Summary

Efficiente protocollo Isolamento da B e T linfociti umani da Palatine Tonsille

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

Il protocollo descrive l'isolamento di cellule linfoidi dal materiale umano paziente e richiede quindi l'approvazione etica. Il lavoro svolto in questo studio è stata concessa dalla Uppsala Ethical Review Board (DNR. 2013/387). 1. Isolamento di cellule mononucleate (MNC) da umani Palatine Tonsille ATTENZIONE: Tutto il materiale non schermati di origine umana come sangue, tessuti o fluidi corporei dovrebbe essere considerato come materiale potenzialmente in…

Representative Results

Una separazione efficace dei tonsillari MNC risultati in sottopopolazioni altamente purificate di linfociti B e T. Ciò è stato confermato mediante analisi FACS utilizzando anti-CD20 e anti-CD2 per rilevare B T linfociti popolazioni, rispettivamente (Figura 3A). Al contrario, una separazione efficiente di MNC risultati in frazioni cellulari che sono una miscela di cellule di entrambe le sottopopolazioni di linfociti B e T (Figura 3B). Il linfociti T <strong…

Discussion

Uno dei fattori più importanti che influenzano il risultato di questo protocollo è l'uso di materiale fresco tonsillare come materiale di partenza. Pertanto, i campioni di tonsille devono essere trattati entro 3 ore dopo l'intervento chirurgico. Tonsille possono essere ottenuti da adulti e bambini. Il materiale tonsillare dai bambini è di solito più piccoli a causa della rimozione chirurgica parziale delle tonsille (tonsillotomy). Pertanto, il numero di multinazionali ottenuti da campioni tonsillotomia (1 x …

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

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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).

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