Roland Scollay

T-Cell Development in the Adult Murine Thymus - Changes in the Expression of the Surface-Antigens Ly2, L3T4 and B2A2 During Development From Early Precursor Cells to Emigrants

Many (perhaps all) of the key events in T cell development take place within the thymus. Thymic lymphocytes are believed to be immature T cells at various stages of development. According to these tenets it should be possible to classify thymocytes into distinct subpopulations, and then order these groups into a sequence representing the intrathymic T cell development pathway. Certain T cell antigens are expressed differentially on the surface of thymocytes, and these have been used to define thymus subpopulations. Recent attempts to classify thymocytes in this way have produced a reassuring degree of agreement between different laboratories (reviewed by Smith 1984). However, there is no such consensus on the ordering of these subpopulations in a developmental pathway, the only point of agreement being that many of the earlier schemes must be wrong. In this review we survey our own work on the definition of thymocyte subpopulations using monoclonal antibodies directed against T cell surface antigens. We begin with a summary of earlier work on antigens used to distinguish cortical and medullary cells (reviewed in detail in Scollay & Shortman 1983), and then present more recent studies on antigens which help to further classify thymocytes

Regulation of TCR α and β gene allelic exclusion during T-cell development

Abstract Early in their development, most T cells become committed to the expression of one, and only one, TCR αβ combination. How do T cells achieve this TCR allelic exclusion? This article discusses the configuration and expression of TCR α and β genes in mature T-cell lines and TCR αβ transgenic mice, and proposes three nonexclusive models to account for the significant occurrence of T cells with two productive α gene rearrangements.

Developmental status and reconstitution potential of subpopulations of murine thymocytes

In this chapter we have summarized our view of the subsets of murine CD4- CD8- thymocytes which can be identified with a range of monoclonal antibodies. We have shown the division rate and turnover time of the main subsets and have listed what we know of the TcR gene rearrangement, and expression at the RNA and protein levels. We have been unable to completely segregate gamma delta-TcR-expressing cells from alpha beta-TcR-expressing cells by any of the markers we have used, although the proportions of the two receptor forms vary widely in the different subsets. Experiments involving intrathymic transfer of the CD4- CD8- subsets are described, which indicate that all the TcR- subsets of the CD4- CD8- thymocytes display some precursor activity and which suggest a progression of at least five stages through the TcR- subpopulations of CD4- CD8- cells. The earliest precursor is a Thy 1 low, HSA low, Pgp-1 high cell which has unrearranged C beta and is non-dividing and which closely resembles the bone marrow prothymocyte. The later precursors are Thy 1 high, HSA high, Pgp-1 low, have rearranged C beta and are rapidly dividing. We tentatively conclude that none of the TcR+ CD4- CD8- cells are precursors of the major thymocyte subsets or of typical peripheral T cells, and we have found no evidence so far of separate precursors for the different mature subsets of thymocytes or peripheral T cells.

Dynamics of early T cells : prothymocyte migration and proliferation in the adult mouse thymus

The object of this review has been to consider precursor cell migration into the normal adult thymus, using the mouse model. We have presented a series of experiments and discussed them in the context of other relevant experiments in the literature. The conclusions, qualified in the text, can be summarized as follows: There is a continual input of precursor cells into the normal undepleted adult thymus. The daily input of precursors under normal circumstances is very low (e.g. several per day). Once a precursor enters the pool of proliferating cells inside the thymus, its proliferation is limited to only several weeks. There is no permanent endogenous stem cell. There are a number of different precursor microenvironments in the thymus with different controls, since the kinetics of early (bone marrow-derived) and late (thymus-derived) precursors is quite different. All of these points require further analysis, and we have presented a minimal model as a basis for further experiment.

Intrathymic events in the differentiation of T lymphocytes: a continuing enigma

The thymus plays an important role in the differentiation of T lymphocytes, but the exact nature of this role remains unclear. As Roland Scollay shows here, a careful analysis of available data shows how poorly we understand the role of the thymic cortex and medulla and how little we know of the exact processes which give rise to thymus-dependent peripheral T cells.

Limit-dilution assay and clonal expansion of all t cells capable of proliferation.

A limit-dilution microculture system is presented in which almost all mature T cells, cultured at a level of about 1 cell/well, grow and expand to clones averaging 60,000 cells over an 8-9 day period. Cloning efficiency is 70-100%, so the set of the expanded clones is representative of the starting T-cell population. T cells of all Lyt phenotypes form clones of progeny cells. The system involves culture in flat-bottom microtitre trays, in the presence of concanavalin A as the initiating stimulus, together with appropriately irradiated spleen filler cells and a supplementary source of soluble T cell growth factors. The resultant clones may be screened for cytolytic function, as described in the accompanying paper. The system may be used to assay the level of T cells capable of expansion or precursor function (PTL-p) by using [3H]TdR uptake as a readout for the presence or absence of proliferating clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of complicating suppressor or helper effects.

