Development of T lymphocytes within the thymus and within thymic nurse cells.
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Biomedical subjects
K Shortman
Publications and source records attributed to K Shortman.
Clonal expansion of T cells: a cytotoxic T-cell response in vivo that involves precursor cell proliferation.
The response of peritoneal exudate lymphocytes to allogeneic tumor cells was used to determine whether the in vivo generation of cytotoxic T cells (CTL) involved the proliferation of precursor cells. Ten days post-injection, both cytotoxic activity and the formation of conjugates between lymphocytes and target cells were shown to be specific for the immunizing tumor alloantigens and to be effected by Ly-2+ cells. A cell-sorting-based procedure was developed to isolate specific conjugates between red-fluorescence-tagged CTL and blue-fluorescence-tagged tumor target cells. When [3H]thymidine was administered during the response, almost all isolated conjugate-forming CTL were 3H-labeled on autoradiography. Thus, the CTL were clearly products of dividing cells, a result that contradicts published data. Reassessment of a previously studied system, which suggested that CTL were not products of cell division, indicated that in that system many of the conjugate-forming cytotoxic cells studied were Ly-2- and nonspecific, and thus perhaps not T cells. We conclude that the clonal selection model is applicable to at least one in vivo T-cell response.
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Thymus hormones do not induce proliferative ability or cytolytic function in PNA+ cortical thymocytes.
A variety of thymus hormone preparations, as well as drugs known to perturb cell differentiation, were tested for their ability to induce nonfunctional cortical thymocytes to become functional precursor cells. Murine cortical thymocytes, defined as the high peanut agglutinin (PNA) binding or as the low H-2K, major [86%] thymocyte subpopulation, were isolated by fluorescence-activated cell sorting. Their function was assessed in a high cloning efficiency, growth factor saturated, concanavalin A-stimulated limit-dilution culture system, determining the number of precursors of extended clones (PTL-p), or determining with a lectin-mediated tumor-lysis readout the number of precursors of cytolytic clones (CTL-p). The hormone preparations tested were crude or partially purified culture supernatants from thymus "epithelial" monolayers (TES), soluble extracts of thymic nonlymphoid tissue (STF), semipure thymus humoral factor (THF), and the pure peptides thymopoietin 32-36 (TP5) and "facteur thymique sérique" (FTS). These preparations were either added directly to the limit dilution cultures, or were first preincubated with the cells, which were then subjected to limit-dilution culture. In no case did the hormone preparations cause any increase in the level of PTL-p or CTL-p in the PNA+ or low H-2K thymocyte population, even though a conversion of only a few percent to functional cells could have been detected. Two possible explanations are considered. One is that the main function of these materials is to control post-thymic peripheral T cells, rather than to induce intrathymic differentiation. Another is that the typical cortical thymocyte is beyond the stage at which thymocytes can be induced by hormones, a view that is strengthened by the failure of either 5-azacytidine or the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate to activate these cells. In this latter explanation the true intrathymic target of hormone action may be an earlier, and very minor, thymus subpopulation.
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Degradation of specificity in cytolytic T lymphocyte clones: two broad specificity, H-2-independent recognition systems, one natural killer-like, develop during culture, in addition to the clonally distributed antigen-specific receptor.
Ly-2+ CBA mouse T lymphocytes stimulated with concanavalin A in limiting dilution culture produce clones of cytotoxic T lymphocytes (CTL) which, although initially specific, eventually lyse a wide range of target cells. The nature of the recognition system for this apparently "nonspecific" cytolysis was examined using a range of tumor cells as labeled targets and as cold target inhibitors. Most syngeneic and allogeneic murine tumor cells were lysed but the degree of lysis varied, even for different sublines of the same tumor. All tumor cells cold target inhibited their own lysis, and cross-inhibited lysis of other targets to varying degrees. The recognition stage of "nonspecific" cytolysis appeared to be independent of target cell H-2 expression; some H-2-negative murine target cells were lysed and some were not, but all gave cold target inhibition of "nonspecific" cytolysis. Xenogeneic tumor cells were resistant to lysis, but some nevertheless gave cold target inhibition of the "nonspecific" cytolysis of murine targets. A study of the specificity of cold target cross-inhibition revealed two distinct patterns of recognition which existed simultaneously in "nonspecific" CTL; one was like that of natural killer cells and was directed to targets such as YAC-1, the other was distinct from that of natural killer cells and was directed to targets such as P815. Thus, murine CTL may express three distinct receptors, the clonally distributed, H-2-restricted, antigen-specific T cell receptor and two different "broad-range" receptors common to most clones.
