid tissue covered by a stratified squamous non-keratinising epithelium. The tonsils have numerous crypts, epithelial invaginations, which penetrate the parenchyma increasing the surface area. Histologically, the palatine tonsils contain numerous lymphoid follicles with germinal centers, which are the sites for B cell maturation and differentiation (B-cell areas). Likewise, the palatine tonsils encompass T cells, which are mainly located in the extrafollicular regions (T-cell areas). In addition to the B and T cells, also various follicular dendritic cells can be detected in palatine tonsils1,2.

Due to their anatomic location, the palatine tonsils are easily accessible by surgical interventions. For example, surgical removal of tonsils, known as tonsillectomy, is routinely carried out worldwide7. In children with tonsillar hyperplasia, a partial surgical removal of the tonsils (tonsillotomy) is sometimes used, causing less postoperative pain to the patients. Considering the accumulation of various pathogens in tonsils, surgically removed tonsils provide a unique opportunity to study the influence of viral and bacterial agents on tonsillar lymphocyte functions2,8. Furthermore it is possible to study if some pathogens prefer to reside in specific cell subpopulations9. In addition, as the tonsils are rich source of B lymphocytes, isolated tonsillar B lymphocytes can be efficiently used to study the activity of different B cell subpopulations10. However, as the palatine tonsils contain a mixture of cell types an efficient method to separate the different cell subpopulations is needed.

Here, we describe a simple method for efficient and rapid isolation of tonsillar B and T cell populations from human palatine tonsils by using a magnetic-activated cell separation technique (Figure 1). The method described here is useful for scientists who want to assess the role of different infectious agents in human lymphoid organs such as palatine tonsils.

Protocol

Het protocol beschrijft de isolatie van lymfoïde cellen van menselijke patiënt materiaal en vereist daarom een ​​ethische goedkeuring. Het werk in het huidige onderzoek werd door de Uppsala Ethische Review Board (DNR. 2013/387) toegekend. 1. Isolatie van mononucleaire cellen (MNC) van Human Palatine Amandelen LET OP: Alle niet-afgeschermde materiaal van menselijke oorsprong, zoals bloed, weefsels of lichaamsvloeistoffen moet worden beschouwd als potentieel be…

Representative Results

Een efficiënte scheiding van tonsillar MNO resulteert in sterk gezuiverde subpopulaties van B- en T-lymfocyten. Dit werd bevestigd door FACS analyse met anti-CD20 en anti-CD2 antilichamen voor B en T-lymfocyt populaties, respectievelijk (figuur 3A) te detecteren. Daarentegen, een inefficiënte scheiding van MNO leidt celfracties die een mengsel van cellen van zowel B- en T-lymfocyt subpopulaties (Figuur 3B). De geïsoleerde tonsillaire B- en T-lymfocyten <s…

Discussion

Een van de belangrijkste factoren die de resultaten van dit protocol is het gebruik van verse tonsillar materiaal als het uitgangsmateriaal. Daarom moet de tonsil monsters wordt binnen 3 uur na de operatie. Amandelen kan worden verkregen van zowel volwassenen als kinderen. De tonsillar materiaal uit de kinderen gewoonlijk kleiner vanwege de gedeeltelijke chirurgische verwijdering van de amandelen (tonsillotomy). Daarom is het aantal MNO verkregen uit tonsillotomy monsters (1 x 10 7 x 10 -1 8) minde…

Declarações

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Swedish Cancer Society (11 0253, 13 0469), the Swedish Research Council (K2012-99X-21959-01-3), Marcus Borgströms Foundation and the Swedish Research Council through a grant to the Uppsala RNA Research Centre (2006-5038-36531-16). We are indebted to the BioVis core facility at Uppsala University for much help with the FACS analysis.

