1Immunology and Diabetes Unit, St. Vincent's Institute of Medical Research, 2Department of Medicine, University of Melbourne
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Necula, A., Chand, R., Albatat, B., Mannering, S. I. Extraction of Tissue Antigens for Functional Assays. J. Vis. Exp. (67), e4230, doi:10.3791/4230 (2012).
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.
1. Preparing Spleen Tissue
2. Preparing Human Islet for Storage
3. Preparing Extract
4. Freeze Drying Extracts
5. Representative Results
Figure 1 shows the staining of a protein gel loaded with extract from spleen and islet depleted pancreatic tissue (labeled acinar) and purified human islets (labeled islets). The results show a good representation of proteins of different molecular weight for each tissue.
The capacity of tissue extracts to stimulate human T-cell proliferation was tested using a CFSE-based proliferation assay7 (Figure 2). The PBMC used in this assay were isolated from an individual with type 1 diabetes. The magnitude of the response is expressed as a ratio of the number of CFSEdim cells per 5,000 CD4+, CFSEbright cells without antigen: of CFSEdim cells per 5,000 CD4+, CFSEbright cells with antigen from triplicate samples7. The results show a weak, but detectable, proliferation in response to acinar (CD1=3.5) and a stronger response to islet extract (6.8). Inactivated influenza virus (CDI= 142.6) is included as a positive control.
Figure 1. Protein gel.
Figure 2. Results from a CFSE-based proliferation assay against acinar and islet extract. Click here to view larger figure.
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.
Using our protocol the response to tissue extracts are not strong. This is expected because we are looking for responses to 'self' antigens; in our case T-cell responses against islet antigens are frequently weak 1. Previously we found that human CD4+ T cell responses to recombinant proinsulin and glutamic acid decarboxylase (GAD), autoantigens in type 1 diabetes, could be detected using our CFSE-based proliferation assay 7,8. We have chosen to use tissue extracts to avoid problems associated with using synthetic peptides 9 and recombinant proteins 10.
We do not routinely add protease inhibitors to our extractions. The presence of protease inhibitors may inhibit antigen processing and presentation 11 and consequently inhibit T-cell responses. Instead we perform the extraction on ice in an attempt to prevent protease-mediated degradation. For other applications the inclusion of protease inhibitors may be beneficial if protein degradation is a problem.
No conflicts of interest declared.
This work is supported by grants from the Australian National Health and Medical Research Council (NHMRC #559007) and the Juvenile Diabetes Research Foundation (JDRF 4-2006-1025) and the Operational Infrastructure Scheme of the Victorian Government. We thank the members of the Tom Mandel Islet Transplantation Program Islet Isolation Team for providing the human tissues. Human tissues were collected and used with local ethical approval (St. Vincent's Hospital HREC-A 011/04 and St. Vincent's Health HREC-A 135/08).
|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|
|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|
Table 1. Specific reagents and equipment.