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The limited immunocompetence of thymocytes within murine thymic nurse cells.
Thymic nurse cells, cortical epithelial cells enclosing 20-200 lymphocytes, were prepared from mouse thymus by enzyme digestion and repetitive sedimentation. Individual nurse cells were then isolated free of any exogenous thymocytes by micromanipulation, and the endogeneous thymocytes released from inside the nurse cells by a brief period of culture. The thymocytes from within individual nurse cells were tested, at the one cell/well level, for their capacity to proliferate in high cloning efficiency mitogen-stimulated limiting dilution cultures. The resultant clones were tested for their cytolytic capacity in a lectin-mediated isotype-release assay. Most intra-nurse cell thymocytes were unresponsive, like typical cortical thymocytes, but an average of 1/30, or around 2-6 lymphocytes/nurse cell, were able to proliferate in response to concanavalin A. The clones produced were of a relatively small size, similar to those characteristic of helper-lineage T cells. No cytolytic clones at all were obtained, despite stringent positive controls showing an efficient cytolytic response from known sources of cytolytic precursor cells. This finding disagrees with earlier studies on nurse cell lymphocytes, where there may have been a possibility of contamination with exogenous thymocytes. These results suggest either that the nurse cell represents a selective environment for helper-lineage T cell differentiation, or that further steps after the nurse cell stage are needed to produce mature cytolytic-lineage T cells.
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The role of the thymic cortex and medulla in T cell differentiation.
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Zonal unit-gravity elutriation. A new technique for separating large cells and multicellular complexes from cell suspensions.
A new and simple technique, zonal unit-gravity elutriation, has been devised for separating very large cells, multicellular complexes, or small organisms from suspensions consisting mainly of small cells. The separation vessel is a conical chamber with an entrance at the lower, narrower part of the cone and an exit at the upper, wider part of the cone via a dome-shaped lid. A baffle at the entrance prevents turbulence from incoming fluid. Chambers of differing widths and wall slopes are chosen depending on the sedimentation rate of the particles to be separated. A small volume of the cell suspension is placed in the chamber on the bench in a cold-room. Medium stabilized by a shallow density gradient is pumped into the base of the chamber and ascends, creating a decreasing velocity gradient. Cells sediment at unit-gravity against this ascending counterstream, and are separated into bands according to sedimentation velocity. By adjusting the flow rate of the medium, different sizes of cells can be separated. Tumor cells can be enriched, and larger blast cells can be separated from small cells in lymphoid cell suspensions. The procedure produces complete separation of thymic nurse cells (epithelial-lymphoid complexes) from free thymocytes in digested thymus suspensions and produces substantial enrichment of thymic rosettes (macrophage-lymphoid complexes). A very favorable situation for applying this technique is the isolation of Taenia taeniaformis larvae, which can be completely purified from infected liver suspensions, representing a 4 X 10(5)-fold enrichment of the parasites, with high recovery, in a single 30 min operation.
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Are any functionally mature cells of medullary phenotype located in the thymus cortex?
Experiments were undertaken to test if thymocytes of "mature" or "medullary" phenotype were restricted to the medullary area of the thymus. A calculation based on direct cell counts on serial sections indicated that 11.5% of adult male CBA thymic lymphoid cells were within the medullary zone. Since only 3-4% of thymocytes were cortisone resistant, the majority of thymocytes within the medulla were, like cortical thymocytes, cortisone sensitive. A series of cell surface antigenic markers, used alone or in pairs, suggested that 13-15% of thymocytes were of medullary phenotype, somewhat more than the number of thymocytes actually present in the medulla. However, much of this discrepancy could be explained by differential death of cortical cells during isolation and staining, and by the existence in the cortex of a subpopulation of early blast cells which shared some, but not all markers with medullary thymocytes. A direct test for mature or medullary phenotype cells in the cortex involved selective transcapsular labeling of outer-cortical cells with fluorescent dyes, followed by multiparameter immunofluorescent analysis of the 10% labeled population. Outer-cortical thymocytes included some cells (mainly early blasts) sharing some markers with medullary thymocytes, but very few (less than 1%) of these cells expressed all the characteristic "mature" markers. Limit-dilution precursor frequency studies showed the level of functional cells in the outer cortex was extremely low. The overall conclusion was that the vast majority of cells of complete "mature" phenotype are confined to the thymic medulla. These findings favor the view that thymus migrants originate from the thymic medulla, but do not exclude a cortical origin. The results also illustrate the need for multiparameter analysis to distinguish medullary thymocytes from early blast cells.
