Studies on CD4-2 positive cells a unique T cell subset of fish

2020 Fiscal Year Annual Research Report

• Project/Area Number: 20J22961
• Allocation Type: Single-year Grants
• Research Institution: Tokyo University of Marine Science and Technology
• Principal Investigator: Lau Lik Ming 東京海洋大学, 大学院海洋科学技術研究科, 特別研究員(DC1)
• Project Period (FY): 2020-10-12 – 2024-03-31
• Keywords: CD4-2 / Ginbuna crucian carp / Cell-mediated immunity

Outline of Annual Research Achievements

The greatest obstacle in developing cell-mediated immunity (CMI)-inducible vaccines is that the mechanism of the CMI responses to intracellular pathogens such as virus or intracellular bacteria remains unclear in fish. CD4 is an important cell surface marker of helper T cells in adaptive immunity of fish. Fish possess two divergent forms of CD4 molecules: CD4-1 resembles the mammalian CD4 while CD4-2 is a unique CD4 homolog in teleost fish. Fish CD4-1 positive cells play an important role in humoral immunity, but not in the CMI. Hence, CD4-2 positive cells may play a key role in the CMI or other functions in adaptive immunity of fish. With the aid of a monoclonal antibody (mAb) against CD4-2 of ginbuna crucian carp (ginbuna), the characteristics of CD4-2 positive cells based on the cell morphology and genes expression analysis were studied. Morphology of cell types recognized by the mAb revealed that ginbuna CD4-2 positive cells had the typical morphology of lymphocytes, while CD4-2 negative cells consist of not only lymphocytes, but also other leukocytes. Genes expression analysis showed that CD4-2 positive cells are entirely T cells, while CD4-2 negative cells contain not only T cells, but also B cells.

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

Due to border enforcement measures to prevent the spread of novel coronavirus (COVID-19) implemented by the Japanese government, I was not able to enter Japan for conducting my research following the original research plan for the fiscal year (FY) 2020. Therefore, gene expression analysis of CD4-2 positive cells as part of the functional analysis in characterization of the cells, was slightly delayed. However, I tried to catch up on the schedule after entering Japan in late December 2020. Magnetic-activated cell sorting (MACS) method was currently used for the separation of CD4-2 positive cells in various tissues. However, the purification of the cells by MACS method was not at a satisfactory level.

Strategy for Future Research Activity

In FY 2021, gene expression analysis of CD4-2 positive cells scheduled for FY 2020 will be continued. Following the original research plan in FY 2021, the distribution of CD4-2 positive cells will be determined in various lymphoid and non-lymphoid tissues of ginbuna. In FY 2022, fluorescence-activated cell sorting (FACS) method will be used instead of MACS method to obtain highly purified CD4-2 positive cells to be used for enzymatic characterization and also transcriptome analysis by Next-Generation Sequencing (NGS).