Immunology. Death in the thymus.
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Biomedical subjects
K Shortman
Publications and source records attributed to K Shortman.
Thymic stem cells in mouse bone marrow.
There is still controversy concerning the nature of the stem cells from bone marrow that colonize the thymus during embryogenesis and continually throughout life. To identify the bone marrow stem cells that home to and populate the thymus, we screened murine bone marrow cells for the presence of a population of surface phenotype similar to the earliest known intrathymic precursor. We have identified a population characterized by expression of an intermediate level of heat stable antigen, a very low level of Thy-1, and high levels of CD44 and class I major histocompatibility complex antigens. It is negative for B-cell, granulocyte, macrophage, and erythrocyte markers (B220, Gr-1, Mac-1, and TER 119). All these markers are common to the intrathymic precursors and bone marrow stem cells. However, this new bone marrow population is Sca-2+, similar to the intrathymic precursor, which makes a clear distinction from the Sca-2- bone marrow hematopoietic stem cells previously characterized. The frequency of the new population in the normal mouse bone marrow is about 0.25%. When transferred intrathymically or intravenously to lethally irradiated mice, it has a higher expansion potential (2 x 10(5)) than has been found for the intrathymic precursors (10(3)), but less than was found for the Sca-2- multipotent stem cell (10(7)). These transfer studies also showed that it was pluripotent, in that its precursor activity was not restricted to the production of T or B lymphocytes. However, it gave a reduced spleen colony number and smaller colonies (day-12 colony-forming unit spleen) when compared with multipotent stem cells. Thus, the cell we have identified appears to be the latest pluripotent cells so far identified in bone marrow and is therefore a good candidate for a bone marrow prothymocyte, but it appears not to be T-cell-committed.
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Small cortical thymocytes are subject to positive selection.
To determine the developmental stages at which positive selection can act to produce mature T cells, CD4+8+3lo thymocytes of large dividing type and of small nondividing type were sorted and transferred into the thymus of nonirradiated Thy-1 congenic recipient mice. In contrast to earlier studies, the small as well as the large thymocytes produced mature CD4+8-3hi and CD4-8+3hi progeny, although production was less efficient from the small cells. The relative efficiency of small cells was increased and was close to that of large cells when bcl-2/anti-HY T cell receptor (TCR) alpha beta transgenic donors were used to improve cell survival, overcome stress effects of the transfer process, and increase the frequency of selectable cells. The results from transferring small CD4+8+3lo thymocytes expressing a TCR transgene from a nonselecting to a selecting thymic MHC environment also confirmed that the small cells were capable of being selected and maturing. Thus the developmental window available for positive selection includes the small CD4+8+3lo thymocytes. The results also showed a striking difference in the kinetics of production of mature progeny from the transferred CD4+8+3lo precursors. CD4+8-3hi cells appeared several days before CD4-8+3hi cells, apparently because the CD4-8+ lineage cells spent several days in transit as CD4+8+3hi intermediates before losing CD4. Most CD4+8- lineage cells on the other hand, either passed very rapidly through this intermediate stage, or lost CD8 before increasing the expression of CD3.
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Assessment of CD4 expression by early T precursor cells and by dendritic cells in the human thymus.
The adult mouse thymus contains a minute population of early lymphoid precursor cells that express moderate levels of CD4. We searched for a corresponding population of early T precursors in the infant human thymus, by first depleting the majority of more mature thymocytes, then using immunofluorescence and flow cytometry to analyze cells bearing a range of early T lineage markers. No discrete population of early T precursors expressing CD4 was observed, in contrast to the murine thymus. Most putative very early human thymocytes were CD4-8-3-1-2lo44+34+7hi class I MHChi class II MHC-. However, a distinct population of human thymic dendritic cells expressing high levels of CD4 was isolated. These were CD4hi8-3-1-2-44+34-7- class I MHChi class II MHChi, and lacked markers of B cells, NK cells, or myeloid cells. They were large cells that exhibited dendritic morphology after brief periods of culture, and they were efficient stimulators of allogeneic T cells. The biologic implications of CD4 expression by thymic dendritic cells are discussed.
