HUMAN CD45RC SPECIFICITY - A NOVEL MARKER FOR T-CELLS AT DIFFERENT MATURATION AND ACTIVATION STAGES

The different isoforms of CD45 seem to play an important role in thymo cyte maturation and T cell activation and function. To investigate the particular contribution of CD45-exon C (exon 6) to human T cell devel opment, we first investigated the expression of the CD45-exon C isofor ms on different human lymphoid cell populations. A wide panel of mAbs against the different isoforms of CD45 was generated by immunizing mic e with purified human CD45. Two of these mAbs (RP1/12 and RP2/19) sele ctively recognized mouse 300-19 pre-B cells that had been transfected with CD45 cDNA that contained exon C, suggesting that epitopes recogni zed by these mAbs are dependent on the expression of exon C. Immunoflu orescence analysis of PBLs showed that CD45 RC is expressed at high le vels on B, NK, and CD8(+) T cells, although a subset of CD4(+) T cells was found to be negative. Activation of PBMCs and purified NK cells r esulted in down-regulation of CD45RC, which took place in a coordinate manner involving both down-regulation of the expression of CD45RA and CD45RB and up-regulation of CD45RO. Two-color immunofluorescence anal yses showed that thymocytes expressing CD45RC were CD1(-to lo) , CD3(h i) and H LA class I-hi, typical markers of mature thymocytes. Accordin gly, immunohistochemical analysis of a normal thymus showed that where as the anti-CD45RA mAb stained only a subset of medullary thymocytes, the anti-CD45RC mAb stained the majority of medullary thymocytes. Thym ocytes that bear CD45RA also express CD45RC, but do not express CD45RO ; a subset of CD45 RA(-) RO(+) thymocytes was also detected. Immunopre cipitation of CD45 molecules from both PBMC and thymocyte cell lysates with mAbs against the different specificities of this molecule reveal ed that the RC determinants are found on CD45 polypeptides of 220 and 205 kDa that also bear RB or both RB and RA determinants, respectively . Together these results support both the existence of a tight regulat ion of the expression of CD45 specificities during lymphocyte activati on and thymocyte maturation and also that CD45RC is selectively expres sed by mature medullary thymocytes during T cell ontogeny.