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Identification of early stages of T lymphocyte development in the thymus cortex and medulla.
Thymocyte subpopulations with a phenotype suggesting they are early stages of T cell development in the adult mouse thymus were characterized and isolated by using multiparameter flow cytometry and sorting, in conjunction with selective killing with antibody and complement (C). The intrathymic localization of these subpopulations was assessed by dipping the thymus in fluorescent dyes to selectively label outer-cortical cells. The main phenotypic markers used were sensitivity to C-mediated lysis by the monoclonal antibody B2A2 (which spares most prothymocytes but kills most thymocytes), the expression of the T cell lineage specific markers Ly-2 and L3T4, and the levels of the common T cell antigens Ly-1 and Thy-1. A preliminary selection for cells lacking Ly-2 and L3T4, or resistant to B2A2 and C, produced a population of large cells, only 5% of all thymocytes and distinct from the typical cortical blast cells. This population of putative early thymocytes was itself heterogeneous, consisting of eight subpopulations separable by phenotype and intrathymic localization. One group of two subpopulations (B2A2-, Ly-1++, Thy-1+ and either Ly-2+ L3T4- or Ly-2- L3T4+) appeared to be of medullary location, and their phenotype suggested they could have been early members of the medullary lineages. Another group of two subpopulations (B2A2-, Ly-1++, Thy-1-, Ly-2-, L3T4- and B2A2-, Ly-1++, Thy-1+, Ly-2- L3T4-) did not show a clear localization pattern and may have represented cells in an earlier stage of transition to medullary phenotype and location. A quite different group of three subpopulations (B2A2++, Ly-1-, Thy-1-, Ly-2- L3T4-; B2A2++, Ly-1-, Thy-1+, Ly-2-, L3T4-; and B2A2++, Ly-1+, Thy-1++, Ly-2- L3T4-) was concentrated in the outer cortex and seemed to represent a series of stages of a cortical pathway, before the typical cortical blast cells. Finally, a very minor subset (0.2% of thymocytes), lacking all these markers, was concentrated in the outer cortex; this fifth group had the phenotype expected of the earliest intrathymic precursor cells. The results suggest that the separate developmental streams of cortical and medullary thymocytes may be traced back, via these minor early blast subpopulations, to common precursor cells in the outer cortex.
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Contribution of thymus lymphocytes to the peripheral lymphoid tissues and the effect of antigen on the rate of cell exit from the thymus.
Some of the important questions concerning the development of T cells in the thymus can be answered by a study of the different thymocyte subpopulations and a comparison of their properties with those of the cells exported to the peripheral lymphoid tissues. What is the relationship between cortical and medullary thymocytes? Why do most cortical cells die? Which subpopulation gives rise to thymus migrants? How many cells are exported from the thymus? Are the exported cells fully mature? Are any of these functions affected by antigen stimulation or other peripheral events? In this paper we review the background to some of these questions and focus on the effect of peripherally administered antigen on the export of cells from the thymus. Experimental data are presented which suggest that the overall rate of emigration is not grossly affected by large doses of intravenous protein antigens. Nor is there any obvious qualitative change, at least in terms of the size of the cells released. The possibility of changes in the specificity of the exported cells is discussed, but as yet there are no data which throw light on this point.
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T-cell development in the absence of a thymus: the number, the phenotype, and the functional capacity of T lymphocytes in nude mice.
