Lineage relationships and developmental kinetics of immature thymocytes: CD3, CD4, and CD8 acquisition in vivo and in vitro.

T lymphocytes develop in the thymus from immunologically naive bone marrow precursors. Based on T cell receptor rearrangement and transcription, and thymic reconstitution potential, we have deduced a developmental sequence among immature thymocytes, before the acquisition of the lineage markers CD3, CD4, and CD8. In the current study, we have followed the ontogenic progression of the latter stages in this sequence, using two different systems: (a) in vivo, by direct injection into the thymus of nonirradiated, congenic recipients; and (b) in vitro, using culture medium without mitogens or cytokines. In vivo, the less mature Pgp-1- interleukin 2 receptor alpha-positive (IL-2R alpha+) CD3-4-8- subset (also heat-stable antigen high) requires 3 d before becoming predominantly IL-2R alpha- CD3lo4+ 8+ typical cortical-type cells, and at least 5 d before the appearance of any mature single-positive cells (CD3hi4+ 8- or CD3hi4-8+). However, these Pgp-1- IL-2R alpha+ precursors do not differentiate further in unstimulated culture. The more mature Pgp-1- IL-2R alpha- CD3-4-8- subset becomes primarily CD3lo4+ 8+ within 1 d after transplantation, and some mature single-positive progeny are evident by day 3. By 5 d, most of these Pgp-1-IL-2R alpha- precursor cells have become CD3hi, and have lost or are downregulating either CD4 or CD8. In culture, these Pgp-1- IL-2R alpha- cells also acquire high levels of CD4 and CD8 within 1 d, and low levels of CD3 by 2 d. However, they do not progress further to mature single positives in vitro, and most of them die by day 3. These experiments directly confirm our previously proposed developmental sequence, and demonstrate the kinetics of T lymphocyte production in a low-stress, steady-state environment.

Developmental potential of the earliest precursor cells from the adult mouse thymus.

A new, numerically minute population of cells representing the earliest T precursor cells in the adult mouse thymus has recently been isolated. This population has been shown to be similar to bone marrow hemopoietic stem cells in surface antigenic phenotype and to express moderate levels of CD4. We now show, by fluorescence-activated cell sorting and intrathymic transfer to irradiated mice, that this apparently homogeneous population differs from multipotent stem cells in expressing the surface stem cell antigen 2 (Sca-2), that it differs from most early B lineage cells in lacking B220 and class II major histocompatibility complex expression, and that it binds rhodamine 123 like an activated rather than a quiescent cell. Irradiated recipient mice differing at the Ly 5 locus were used to compare the developmental potential of these early intrathymic precursors with bone marrow stem cells. Only T lineage product cells were detected when the intrathymic precursor population was transferred back into an irradiated thymus. However, when the intrathymic precursor population was transferred intravenously, it displayed the capacity to develop into both B and T lymphoid cells in recipient bone marrow, spleen, and lymph nodes, but no donor-derived myeloid cells were detected. The absence of myeloid and erythroid precursor activity was confirmed by showing that the intrathymic precursor population was unable to develop into myeloid or erythroid spleen colonies on intravenous transfer or to form colonies in an agar culture. These findings indicate that this earliest intrathymic precursor population has become restricted (or strongly biased) to lymphoid lineage development, but not exclusively to T lymphocytes.

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.

Introduction: homeostasis in the immune system

No abstractCopyright 1997 Academic Press Limited Copyright 1997Academic Press Limited

High doses of purified stem cells cause early hematopoietic recovery in syngeneic and allogeneic hosts.

