Immune Regulation Lab

Various investigations are performed in the Immune Regulation Lab including research about different subtypes of lymphocytes and their function, production of hybrid cells and also immunohistochemistry based evaluations on tumor tissues.

 One of our research areas in this lab is focused on lymphocytes derived from tumor draining lymph nodes of patients with different cancers such as breast, head and neck and colorectal cancer. Lymph nodes are important places for the formation of immune responses, and the immune responses change during malignancy. Therefore, different surface and intracellular markers are assessed in B and T lymphocytes using flow cytometry technique. In addition, the association between clinico-pathologic characteristics of patients and various markers’ expression is evaluated.

Another common method in our lab is hybrid cells production. In this vein intended antigens (specific antigen, lysate or cells) inject into laboratory animal, collecting an antibody-producing cell from the mouse's spleen (B-cells), and fusing it with a tumor cell called a myeloma cell by polyethylene glycol. Then hybrid cells screened and isolate to get polyclonal or mono clonal antibody by ELISA and flow cytometry. Then purification and MW determination are done by HPLC chromatography and Western-blot respectively. Functionality of selected Abs are examined by multiple method such as Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC), and Complement Dependent Cytotoxicity (CDC) and MTT assay. In addition to immunophenotyping of cells, the aim of Ab production is identification of unknown tumor antigens. For this purpose, unknown antigens (recognized by antibodies) are isolated by 2D blot or immunoprecipitation and then sent for identification by mass spectrometry.

Immunohistochemistry (IHC) is one the methods that plays a pivotal role in both diagnostic and research areas. This method, which is based on the antigen-antibody (Ab) interactions, is used to detect the presence or absence of the antigen of interest in a specific tissue. In this regard, 3 to 4 micrometer tissue sections will be prepared and fixed on positively-charged IHC slides. After deparafinization and rehydration and before adding primary Ab, to prevent nonspecific interactions and reduce the background stain, endogenous proxidases and hydrophobic protein-protein interactions will be blocked. In next step HRP-conjugated secondary Ab which is against the Fc region of primary Ab will be added. After that to visualize the location of target antigen, we use DAB chromogene on the effect of enzyme-substrate interaction will be changed to a water insoluble brown precipitate. Finally slides will be counterstained with hematoxylin, dehydrated in increasing graded ethanol and mounted with mounting medium.

It should be mentioned that it is only one of the possible ways of doing IHC. However there are several possible other ways with different enzymes and substrates.

Publications:

1.Norouzian M, Mehdipour F, Balouchi Anaraki S, Ashraf MJ, Khademi B, Ghaderi A. Atypical Memory and Regulatory B Cell Subsets in Tumor Draining Lymph Nodes of Head and Neck Squamous Cell Carcinoma Correlate with Good Prognostic Factors. Head and neck pathology. 2019. doi: 10.1007/s12105-019-01095-1

2. Arabpour M, Rasolmali R, Talei AR, Mehdipour F, Ghaderi A. Granzyme B production by activated B cells derived from breast cancer-draining lymph nodes. Molecular immunology. 2019;114:172-8.

3.Ghods A, Ghaderi A, Shariat M, Talei AR, Mehdipour F. TNFR2 but not TNFR1 is the main TNFR expressed by B and T lymphocytes in breast cancer draining lymph nodes. Immunology letters. 2019;209:36-44.

4.Mehdipour F, Razmkhah M, Faghih Z, Bagheri M, Talei AR, Ghaderi A. The significance of cytokine-producing B cells in breast tumor-draining lymph nodes. Cellular oncology (Dordrecht). 2019;42(3):381-95.

5.Ahmadvand S, Faghih Z, Montazer M, Safaei A, Mokhtari M, Jafari P, et al. Importance of CD45RO+ tumor-infiltrating lymphocytes in post-operative survival of breast cancer patients. Cellular oncology (Dordrecht). 2019;42(3):343-56.

6.Ghaderi F, Ahmadvand S, Ramezani A, Montazer M, Ghaderi A. Production and characterization of monoclonal antibody against a triple negative breast cancer cell line. Biochemical and biophysical research communications. 2018;505(1):181-6.