Adolescent↗
CD4 and CD8 expression by human and mouse thymic dendritic cells.
Dendritic cells (DC) from human and mouse thymus were compared. DC from both sources were isolated by digestion with collagenase, disruption of cellular complexes with a chelating agent, selection of light density cells, immunomagnetic bead depletion of other cell types (without depletion with anti-CD4 or anti-CD8) and finally sorting for cells expressing high levels of class II MHC. Yields of DC from human and mouse thymus were comparable (around 1 DC/10(3) thymocytes), they displayed similar DC morphology, and both showed strong expression of CD11c. DC from the human thymus all expressed very high levels of CD4 but low levels of CD8. In contrast, DC from the mouse thymus expressed high levels of CD8 but only low levels of CD4. Human thymic DC were also substantially larger than mouse thymic DC. The biological significance of CD4 and CD8 expression by DC is discussed in view of this major species difference and the possibility that human thymic DC may be targets for HIV infection.
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Intrathymic lymphoid precursor cells during fetal thymus development.
Our previous studies have demonstrated the presence, in the adult mouse thymus, of a population of early precursor cells able to give rise to T and B lymphocytes but not myeloid cells. This population of cells expresses low levels of CD4 and has been termed the "low CD4 precursors." All these precursors were found to be c-kit positive, and they precede the better known CD4-CD8- precursor stage. In this study, embryonic and neonatal thymuses were examined to see whether a similar low CD4 precursor was part of the pathway of T cell development during ontogeny. A population with the phenotypic characteristics of the adult low CD4 precursor was found from day 15 of embryonic development, although the expression of low levels of CD4 was apparent only from embryonic day 17. Functional tests of these putative precursors showed they had no thymus-reconstituting ability when isolated from thymuses at any time during embryonic life, and very low reconstituting ability even 24 days after birth. These results raise questions about the adult low CD4 precursor as an obligatory stage in the development of T cells in the thymus.
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Multiple rearrangements in T cell receptor alpha chain genes maximize the production of useful thymocytes.
Peripheral T lymphocytes each express surface T cell receptor (TCR) alpha and beta chains of a single specificity. These are produced after random somatic rearrangements in TCR alpha and beta germline genes. Published model systems using mice expressing TCR alpha and/or beta chain transgenes have shown that allelic exclusion occurs conventionally for TCR-beta. TCR alpha chain expression, however, appears to be less strictly regulated, as endogenous TCR alpha chains are often found in association with transgenic TCR beta chains in TCR alpha/beta transgenic mice. This finding, coupled with the unique structure of the TCR alpha locus, has led to the suggestion that unlike TCR beta and immunoglobulin heavy chain genes, TCR alpha genes may make multiple rearrangements on each chromosome. In the current study, we demonstrate that the majority of TCR-, noncycling thymocytes spontaneously acquire surface expression of CD3/TCR. Further, we show that cultured immature thymocytes originally expressing specific TCR alpha and beta chains may lose surface expression of the original TCR alpha, but not beta chains. These data provide evidence that not only must multiple rearrangements occur, but that TCR alpha gene rearrangement continues even after surface expression of a TCR alpha/beta heterodimer, apparently until the recombination process is halted by positive selection, or the cell dies. Sequential rearrangement of TCR alpha chain genes facilitates enhanced production of useful thymocytes, by increasing the frequency of production of both in-frame rearrangements and positively selectable TCR alpha/beta heterodimers.
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CD4+8- and CD4-8+ mature thymocytes require different post-selection processing for final development.