In humans, autologous transplants derived from bone marrow (BM) usually engraft more slowly than transplants derived from mobilized peripheral blood. Allogeneic BM transplants show a further delay in engraftment and have an apparent requirement for donor T cells to facilitate engraftment. In mice, Thy-1.1(lo)Lin-/loSca-1+ hematopoietic stem cells (HSCs) are the principal population in BM which is responsible for engraftment in syngeneic hosts at radioprotective doses, and higher doses of HSCs can radioprotect an allogeneic host in the absence of donor T cells. Using the mouse as a preclinical model, we wished to test to what extent engraftment kinetics was a function of HSC content, and whether at high doses of c-Kit+Thy-1.1(lo)Lin-/loSca-1+ (KTLS) cells rapid allogeneic engraftment could also be achieved. Here we demonstrate that engraftment kinetics varied greatly over the range of KTLS doses tested (100-10,000 cells), with the most rapid engraftment being obtained with a dose of 5,000 or more syngeneic cells. Mobilized splenic KTLS cells and the rhodamine 123(lo) subset of KTLS cells were also able to engraft rapidly. Higher doses of allogeneic cells were needed to produce equivalent engraftment kinetics. This suggests that in mice even fully allogeneic barriers can be traversed with high doses of HSCs, and that in humans it may be possible to obtain rapid engraftment in an allogeneic context with clinically achievable doses of purified HSCs.

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.

Association between alphabetaTCR+CD4-CD8- T-cell deficiency and IDDM in NOD/Lt mice.

NOD mice develop spontaneous IDDM as a result of T-cell-mediated autoimmune destruction of pancreatic beta-cells. It is not known why these T-cells become autoreactive, nor is it clear whether the breakdown in self-tolerance reflects a general problem in T-cell development or a selective defect in an as yet undefined regulatory cell population. In this study, we showed that NOD mice, although relatively normal with regard to most thymocyte subsets, exhibit a marked deficiency in alphabetaTCR+CD4-CD8- (alphabeta+DN) T-cells in the thymus and, to a lesser extent, in the periphery. These T-cells have been termed NKT cells (NK1.1+-like T-cells) because they share some cell surface markers with conventional natural killer (NK) cells. To examine the role of these cells in the pathogenesis of IDDM, semiallogeneic or syngeneic double-negative (DN) thymocytes, enriched for NKT cells, were transferred into intact 4-week-old NOD recipients; the onset of diabetes was then monitored over the ensuing 30 weeks. Mice receiving NKT-enriched thymocytes did not develop diabetes, whereas mice receiving unfractionated thymocytes or phosphate-buffered saline developed diabetes at the normal rate. NKT cells represent a distinct T-cell lineage that has been shown to play a role in immunoregulation in vivo. The deficiency of these cells observed in NOD mice may therefore contribute to destruction of pancreatic islet cells by conventional T-cells.

Specific demethylation of the CD4 gene during CD4 T lymphocyte differentiation.

The expression of CD4 during T cell development is a highly regulated process. Numerous regulatory elements have been identified including a promoter, two distinct enhancers and a silencer. Here we report a methylation site in the first intron of the CD4 gene that is specifically demethylated in cells which have previously, or are currently expressing CD4. In addition, this site becomes progressively demethylated as T lymphocytes differentiate from double-negative to double-positive to CD4 single-positive thymocytes, and finally to CD4 single-positive peripheral T lymphocytes. This specific and progressive demethylation suggests that this site represents another potential control region for the regulation of CD4.

A simplified method for enrichment of mouse hematopoietic stem cells.

