Eur. J. Immunol. 1993. 23: 103-107 Re-expression of CD45RC zy in zyx vitro zy 103 Rapid re-expression of CD45RC on rat CD4 Tcells in vitro correlates with a change in function* Sally R. Sarawar., Sheila M. Sparshott, Philip Sutton, Chun-ping Yang, Ian V. Hutchinson and Eric B. Bell Immunology Research Group, Department of Cell and Structural Biology, University of Manchester Medical School, Manchester Rat CD4+ T cells are divided phenotypically by the anti-CD45RC monoclonal antibody OX22 into subsets with contrasting functions. Stimulation of T cells in vitro is known to induce a change in isoform from CD45RC+ to CD45RC-. We have investigated the in vifro conditions which promote a switch in isoform in the opposite direction. We observed that a majority of CD45RC- CD4 Tcells z (> 90%) spontaneously re-expressed CD45RC during the first 1-3 days of culture in both the presence and absence of alloantigen. The Tcells remained CD45RC+ when cultured for 7 days in serum-free growth medium. However, alloantigen- activated lymphocytes, expressing the interleukin-2 receptor (IL-2R), down- regulated CD45RC by day 4 and remained CD45RC- during the course of the experiment. Using mixtures of allotype-marked CD45RC+ and CD45RC- T cells, it was demonstrated that each subset showed comparable survival, IL-2R expression and time courses of activation in response to alloantigen. The repertoire of neither subset was, therefore, deficient in terms of allorecognition. The rapid re-expression of CD45RC in culture was accompanied by a change in function: CD45RC+ “converts”, obtained by overnight culture of CD45RC- T cells, induced significantly higher graft-versus-host responses. Thus, the transition in culture from CD45RC- to CD45RC+ reflects a major functional reprogramming of the cell and not a trivial modulation of a surface antigen. 1 Introduction The leukocyte common antigen, CD45, can exist in a number of isoforms generated by alternative splicing of three (or possibly more) variable exons A, B and C [l-41. While B cells express a CD45 molecule encoding all three exons, CD45R isoforms are differentially expressed on T lymphocytes. Thus, higher molecular weight isoforms may be the products of different combinations of the three exons. A low molecular mass (180 kDa) isoform is gener- ated by splicing out all three variable exons. In humans, rats, mice and sheep, the expression of different isoforms of CD45R defines functionally distinct subsets of CD4 T cells [5-111. This was first documented in the rat using the mAb OX22 [8] which identifies an epitope of the CD45RC exon product [12]. CD45RC+ CD4 T cells produced more IL-2 [9], responded well to alloantigen and mitogen in vifro [8,9], induced high graft-versus-host (GVH) responses [8, 131 and were the most active in inducing skin allograft rejection in vivo [14]. In contrast, CD45RC- CD4 Tcells provided help for B cells in secondary antibody responses [9, 151, produced higher levels of IL-4 [16] and were deficient in inducing allograft rejection [ 141. It was not clear whether the different responses induced by CD45RC+ and [I 105721 Present address: Immunology Department, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA. * This work was supported by a grant to EBB from the Medical Research Council. C-pYang was supported by a grant from the British Heart Foundation. Correspondence: Eric B. Bell, Immunology Research Group, Medical School, Manchester M13 9PT. GB Abbreviations: TDL: Thoracic duct lymphocytes zyxwvut PLN: Popli- teal lymph node Key words: CD45RC re-expression / CD4 T cells / Cell function zyxwvu 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1993 CD45RC- CD4 Tcells to GVH and graft rejection repre- sented differences in allorecognition repertoires (a TcR deficiency) or in changes in the physiology of the Tcell. Many groups have shown that T cells, when stimulated in vitro, lose the high molecular mass isoforms (CD45RA, human; CD45RB, mouse) and synthesize the 180-kDa (CD45RO) molecule [6, 17-20]. It was suggested that this change in isoform expression represented a unidirectional transition from naive to memory Tcells [21-231. There are instances in vivo, however, when a switch in isoform occurs in the opposite direction. Recent work in the rat showed that CD4 Tcells leaving the thymus as recent thymic emigrants were CD45RC- and re-expressed CD45RC in the periphery within 7 days [14]. In addition, adoptive transfer to athymic nude rats established that the progeny of CD45RC- T cells re-expressed CD45RC some months later [ 13,241.These observations indicated that the expres- sion of CD45RC could be cyclical in vivo [25, 261 and not unidirectional as suggested by in vitro studies. In view of this apparent discrepancy, we have re-examined CD45RC isoform changes in vifro. Were there, for example, conditions that could be defined which favored a change from CD45RC- to CD45RC+? We examined the expres- sion of this isoform in both the presence and absence of alloantigen. The investigation documents a rapid in vitro switch of CD4 Tcells from CD45RC- to CD45RC+ and links this change to the acquisition of functional properties, demonstrable in vivo, associated with the higher molecular weight isoform. 2 Materials and methods 2.1 Animals The following inbred and congenic strains of rat were bred and maintained under conventional conditions in the OO14-2~80/93/0101-0103$3.50 + .25/0