T lymphocytes express multiple forms of the leukocyte common antigen CD45, transcribed by alternative usage of leukocyte-common antigen exons 4-6. Species-specific monoclonal antibodies against restricted epitopes (CD45R) of the antigen subdivide CD4 T cells into reciprocal subsets expressing either the high molecular weight isoforms CD45RA or RB or a molecule in which exons 4-6 have been spliced out (CD45R0). CD45R+ or RB+ CD4 T cells are potent in graft-versus-host reactions, and interleukin-2 related activities, whereas the CD45R0+ subset responds in vitro to recall antigens and provides help for antibody synthesis. It is unclear whether CD45R subsets derive from separate lineages, or are products of unidirectional or reversible differentiation. We show by transferring CD45R+ or CD45R- allotype-marked CD4 T cells into athymic nude rats that both subsets routinely generate cells of the opposite phenotype with a function that follows phenotype, not parentage. The recent equation of CD45R subsets as maturation stages representing 'naive' and 'memory' T cells is difficult to reconcile with this finding.

No abstract

Although the thymus is primarily noted for the export of T cells to the periphery, a small influx of cells has also been observed. It is still a matter of debate whether entry into the thymus depends on prior activation. The phenotypes, sources and degree of immigration are largely unknown. We monitored by quantitative immunohistochemistry the entry of cells from the periphery into the rat thymus in three experimental models. We injected i.v. recirculating, small, nonactivated CD4+ T cell subsets, often referred to as naive (CD45RC+) and memory or antigen-experienced (CD45RC-) cells, purified from thoracic duct lymph of allotype-marked donors, allotype-marked leukocytes released from spleen or lung transplants, or leukocytes labeled in the periphery for 12 weeks during the S-phase of the cell cycle by oral application of 5-bromo-2-deoxyuridine (BrdUrd). Early after i.v. injection (0.5 h), significantly more antigen-experienced (CD45RC-) CD4+ T cells entered the thymus, and by 24 h four times as many cells from the CD45RC- subset as from the CD45RC+ subset had entered the thymus and localized to the medulla. None of the thymic entrants expressed the interleukin (IL)-2 receptor. Following spleen transplantation approximately 40% of donor cells entering the thymic medulla were T cells and approximately 55% were B cells. In contrast, from a lung transplant, approximately 85% of peripheral immigrants were T cells and approximately 10% were B cells. After both procedures, a small number of NK cells and monocytes/macrophages were found among the immigrants (< 5%). Rats were fed BrdUrd continuously for 12 weeks, a procedure which labeled approximately 30% of peripheral lymphocytes but not cortical thymocytes. BrdUrd-labeled cells were localized almost exclusively to the thymic medulla and represented approximately 10% of medullary cells. Of the thymic immigrants approximately 50% were T cells, approximately 30% were B cells (including approximately 15% IgD+ cells), approximately 15% were NK cells and the remainder (approximately 5%) were monocytes/macrophages. Only a quarter of BrdUrd-labeled cells expressed the IL-2 receptor. The thymus is continuously infiltrated by both activated and nonactivated leukocytes from the periphery, including T cells, B cells, NK cells and monocytes. These immigrants are supplied by lymphoid and nonlymphoid organs in a characteristic subset composition. Their entry is facilitated by prior antigen experience or activation. Thus, the participation of the thymic medulla in general leukocyte traffic suggests a mechanism by which the T cell repertoire could potentially be modulated by the peripheral tissues.

The present investigation examines the localization and migration of purified T cell subsets in comparison with B cells, CD8 T cells and CD4+ CD8- single-positive thymocytes. CD4 T cell subsets in the rat are defined by mAb MRC OX22 (anti-CD45RC), which distinguishes resting CD4 T cells (CD45RC+) from those (CD45RC-) which have encountered antigen in the recent past--subpopulations often referred to as 'naive' and 'memory'. Purified, 51Cr-labelled CD45RC+ CD4 T cells broadly reflected the migration pattern of CD8 T cells and B cells. Early localization to the spleen was followed by a redistribution to mesenteric lymph nodes (MLN) and cervical lymph nodes (CLN), B cells migrating at a slightly slower tempo. There was almost no localization of these subpopulations to the small or large intestine [Peyer's patches (PP) excluded]. In contrast, CD45RC- CD4 T cells (indistinguishable in size from the CD45RC+ subset) localized in large numbers to the intestine; they were present here at the earliest time point (0.5 h), persisted for at least 48 h but did not accumulate, indicating a rapid exit. Numerically, localization of CD45RC- CD4 T cells in the MLN could be accounted for entirely by afferent drainage from the intestine. Unexpectedly, CD45RC- CD4 T cells (but not other subsets) localized and accumulated in the thymus. In vivo treatment with mAb HP2/1 against the integrin alpha 4 subunit inhibited almost entirely CD45RC- CD4 T cell migration into the PP (98.1%), intestine (87.1%), MLN (89.1%) and thymus (93.5%); migration into the CLN was only reduced by half. To distinguish between recognition of MAdCAM-1 and VCAM-1 by alpha 4-containing integrins, recipients were treated with mAb 5F10 against rat VCAM-1. Except for the thymus and a small reduction in CLN, localization of CD45RC- CD4 T cells was unaffected; entry to the thymus was almost completely blocked (92.3%) by anti-VCAM-1. The results indicated (i) that CD45RC- CD4 T cells alone showed enhanced localization to the gut and PP, probably via alpha 4 beta 7-MAdCAM-1 interaction; (ii) that many CD45RC- cells entered non-mucosal LN independently of alpha 4 integrin or VCAM-1; and (iii) that entry of mature recirculating CD45RC- CD4 T cells into the thymus across thymic endothelium was apparently regulated by alpha 4 integrin-VCAM-1 interaction.

CD4 T cells bearing high (240-190 kDa) and low (180 kDa) molecular mass isoforms of the leukocyte common antigen CD45 define functionally distinct subsets which have been equated with naive and memory T cells. In the rat, CD4 T cells expressing a high molecular mass isoform [identified by monoclonal antibody MRC-OX22 (anti-CD45RC)] exchange this for the 180 kDa molecule (CD45RC-) when stimulated by antigen. Here we show, by transferring mature allotype-marked CD45RC- CD4 T cells (depleted of immature Thy-1+ CD45RC- recent thymic emigrants) into normal euthymic recipients, that many T cells re-express the high molecular mass isoform in less than 6 h. By 24 h, 30-60% of CD45RC- CD4 T cells became CD45RC+; within a week the entire cohort appeared to exchange the low for the high molecular mass isoform. Isoform exchange was dynamic and many CD4 T cells returned once again to the CD45RC- state. CD45RC- CD4 T cells declined in number more rapidly than the CD45RC+ subset after transfer. The results suggest that CD45R isoforms distinguish between resting T cells (CD45RC+) and those which have encountered antigen in the recent past. CD45R isoforms would appear to be unsuitable markers of naive and memory T cells.