CHARACTERIZATION OF MOUSE CD53 - EPITOPE MAPPING, CELLULAR-DISTRIBUTION AND INDUCTION BY T-CELL RECEPTOR ENGAGEMENT DURING REPERTOIRE SELECTION

The pan-leukocyte antigen CD53 is a member of the poorly understood tr ansmembrane 4 superfamily (TM4SF) of cell membrane glycoproteins. CD53 is proposed to play a role in thymopoiesis, since rat CD53 is express ed on immature CD4(-)8(-)thymocytes and the functionally mature single -positive subset, but is largely absent from the intermediate CD4(+)8( +) cells. We have characterized CD53 in the mouse through the producti on of two new monoclonal antibodies, MRC OX-79 and OX-80, which were r aised against the RAW 264 cell line and screened on recombinant CD53 f usion proteins. The epitopes recognized by both antibodies are depende nt on disulfide bonding and map to the major extracellular region of C D53, requiring the presence of a single threonine residue at position 154. Mouse CD53 has a molecular mass of 35-45 kDa and is expressed on virtually all peripheral leukocytes, but not on cells outside the lymp hoid or myeloid lineages. CD53 expression distinguishes subpopulations of thymocytes in the mouse and resembles the expression pattern of ra t CD53. Amongst the immature CD4(-)8(-) thymocytes. mouse CD53 is clea rly detectable on the earliest CD44(high)25(-) subset, but down-regula ted on the later CD44(high)25(+), CD44(low)25(+) and CD44(low)25(-) st ages. Also, the subsequent transient TcR(-flow) CD4(-)8(+) cells and m ost CD4(+)8(+) thymocytes express little or no CD53. This is consisten t with the idea that cells which are committed to enter the selectable CD4(+)8(+) compartment switch off CD53. The effect of T cell receptor (TcR) engagement on the reexpression of CD53 on CD4(+)8(+) thymocytes was studied both ex vivo and in vitro using F5 mice; transgenic for t he H-2(b)/influenza nucleoprotein-peptide-specific TcR, back-crossed o nto an H-2(q) or H-2(b) background of RAG-2-deficient mice. CD4(+)8(+) thymocytes from non-selecting H-2(q) F5 mice are CD53 negative, but i n vitro stimulation through the TcR dramatically induces CD53 expressi on. In contrast: a fraction of CD4(+)8(+) thymocytes from positively s electing H-2(b) F5 transgenic mice express CD53. Therefore TcR engagem ent by selecting major histocompatibility complex peptide complexes, o r surrogate ligands, induces CD53 expression on otherwise CD53-negativ e, non-selected CD4(+)8(+) thymocytes. Whether CD53 itself participate s as a signaling molecule in further stages of thymic selection is sti ll a matter of speculation.