Resistance to cutaneous leishmaniasis in nude mice injected with L3T4 + T cells but not with Ly-2 + T cells

The importance of T cells in resistance to infection with the intracellular protozoan parasite Leishmania major is substantiated by the susceptibility to infection of athymic nude mice of both resistant and susceptible strains. However, the relative roles of different T cell subpopulations remain controversial. In order to address this issue, selected L3T4+ Ly‐2− or L3T4− Ly‐2+ T cell subpopulations from normal mice were adoptively transferred into athymic nude recipients of the same strain, and their capacity to mediate host‐protective immunity against infection with L. major promastigotes was determined. In experiments with mice of different inbred strains, reconstitution with L3T4+ Ly‐2− cells rendered the nude mice completely resistant to cutaneous leishmaniasis, whereas L3T4− Ly‐2+ cells failed to do so. Partial protection in some recipients of large numbers of Ly‐2+ cells could be ascribed to contamination of the transferred inoculum with L3T4+ cells. Thus, resistance to L. major infection in reconstituted nude mice can be promoted by L3T4+ T cells in the absence of detectable Ly‐2+ T cells.

T-cell subset relationships in thymocyte development.

During the past couple of years there has been significant progress in our understanding of the development of different lineages of T cells within the thymus. Pathways, subpopulations and cellular dynamics are all becoming clearer. Signal transduction through primary and accessory receptors is also beginning to be understood. However, the exact nature of the events that lead uncommitted cells to choose a particular lineage (either alpha beta/gamma delta or CD4+/CD8+) has still not been determined.

Semi-automated limit-dilution assay and clonal expansion of all t-cell precursors of cytotoxic lymphocytes.

Abstract A limit-dilution microculture system is described, where almost all precursor T cells of the cytotoxic lineage (CTL-p) develop into extended clones of cytotoxic T cells (CTL), which are then detected with a new radio-autographic 111 In-release assay. The principle is to polyclonally activate all T cells with concanavalin A, to expand the resultant clones over an 8–9 day period in cultures saturated with growth factors, then to detect all clones with cytotoxic function by phytohaemagglutinin mediated lysis of P815 tumour cells. The key variables for obtaining high cloning efficiency are the use of flat-bottomed 96-well culture trays, the use of appropriately irradiated spleen filler cells, and the inclusion of a T-cell growth factor supplement. Cultures are set up at input levels of around one T cell per well. Forty percent of T cells then form CTL clones readily detected by the cytotoxic assay. The lytic activity of the average clone is equivalent to 3000 CTL, but clone size appears to be much larger. The precursor cells are predominantly if not entirely from the Lyt 2 + T-cell subclass and almost all cells of this subclass form cytolytic clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of the CTL-p frequency estimates being distorted by helper or suppressor effects. The assay system can be automated by the use of 96-channel automatic pipettors throughout, so large numbers of cultures can be set up for accurate CTL-p frequency determinations. The method is suitable for accurately estimating the total number of functional T cells of the cytotoxic class in developing T-cell populations, and as a future development for scanning their specificity repertoire.

CD4+ CD8+ cells are rare among in vitro activated mouse or human T lymphocytes

The predominant cell type in the thymus expresses both of the function-associated T cell surface markers, CD4 and CD8, but CD4+ CD8+ cells are rare or absent outside the thymus. Double expression has therefore been assumed to be an indication of immaturity. However, recent reports have suggested that CD4+ CD8+ cells can appear in cultures of activated mature cells. We have therefore activated human peripheral blood lymphocytes and mouse spleen cells, lymph node cells, and cortisone-resistant thymocytes using a number of different stimulation regimes, and we have analyzed them at various times for CD4 and CD8 expression. In all cases, upon analysis of cultured cells by flow cytometry, CD4+ CD8+ cells were rare. A combination of microscopic analysis, cell sorting followed by microscopic analysis, and careful staining controls demonstrated that even when flow cytometry showed some CD4+ CD8+ cells, most of these were artifacts in the form of doublets or clumps of single positive cells or dead cells. Taking this into account, CD4+ CD8+ cells made up less than 1% in the mouse and less than 3% in the human T cell cultures at any time periods. We therefore found no evidence for the generation of large numbers of CD4+ CD8+ cells in cultures of mouse or human T cells.