Two primary types of TCR-alpha/beta+ T cells are found in the peripheral lymphoid system; CD4+8- T cells, with MHC-class II restricted TCR, and CD4-8+ T cells, which are MHC-class I restricted. Both lineages develop in the thymus from a series of common precursors. However, the precise stage at which they diverge, and the combination of factors that regulates such divergence, are not well defined. The up-regulation of CD3/TCR to high mature levels is thought to be an early event associated with positive selection for self-MHC recognition. Using purified cells from bcl-2 transgenic mice in order to overcome the limitations imposed by cell death on normal thymocytes, we find that a minor subset of CD4+8+ thymocytes expressing high levels of CD3/TCR gives rise to both CD4+8- and CD4-8+ mature cells upon intrathymic transplantation, but only to CD4-8+ in culture. Thus, in addition to demonstrating the dual lineage potential of this subset, these findings show that additional post-selection processing events are required for the production of mature thymocytes, and that CD4+8- and CD4-8+ subsets differ in the types of processing required.
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Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population.
Dendritic cells, a minor cell population in lymphoid tissues, are specialized for presentation of antigenic peptides to T lymphocytes. Thymic dendritic cells are involved in the deletion of self-reactive T lymphocytes. Although all dendritic cells are ultimately of bone-marrow origin, it has not been clear whether thymic dendritic cells are produced in the adult thymus from a precursor cell or whether they migrate there preformed from the periphery. Recently we isolated from adult mouse thymus a population of early T precursors that could still form B lymphocytes, but not erythroid or myeloid cells, when transferred intravenously. Here we show that these thymic lymphoid precursor cells, as well as bone-marrow haematopoietic stem cells, are able to form both dendritic cells and T-cell progeny when transferred into an irradiated thymus. Such linked development may ensure that developing T cells are negatively selected predominantly by self antigens presented on newly formed thymic dendritic cells.
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Surface antigens of human thymocyte populations defined by CD3, CD4 and CD8 expression: CD1a is expressed by mature thymocytes but not peripheral T cells.
Three colour flow cytometric analysis has been used to analyze the expression of a series of surface antigens on human thymic and peripheral T-cell populations. CD4, CD8 and CD3 were used to divide the populations into the conventional major categories, and the distribution of CD1a, CD2, CD7, CD34, CD44, class I MHC and class II MHC was then determined. Some characteristics of 'single positive' (CD4+8- and CD4-8+) T-lineage cells were unexpected. Amongst thymocytes, some 'immature single positives' were delineated as larger sized cells lacking cell surface CD3 and expressing low levels of class I MHC; however, in contrast with murine thymocytes, these were all CD4+8-, rather than being predominantly CD4-8+. Amongst peripheral T cells, a small proportion of CD7- cells were detected, within both the CD4+8-3+ and the CD4-8+3+ categories. Finally, in contrast to previous conclusions, CD1a was expressed at high levels on mature (CD4+8-3+ and CD4-8+3+) human thymocytes, although in agreement with previous reports it was absent from peripheral T cells. CD1a is therefore a useful marker of post-selection, post-thymic T-cell maturation.
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Mouse thymic dendritic cell subpopulations.
Mouse thymic dendritic cells (DC) have been isolated after collagenase digestion, selection of the low-density cell fraction, then depletion of T-lineage cells and other non-DC by treatment with specific monoclonal antibodies (mAb) and removal with anti-Ig-coated magnetic beads. The resulting DC preparation represented 0.1-0.2% of total thymic cells and contained 70-80% DC. Flow cytometry analysis of MHC class II (MHC II) expression by DC showed that 40% of DC expressed intermediate levels of MHC II, and 60% expressed high levels of this marker. Moreover, immunofluorescent 2-colour staining allowed the characterization of two clearly distinguishable DC subpopulations: MHC IIinter DC were CD45hi, CD44hi, HSAhi, whereas MHC IIhi DC were CD45lo, CD44lo, HSAlo. These results are discussed with regard to the functional significance of MHC IIinter and MHC IIhi DC subpopulations in the mouse thymus.
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The CD44 expressed on the earliest intrathymic precursor population functions as a thymus homing molecule but does not bind to hyaluronate.
