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Updated: Aug 10, 2026
Isolation and Activation of Murine Lymphocytes
Published on: October 30, 2016
Two distinct P70 interleukin-2 receptors on a murine large granular lymphocyte clone Y479
1Shionogi Research Laboratories, Shionogi & Co., Ltd., Osaka, Japan.
Insights
Researchers identified two distinct 70 kDa interleukin-2 receptors (IL-2R) on mouse Y479 cells. These receptors bind IL-2 despite lacking the typical p55 IL-2R subunit, offering new insights into IL-2 signaling.
Area of Science:
- Immunology
- Cell Biology
Background:
- Interleukin-2 (IL-2) is crucial for T cell proliferation and function.
- IL-2 signaling is primarily mediated by the IL-2 receptor (IL-2R), composed of alpha (CD25, p55), beta (p70-75), and gamma (p64) subunits.
- Understanding IL-2R heterogeneity is vital for deciphering immune responses.
Purpose of the Study:
- To characterize the IL-2 binding proteins on the Y479 cloned cell line.
- To investigate the structure and epitopes of IL-2 receptors on Y479 cells.
- To determine if Y479 cells express the canonical p55 IL-2R subunit.
Main Methods:
- Establishment of a continuous cloned cell line (Y479) from mouse spleen cells cultured in high IL-2.
- Phenotypic analysis of Y479 cells using flow cytometry.
- IL-2 binding assays and immunoprecipitation using radioiodinated IL-2 and monoclonal antibodies against human IL-2.
- Limited trypsin digestion of IL-2-cross-linked cells.
Main Results:
- Y479 cells, a large granular lymphocyte line, expressed IL-2 binding proteins but lacked detectable p55 IL-2R.
- Both high and low affinity IL-2 binding were mediated by a 70 kDa protein.
- Monoclonal antibodies recognized different epitopes on the 70 kDa IL-2R.
- Trypsin digestion yielded a 45 kDa fragment, suggesting distinct structural domains within the 70 kDa IL-2R.
Conclusions:
- Y479 cells possess at least two distinct types of 70 kDa IL-2 receptors.
- These receptors mediate IL-2 binding independently of the p55 IL-2R subunit.
- The findings reveal novel IL-2 receptor heterogeneity and potential alternative IL-2 signaling pathways.
Abstract:
A continuous cloned cell line (Y479) was established by culturing normal mouse spleen cells in a high concentration of interleukin-2 (IL-2). Y479 cells showed morphological characteristics of large granular lymphocyte with the phenotypes of Thy-1.2+, T3+, Lyt-1-, Lyt-2-, L3T4-, B220-, AsGM1+, LFA-1+, and TcRV beta 8-. The Y479 cells required a high concentration of IL-2 for their growth but did not express detectable p55 IL-2 receptor (IL-2R) although they bound IL-2 with high and low affinities. Analysis of the IL-2 binding proteins on the Y479 cells revealed that both the high and low affinity receptors consisted only of 70 kDa protein. Analysis of the 70 kDa protein was performed using five monoclonal antibodies (L15, L20, L23, L34, and L61) against human recombinant IL-2. Although they recognized different epitopes, all monoclonal antibodies immunoprecipitated 70 kDa IL-2R that was cross-linked with radioiodinated IL-2. The supernatant after immunoprecipitation with L61 still contained IL-2/IL-2R complex that was L23-reactive, and the supernatant after immunoprecipitation with L23 contained L61-reactive IL-2/IL-2R complex, whereas L15 immunoprecipitated almost all the complex. Limited digestion of IL-2-cross-linked Y479 cells with trypsin caused the liberation of 45 kDa IL-2R fragment cross-linked with IL-2. This complex was immunoprecipitated by L15 or L61 but not by L23. These results suggest that there are at least two distinct 70 kDa IL-2R on the surface of Y479 cells.