A small but definite proportion of T-lymphocyte-like cells have been reported in nu/nu (nude) mouse spleen despite the congenital absence of a thymus in these animals. We have determined the number and the characteristics of such cells using flow cytometry. The level of T-like cells increased with age. In 4-month-old nu/nu CBA spleen, 14% of all cells expressed some Thy 1 antigen. However, only 4% expressed mature T-cell levels, and only the 2% with the highest Thy 1 also showed a normal distribution of Ly 1 and Ly 2 antigens. These T-like cells were slightly larger than normal nondividing T lymphocytes. We have assessed the total functional capacity of T-like cells in nu/nu CBA spleen using a high-cloning-efficiency limit-dilution culture system. Almost all precursor cells capable of forming clones when stimulated with concanavalin A in the presence of irradiated spleen cells and growth factors, and almost all precursors of those clones that were cytolytic in a lectin-mediated tumor-cell-lysis assay, were within this 2% subpopulation of nu/nu spleen cells with mature T-cell markers. Increased levels of purified interleukin 2 failed to induce further precursor function, indicating that maturation of pre-T cells was not obtained. However the nu/nu spleen cells bearing mature T-cell markers displayed only 10-30% of the cloning efficiency of normal splenic T cells. The majority of nu/nu spleen T-like cells, even within this phenotypically "normal" subset, appeared to be nonfunctional. We conclude that the absence of a thymus leads to qualitative, as well as quantitative, deficiencies in the T-cell population, and various interpretations are discussed.
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Degradation of specificity in cytolytic T lymphocyte clones: mouse strain dependence and interstrain transfer of nonspecific cytolysis.
Limiting-dilution culture of murine Ly-2+ T cells with concanavalin A (Con A) and irradiated spleen filler cells produces, with high efficiency, cytolytic T lymphocyte (CTL) clones. With most mouse strains (including CBA and C57BL/6) the specificity of these CTL clones drops after day 6 of culture, so that by day 9 the majority of clones can lyse most murine target cells, whether syngeneic or allogeneic. The rate of specificity degradation and relative target cell preference varies with the mouse strain. Some strains (e.g. BALB/c) do not show this effect and CTL clones remain specific to day 9. Many low natural killer (NK) cell strains (e.g. C57BL/6J.bg) maintain CTL specificity in such cultures, but the correlation between CTL specificity and NK status is not absolute. Growth of BALB/c precursor cells on CBA filler cells leads to specificity degradation in the BALB/c CTL clones; however, the specificity of CBA-derived CTL clones is not maintained by growth on BALB/c fillers. The results suggest that specificity degradation is induced in the developing CTL-clone by factors in the culture environment, perhaps a soluble lymphokine (a differentiation factor) or by an infectious agent (an endogenous mouse virus). Although such CTL specificity loss may render many limiting dilution studies of the CTL specificity repertoire invalid, the problem may be bypassed by an appropriate choice of mouse strain.
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Analytical and preparative equilibrium density separation of lymphoid cells on albumin and metrizamide.
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Ly 2 positive cytotoxic T lymphocytes, whether specific or non-specific in lytic activity, may express a large, granular, vacuolated lymphocyte morphology.
Cytotoxic clones consisting entirely of large, vacuolated granular lymphocytes (LGL) are produced with high frequency when individual purified mature Ly 2+ T cells are cultured at limit dilution in the presence of concanavalin A and irradiated spleen filler cells. Similar cells are produced in cultures of Ly 2- T cells but in lower proportion, the level of granular lymphocytes amongst the largely non-cytotoxic, Ly 2- L3T4+ product cells being only 20%. A proportion (15%) of LGL are also found in conventional mixed lymphocyte cultures. In the limit-dilution cultures of Ly 2+ T cells the LGL originate from the single precursor cell and not from the irradiated filler cells. They bear the markers expected of active cytotoxic T lymphocytes (Thy 1++; PNA++; Ly 1+; Ly 2++; L3T4-). Their DNA and chromosome complement appear normal. The granules within the cells are heterogeneous in form, and some resemble in ultrastructure those described in certain cytotoxic, suppressor and NK-cell lines. The granules do not show mast cell staining characteristics. The LGL are not phagocytic. Although LGL are associated with nonspecific cytotoxicity late in culture, the first granular cells appear early (day 3) and dominate in the clones at times when cytolysis is specific. LGL appear to be the normal morphological form of cytotoxic T lymphocytes when grown under these conditions.
