Extraction of Tissue Antigens for Functional Assays

Summary

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

Abstract

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined ¹. This is particularly true in autoimmune diseases and cancer². Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes ^1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays ⁶. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

Protocol

1. Preparing Spleen Tissue

1. Note- all human material should be treated as potentially infectious and all procedures should be conducted in a Class II Laminar Flow Cabinet. Using sterile scissors and forceps, remove fat and fibrous tissue from spleen sections (~1-2 cm in size) and trim off as much of the outer capsule material as possible.
2. Cut a small piece (1-2 cm³) of spleen tissue and place each piece into a sterile 50 ml Falcon tube.
3. Snap-freeze the pieces of tissue by immersion in liquid N₂.
4. Store at -80 °C. A similar protocol is suitable for other tissue(s).

2. Prepari....

Discussion

This protocol was developed because we wanted to generate an extract from human tissue that was free from toxic chemicals such as detergents. Specifically we have used it to prepare extracts of human tissue that can be used in assays of human immune function in vitro. Extracts prepared using this protocol can equally be reconstituted in any buffer and used for many biochemical analyses, such as western blotting or liquid chromatography. This makes this technique applicable to many downstream applications.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5 ml 12 x 75 mm sterile polystyrene tubes	BD Falcon	352054
Caps for tubes polystyrene tubes (above)	BD Falcon	352032
50ml sterile tubes	Becton Dickinson	352070
Acetonitrile	Mallinckradt Chemicals	2856-10
Butan-1-ol	Sigma Aldrich	537993-IL
Homogenizer: PRO200	Bio-strategy	01-01200	10 x 115 mm saw-tooth generator
Lyophilizer			Virtis, Benchtop 4K
Sterile Needle 18-20 gauge	Becton Dickinson	REF 302032
CMRL-1066 Medium	Sigma	C0422
PBS	Sigma	D8537
Table 1. Specific reagents and equipment.