A minute population of cells representing the earliest lymphoid-restricted precursor cells in the adult mouse thymus has been isolated recently in our laboratory. This population expresses low levels of CD4, very high levels of CD44 and has a surface antigenic phenotype similar to that of bone marrow hemopoietic stem cells. To examine the role of CD44 on these cells, and to ascertain its ligand, we analysed the ability of the early thymic precursor cells to bind hyaluronate (HA) which functions as a cell adhesion molecule and a ligand for CD44. We also examined if anti-CD44 antibodies (clones IM7.8.1 and KM201) can block the thymic precursor activity. The majority of the thymic precursor cells did not show specific HA binding, indicating that HA is not the ligand for the CD44 molecules expressed on these precursor cells. HA and CD44 interactions are therefore unlikely to play a role in development of cells at this early intrathymic precursor stage. When the purified intrathymic precursor cells were incubated with anti-CD44 antibodies before intravenous transfer into recipients, very few progeny cells were detected in the thymus, although progeny were found in the spleen and lymph nodes. Coating the cells with other isotype-matched antibodies did not have this effect. However, when the same anti-CD44-coated cells were transferred directly into the recipient thymus, normal levels of progeny cells were detected in this organ. This suggests that the CD44 on the intrathymic precursor cells is a homing molecule, able to direct cells to the thymus but utilizing some ligand other than HA.
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Specificity variants in monoclonal antibodies reactive with peptide epitopes of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum.
It has been suggested that repeat sequence antigens of Plasmodium falciparum may serve the parasite in immune evasion by modifying the host antibody response and impairing the development of protective immunity. According to this proposal networks of cross-reactive, repeat sequence malarial antigens have the ability to stimulate a high proportion of all somatically mutated B cells with altered antibody specificity, and thus to hinder the normal process of antibody affinity maturation. To determine the rate at which immunoglobulin mutations produce new reactivities with repeat sequence antigens, hybridoma cell lines specific for the ring-infected erythrocyte surface antigen (RESA) were examined for the incidence of specificity variants that arose naturally or as a result of treatment with the chemical mutagen ethylmethane sulphonate (EMS). From one of the cell lines variants were readily isolated having reactivity towards a very closely related repeat sequence epitope within the same RESA antigen. However, the other hybridoma/antigen combinations revealed no variants. In general, mutations giving rise to antibodies with altered specificity for related repetitive antigens were not readily induced and only limited support of the hypothesis was obtained.
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Characterization of thymic nurse-cell lymphocytes, using an improved procedure for nurse-cell isolation.
Thymic nurse cells (TNC), multicellular complexes consisting of lymphoid cells enclosed within cortical epithelial cells, were isolated from mouse thymus by a modified procedure allowing immunofluorescent labeling and flow cytometric analysis of their lymphoid contents (TNC-L). Collagenase was the only protease used for tissue digestion, to ensure that surface antigen markers remained intact. Zonal unit-gravity elutriation was used to enrich the TNC on the basis of their high sedimentation rate, followed by immunomagnetic bead depletion to remove residual mononuclear cell contaminants and a density separation to remove debris. The TNC-L were then released from inside TNC by a short period of culture. The measured contamination of TNC-L with exogenous thymocytes was around 0.5%. Three-color immunofluorescent labeling revealed that TNC-L included, as well as a majority of immature CD4+8+3low thymocytes, about 12% of apparently mature CD4+8-3high and CD4-8+3high thymocytes. TNC are located in the cortex, where mature cells are rare; the occurrence of mature phenotype cells within these structures suggests that they represent a microenvironment for the selection and generation of mature T cells.
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The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells.
