A study on CD45 isoform expression during T-cell development and selection events in the human thymus

CD45 molecules are known to appear as various isoforms generated by alternative splicing of variable exons 4, 5, and 6, but the detailed profile of CD45 isoform expression during thymocyte development has not been revealed. We examined the CD45 isoforms expressed in the various human thymocytes' subsets defined by CD3, CD4, and CD8 expressions using RT-PCR and 4-color flow cytometry. RT-PCR study revealed that RABC, RAB, RBC, RB, and R0 isoforms were expressed in thymocytes while any of RAC, RA, or RC isoforms were not detected. RABC, RAB and RBC isoforms were expressed at CD3^-CD4^-CD8^- and CD3⁺CD4⁺CD8^- stages, but were barely detectable at CD3^-CD4⁺CD8⁺ stage. RB isoform was consistently expressed at a relatively high level at all stages. R0 isoform was expressed at a low level at CD3^-CD4^-CD8^- and CD3^-CD4⁺CD8^- stages but upregulated at CD3⁺CD4⁺CD8⁺ and CD3⁺ CD4⁺CD8^- stages. In combination with the results obtained by 4-color flow cytometric study, CD45 isoform expression on human thymocytes were determined to be RABC⁺RAB^±RBC⁺RB⁺R0 ^± at CD3^-CD4^-CD8^- stage, RABC^-RAB^-RBC^-RB⁺R0⁺ at CD3^-CD4⁺CD8^- and CD3^-CD4⁺CD8⁺ stages, RABC^±RAB⁺RBC⁺ RB⁺⁺R0⁺⁺ at CD3⁺CD4⁺CD8⁺ stage, and RABC⁺RAB⁺RBC⁺RB⁺⁺R0⁺ at CD3⁺CD4⁺CD8^- stage. Bcl-2 expression was upregulated between CD3^-CD4⁺CD8⁺CD45R0⁺ and CD3⁺ CD4⁺CD8⁺CD45R0⁺ stages. Expression of CD45R0 epitope was upregulated between CD3^- CD4⁺ CD8⁺ CD69^- and CD3⁺ CD4⁺ CD8⁺CD69⁺ stages while CD45RA epitope expression was unchanged. Thus, when thymocytes are positively selected, CD45R0 isoform expression seems to be upregulated while CD45RABC isoform expression stays at a very low level. In summary, various isoforms of CD45 were shown to be tightly regulated during thymocyte development and through the selection process.