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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.
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The functional capabilities of cells leaving the thymus.
There has been a controversy for many years over the functional status of cells that leave the thymus (thymus migrants) to populate the peripheral lymphoid organs. Are they immunoincompetent like cortical thymocytes and so probably derived from them, or are they functionally mature like medullary thymocytes? Until recently the techniques used to assess putative thymus migrants have been indirect, but it is now possible to measure the function of recent thymus migrants directly. We used intrathymic injection of a solution of fluorescein isothiocyanate to label thymocytes, and used electronic cell sorting to purify the fluorescent cells that accumulate in the periphery over the following 3 to 4 hr. The migrants have been enriched from an original frequency of about 1:1000 in lymph nodes and spleen, to greater than 98% purity. These cells have been compared with normal peripheral T cells for proliferative and cytotoxic precursor activity in a high cloning efficiency, lectin-induced, limit dilution culture system and in an allospecific limit dilution system. The frequency of precursors of proliferative lymphocytes and cytotoxic lymphocytes and the size of the clones produced is the same for recent migrants and peripheral T cells. Thus by the criteria of proliferation and cytotoxic responses to mitogens and generation of allospecific CTL, thymus migrants, a few hours after their emigration from the thymus, are fully immunocompetent; we therefore see no evidence of a post-thymic precursor-type cell that requires major maturation steps after leaving the thymus.
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Loss of specificity in cytolytic T lymphocyte clones obtained by limit dilution culture of Ly-2+ T cells.
Nonspecific cytotoxicity developed reproducibly and with high frequency in limit dilution cultures consisting of low numbers of murine cells stimulated with concanavalin A in the presence of growth factors and irradiated filler cells. The individual clones in cultures showing nonspecific killing were all derived from single, Thy-1+, Ly-2+ cells. At early times of culture (day 5 or 6), clones appeared to be specific in their lytic activity, as expected of cytolytic T lymphocytes (CTL). On continued culture (day 8 or 9), most of the originally specific CTL clones became nonspecific, killing a range of murine target cells, both syngeneic and allogeneic. The lack of specificity was observed at all effector cell doses. The effector cells responsible for the nonspecific cytolysis were Thy-1+ and Ly-2+, as were most cells in the cultures. The effector cells had the normal DNA content for a dividing T cell population, and most cells in the cultures had a normal chromosome complement. In mixed cultures in which the responder cells and the irradiated filler cells were from different mouse strains, the nonspecific killers displayed the Thy-1 and H-2 allotypes of the responder, and not of the filler cells. The development of a broad cytotoxic potential appears to be a normal and rapid event when Ly-2+ T cell-derived CTL-clones are grown under these conditions; this is a caveat for the use of limit dilution cultures to determine the T cell specificity repertoire. The relationship between these nonspecific CTL, activated lymphocyte killers, and natural killer cells is discussed.
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Thymus cell migration: analysis of thymus emigrants with markers that distinguish medullary thymocytes from peripheral T cells.
Peripheral T cells and cells from the thymic medulla have many phenotypic characteristics in common, and both populations are quite distinct from cortical thymocytes. The cells that migrate out of the thymus (thymus migrants) may be detected in the periphery as fluorescent cells shortly after intrathymic injection of fluorescein isothiocyanate. Our previous work has established that they resemble T cells and medullary thymocytes rather than cortical thymocytes, and are fully functional. In this report, we consider several phenotypic characteristics that differ between medullary thymocytes and peripheral T cells, namely size, buoyant density, and sensitivity to a new monoclonal antibody (B2A2) and complement. By these criteria, the fluorescence-labeled thymus migrants in spleen and lymph node tend to resemble medullary thymocytes and are distinct from the majority of T cells that surround them. This suggests, but does not prove, that migrants originate in the medulla. After adult thymectomy, the small population of medullary-thymocyte-like cells disappears from spleen and lymph node, further supporting the idea that cells leave the thymus with a medulla-like phenotype, and acquire normal peripheral T cell phenotype only after their arrival in the periphery. Thus, although thymus migrants appear to leave the thymus in an immunocompetent state and are phenotypically mature by most criteria, there are a few final maturation steps that occur after arrival in the periphery.
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