A new procedure for rapid isolation of dendritic cells (DC) was devised, involving collagenase digestion of tissues, dissociation of lymphoid-DC complexes, selection of light-density cells, then depletion of lymphocytes and other non-DC by treatment with a mixture of lineage-specific monoclonal antibodies (mAbs) and removal with anti-immunoglobulin-coupled magnetic beads. This enriched population (approximately 80% DC) was further purified when required by fluorescence-activated cell sorting for cells expressing high levels of class II major histocompatibility complex (MHC). The isolated DC were characterized by immunofluorescent staining using a panel of 30 mAbs. Thymic DC were surface positive for a number of markers characteristic of T cells, but they were distinct from T-lineage cells in expressing high levels of class II MHC, in lacking expression of the T cell receptor (TCR)-CD3 complex, and having TCR beta and gamma genes in germline state. Splenic DC shared many markers with thymic DC, but were negative for most T cell markers, with the exception of CD8. A substantial proportion of DC from both thymus and spleen expressed CD8 at high levels, comparable with that on T cells. This appeared to be authentic CD8, and was produced by the DC themselves, since they contained CD8 alpha mRNA. Thymic DC presented both the CD8 alpha and beta chains on the cell surface (Ly-2+3+), although the alpha chain was in excess; the splenic DC expressed only the CD8 alpha chain (Ly-2+3-). It is suggested that the expression of CD8 could endow certain antigen-presenting DC with a veto function.
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Cell surface marker analysis of mouse thymic dendritic cells.
Cell surface markers of mouse thymic dendritic cells have been studied by flow cytometry after isolation by collagenase digestion, separation of the low-density cell fraction and differential adherence. The dendritic cell preparation had a purity of greater than 90%, the contaminating population being essentially composed of thymocytes, macrophages constituting less than 1%. Dendritic cells displayed high forward and low-intermediate side angle scatter, and expressed high levels of major histocompatibility complex (MHC) class I and class II molecules, the heat-stable antigen (HSA), the adhesion molecules Pgp-1 (CD44), LFA-1, ICAM-1 and low levels of Mac-1 and the leukocyte common antigen CD45. Thymic dendritic cells are negative for the stem cell antigen-2 (Sca-2), the B cell-specific form of CD45 (B220), the mouse macrophage markers Fc receptor and F4/80, and the granulocyte marker Gr-1. However, although they do not express the T cell markers Thy-1, CD2, CD3, CD4 and CD5, 20%-30% of dendritic cells are positive for the interleukin 2 receptor alpha chain (CD25), and about 30% express intermediate levels of CD8. These results are discussed with regard to the functional significance of the expression of CD8 by thymic dendritic cells, and the existence of different dendritic cell subpopulations in the murine thymus.
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Commitment to the T cell receptor-alpha beta or -gamma delta lineages can occur just prior to the onset of CD4 and CD8 expression among immature thymocytes.
Two types of T lymphocytes, distinguishable by their surface expression of either the gamma delta or the alpha beta T cell receptor (TcR) for antigen, populate the periphery in the adult. In addition, immature precursors of both T cell types can be found in the thymus. While it is generally accepted that these two cell types represent distinct lineages, it is not known at which developmental stage these lineages diverge. The most mature thymocyte precursor population not yet expressing T lineage-specific surface markers (i.e. CD3, CD4, and CD8) is known to be capable of generating TcR-alpha beta T cells, and has been thought to be preprogrammed into the TcR-alpha beta lineage at an earlier developmental stage. We now show that this late-stage precursor is capable of giving rise to cells of both the TcR-alpha beta and -gamma delta lineages, both in vitro after intrathymic transplantation, and in vitro in simple culture medium or medium with cytokines. Thus it appears that the divergence of TcR-alpha beta and -gamma delta cells can occur at a relatively late stage of intrathymic development, just prior to the onset of CD4 and CD8 expression in most cells.
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Cellular aspects of early T-cell development.
Although the nature of the precursor cells seeding the thymus is still uncertain, their immediate progeny in the adult murine thymus have now been isolated. These lymphoid-restricted, prothymocyte-like cells express CD4, but neither CD4 nor CD8 seem to be involved in the early steps of T-cell development. Cytokines produced by stromal cells are likely to be involved in intrathymic T-cell development, but interleukin-2 and interleukin-4 do not appear to be required. There is still no satisfactory cell-culture model of intrathymic T-cell development. Current culture systems reflect only fragments of the process, or are models of extrathymic developmental pathways.
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