Materials

Hanks balanced salt solution (HBSS)  Gibco 14175-053 Contains 5% fetal bovine serum (FBS), 10 mM Glutamine, 
 0.05 mg/ml Gentamicin and 1% Antibiotic-Antimycotic mix
Freezing medium 90% FBS and 10% DMSO
MACS buffer PBS (pH 7.2), 0.5% BSA and 2mM EDTA
PBSA  PBS containing 0.2% BSA
PFA PBS containing 1% paraformaldehyde (PFA). Make fresh.
PFA is suspected carcinogen. Wear gloves and goggles.
Antibiotic-Antimycotic mix Gibco 15240-062
100-mm petridish Nunc 172958
Dissecting foreceps Fisher Scientific 1381241
Straight iris scissors  Fisher Scientific 12912055
disposable scalpels Swann-Morton REF 0501
100μm plastic cell strainer Corning Life Sciences 352360
40μm plastic cell strainers  Corning Life Sciences 352340
2-ml plastic syringe BD Biosciences  300185
15-ml conical centrifuge tubes SARSTEDT 62554502
50-ml conical centrifuge tubes SARSTEDT 62547254
Low-speed centrifuge with fixed-angle or swinging-bucket rotor Thermo Scientific Heraeus Megafuge 16R
Ficoll–Hypaque Sigma-Aldrich F5415-50ML Ficoll solution
Fetal calf serum Biological industries 040071A
Dimethyl sulphoxide (DMSO) SIGMA D2650-5X5ML
MACS MS columns  Miltenyi Biotec 130-042-201
MACS human CD3 MicroBeads Miltenyi Biotec 130-092-881
MACS separator (Octo MACS) Miltenyi Biotec 130-042-109
Bovine Serum Albumin (BSA) Merck Millipore 1120180100
EDTA AnalaR NORMAPUR 20302.293
CD2 conjugated to allophycocyanin (CD2-APC)  BD Biosciences  560642
CD20 conjugated to fluorescein isothiocyanate (CD20-FITC)  BD Biosciences  556632
Human serum  Rockland Immunochemicals D119-0100
Phusion High-Fidelity DNA Polymerase Thermo Scientific F-530L
FACS tubes BD Falcon 352003
Cryotube SARSTEDT 72379
BD LSRII flowcytometer BD Biosciences 
BD FACSDiva 4.1 software BD Biosciences 
Nucleospin Blood  Macherey-Nagel 740951.50 DNA isolation kit
TRIzol  reagent Life technologies 15596 RNA isolation reagent
Hemocytometer The Paul Marienfeld GmbH & Co. KG 0610030 cell counter device
GelRed Biotium 41003 Nucleic acid gel stain 

Referências

  1. Nave, H., Gebert, A., Pabst, R. Morphology and immunology of the human palatine tonsil. Anat Embryol (Berl). 204, 367-373 (2001).
  2. Perry, M., Whyte, A. Immunology of the tonsils. Immunology today. 19, 414-421 (1998).
  3. Alkhalaf, M. A., Guiver, M., Cooper, R. J. Prevalence and quantitation of adenovirus DNA from human tonsil and adenoid tissues. J Med Virol. 85, 1947-1954 (2013).
  4. Brandtzaeg, P., Halstensen, T. S. Immunology and immunopathology of tonsils. Adv Otorhinolaryngol. 47, 64-75 (1992).
  5. Imai, S., et al. Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection. Blood. 87, 1446-1457 (1996).
  6. Veen, J., Lambriex, M. Relationship of adenovirus to lymphocytes in naturally infected human tonsils and adenoids. Infect Immun. 7, 604-609 (1973).
  7. Friedman, M., Wilson, M., Lin, H. C., Chang, H. W. Updated systematic review of tonsillectomy and adenoidectomy for treatment of pediatric obstructive sleep apnea/hypopnea syndrome. Otolaryngol Head Neck Surg. 140, 800-808 (2009).
  8. Garnett, C. T., et al. Latent species C adenoviruses in human tonsil tissues. J Virol. 83, 2417-2428 (2009).
  9. Garnett, C. T., Erdman, D., Xu, W., Gooding, L. R. Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes. J Virol. 76, 10608-10616 (2002).
  10. Perez, M. E., Billordo, L. A., Baz, P., Fainboim, L., Arana, E. Human memory B cells isolated from blood and tonsils are functionally distinctive. Immunol Cell Biol. 92, 882-887 (2014).
  11. Cooper, R. J., Yeo, A. C., Bailey, A. S., Tullo, A. B. Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis. Invest Ophthalmol Vis Sci. 40, 90-95 (1999).
  12. Zhang, Y., Huang, W., Ornelles, D. A., Gooding, L. R. Modeling adenovirus latency in human lymphocyte cell lines. J Virol. 84, 8799-8810 (2010).
  13. Chomczynski, P., Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. biochem. 162, 156-159 (1987).
  14. Watanabe, T., Yoshizaki, K., Yagura, T., Yamamura, Y. In vitro antibody formation by human tonsil lymphocytes. J Immunol. 113, 608-616 (1974).

Play Video

Citar este artigo
Assadian, F., Sandström, K., Laurell, G., Svensson, C., Akusjärvi, G., Punga, T. Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils. J. Vis. Exp. (105), e53374, doi:10.3791/53374 (2015).

View Video