TRANSCRIPTION OF GRANZYME-A AND GRANZYME-B GENES IS DIFFERENTIALLY REGULATED DURING LYMPHOID ONTOGENY

During development, thymocytes express a number of genes typical for activated peripheral T lymphocytes, including granzymes. We have now analyzed by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and cytochemistry fetal liver cells and thymocytes at various developmental stages for the expression of granzyme A-G genes. At days 13-17 of gestation, only granzyme B but none of the other granzymes is expressed in fetal liver. In the most immature, Pgp-1(+)IL2R alpha(-), thymocyte subpopulation mRNAs for granzymes A-C but not for granzymes D-G are detectable. Upon further differentiation via Pgp-1(-)IL-2R alpha(+) into more mature Pgp-1(-)IL-2R alpha(-) thymocytes the level of expression of granzymes A, B, and C gradually declines reaching its lowest level at the CD4(+)8(+) double positive stage. In fetal thymic lobes depleted of lymphoid cells by treatment with deoxyguanosine, no transcripts for granzymes A, B, and C were found indicating that the PCR signals are derived exclusively from early precursor T/natural killer (NK) lineage cells rather than from residual stromal elements. In mature CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes, granzyme B mRNA is found at similar levels in both subsets whereas granzyme A mRNA is expressed selectively in the CD4(-)CD8(+) subset. Enzymatic activity of granzyme A was only seen in a fraction of CD4(-)CD8(+) thymocytes negative for heat stable antigen (HSA) but not in the more immature HSA(+) fraction of CD4(-)CD8(+) thymocytes. The data suggest that (a) granzyme B is a pro-thymocyte marker for all T/NK lineage cells; (b) granzyme A transcripts are associated with thymocytes with the potential to develop into the CD8(+) lineage; and (c) granzyme A enzymatic activity is only expressed in the most mature CD4(-)CD8(+) stage, suggesting that granzyme proteins are not involved in early stages of thymocyte development.