A simplified method for enriching mouse hematopoietic stem cells using standard two-color fluorescence-activated cell sorting (FACS) has been developed. By eliminating one fluorescence parameter from a previously described four-parameter (three-color) method, FACS enrichment of mouse hematopoietic stem cells to a purity within twofold of that accomplished by the more complex method could be achieved using a single-laser, two-color FACS instrument. The method involves positive selection of mouse bone marrow cells expressing the Ly-6A.2 molecule (previously termed stem cell antigen-1, or Sca-1) and negative selection for expression of a number of cell surface markers characteristic of differentiated cells of hematolymphoid lineages (Lin-). Cell populations selected by this method contain 480 +/- 100 day-13 splenic colony-forming units (CFUs-13) per 10(4) cells, whereas the day-8 splenic colony-forming unit (CFUs-8) content is about tenfold lower. The frequency of thymic colony-forming units (CFUt) is about one in ten cells. Long-term hematopoietic repopulation of irradiated animals was observed following the transfer of 60-100 cells. However, as few as 20 cells could mediate radioprotection of lethally irradiated mice. An analysis of the cell surface phenotype of isolated Ly-6A.2+Lin- cells showed that 30%-50% expressed low levels of the Thy-1 antigen and that the CFUs-13 activity was predominantly associated with the Thy-1lo cells. The Ly-6A.2+Lin- cells expressed intermediate levels of phagocyte glycoprotein-1 (Pgp-1), low levels of heat-stable antigen (HSA), and high levels of class I major histocompatibility antigens (H2 K/D), leukocyte common antigen (Ly-5), and carbohydrate binding sites for the lectin wheat-germ agglutinin (WGA). By these criteria, Ly-6A.2+Lin- cells are phenotypically similar to mouse hematopoietic stem cells isolated by other methods.

CD34+ cells from mobilized peripheral blood retain fetal bone marrow repopulating capacity within the Thy-1+ subset following cell division ex vivo.

Ex vivo cell cycling of hematopoietic stem cells (HSC), a subset of primitive hematopoietic progenitors (PHP) with engrafting capacity, is required for transduction with retroviral vectors and to increase transplantable HSC numbers. However, induction of division of HSC ex vivo also may lead to differentiation and loss of in vivo marrow repopulating potential. We evaluated mobilized peripheral blood (MPB) PHP for maintenance of stem cell function after ex vivo culture under conditions that we show can induce cycling of a majority of PHP with minimal differentiation. The following methods were combined: cell labeling with the division tracking dye carboxyfluorescein-diacetate succinimidylester (CFSE), analysis of primitive cell surface marker expression, an ex vivo PHP assay, and an in vivo marrow repopulating assay. MPB-purified CD34+ Thy-1+ cells were labeled with CFSE dye and cultured for 112 hours in serum-deprived medium in the presence of the cytokine combinations of thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL), or TPO, FL, and interleukin 6 (IL-6). Both cytokine combinations supported division of greater than 95% of cells within 112 hours with an average 2.1-fold (TPO, FL, KL) or 1.3-fold (TPO, FL, IL-6) increase in total cell numbers. An average of 21.6% (TPO, FL, KL) and 27.4% (TPO, FL, IL-6) of the divided cells still expressed the Thy-1 marker after 112 hours. Functional assays were performed to compare cultured and uncultured cells. CD34+ Thy-1+ CFSElo (post division) cells showed maintenance of cobblestone area-forming cell (CAFC) frequency (a mean of 1/9.0) relative to the starting population of uncultured CD34+ Thy-1+ cells (a mean of 1/8.4). In contrast, CD34+ cells that had lost Thy-1 expression during culture (CD34+ Thy-1 CFSElo) showed a mean 5.8-fold reduction in CAFC frequency (a mean of 1/52.5). Only the Thy-1-expressing fraction of cells post culture could engraft in vivo in the SCID-hu bone assay. Because the majority of HSC functional activity post culture was found in the CD34+ Thy-1+ fraction, we focused on this fraction for subsequent analysis. CFSE labeling allows segregation and purification by flow cytometry of cells having undergone discrete numbers of divisions during culture. Very few cells that divided more than four times in culture still expressed Thy-1. Cells that retained expression of Thy-1 during culture retained CAFC activity relative to fresh CD34+ Thy-1+ cells, after undergoing at least two divisions. CAFC frequency decreased after four divisions in culture with TPO, FL, and KL or after three divisions in TPO, FL, and IL-6. We then compared populations of Thy-1+ cells that had undergone sequential numbers of divisions in culture for their ability to engraft in the SCID-hu bone assay. Engrafting ability was retained throughout four divisions in both cytokine combinations. These data demonstrate that primitive MPB CD34+ cells maintain HSC function coincident with Thy-1 expression while undergoing two to four divisions under these culture conditions. Essentially all CD34+ Thy-1+ cells divided under the conditions tested, promoting susceptibility to retroviral transduction.

Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34+ cells and recruit primitive CD34+ Thy-1+ cells into rapid division.

Various combinations of cytokines have profoundly different effects on inhibition of apoptosis and stimulation of self-renewal division of hematopoietic stem cells (HSC) in short-term, ex vivo culture. Our goal was to quantitate expansion of cells with a primitive CD34+ Thy-1+ phenotype, as well as cell cycling, division history, differentiation, and apoptosis of CD34+ cells enriched from normal donor mobilized peripheral blood (MPB) cells. The balance of these parameters determines the net number of transplantable HSC produced in ex vivo cultures. Comparing several different combinations of cytokines added to 90-hour cultures of MPB CD34 cells, thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL) gave the best result, with the lowest percentage of apoptotic cells and a mean 1.2-fold increase in the number of CD34+ Thy-1+ cells. A combination of interleukin 3 (IL-3), interleukin 6 (IL-6), and leukemia inhibitory factor (LIF) gave the worst outcome, including a decrease of CD34+ Thy-1+ cell number to a mean of 30% of the starting cell number. Cell division history was tracked using the dye 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE). Division of CD34+ Thy-1+ cells was faster and more synchronous in TPO, FL, and KL than in IL-3, IL-6, and LIF, which left a significant proportion of CD34+ cells undivided. Such detailed analyses of short-term, ex vivo cultures generated "replication scores," which allowed prediction of a sixfold improvement of the efficiency of gene transduction of primitive hematopoietic progenitors from MPB, using TPO, FL, and KL to replace IL-3, IL-6, and LIF. Analysis of retroviral transduction efficiency confirmed the increase of transgene expression from MPB primitive hematopoietic progenitors assayed after stromal culture was fivefold, validating the usefulness of multiparameter analysis of short-term cultures for survival and replication of CD34+ Thy-1+ cells.

Inconsistencies detected by flow cytometry following immunofluorescence staining with anti-Thy 1 antibodies.

A number of inconsistencies have been observed when anti-Thy 1 reagents are used for immunofluorescence. (1) When indirect staining procedures are used, the differences in staining between high and low Thy 1 populations may be obscured, (2) with both direct and indirect staining methods, variable peaks of 'low Thy 1' cells can be seen, apparently derived from the high Thy 1 population by loss of antigen and/or antibody during the staining procedure. This disparity is quite variable, appears to occur at random and is independent of a large number of technical variables tested. Similar inconsistencies are not apparent with other lymphocyte markers (e.g., Lyt 1, Lyt 2, TLa, peanut agglutinin, H2, Ig).

Direct fluorescent labeling of cells with fluorescein or rhodamine isothiocyanate. II. Potential application to studies of lymphocyte migration and maturation.

The effect of direct cell labeling with fluorescein or tetramethyl rhodamine isothiocyanate on lymphocyte migration is examined. In vitro conditions of labeling are defined which (1) do not significantly affect immediate or long term viability of lymphocytes (up to 2 weeks after transfer in vivo), (2) do not alter normal lymphocyte migration, (3) do not affect expression or detectability of surface antigens, and (4) permit direct visualization and counter-staining with fluorescent antibody reagents for days after intravenous injection. The potential application of this method to studies of lymphocyte migration and maturation is discussed.

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.

Differences in the graft-versus-host reactivity of cells migrating through nonlymphoid tissue or lymph nodes.

Normal lymphocyte transfer reactions have been used to test the alloreactivity of lymphocytes which have migrated through lymph nodes (i.e., in efferent lymph) or nonlymphoid tissues (i.e., in afferent lymph) in sheep. Lymphocytes from afferent lymph are much less efficient at inducing a normal lymphocyte transfer lesion than lymphocytes from efferent lymph. Afferent cell preparations (which contain about 10% macrophages) do, however, cause a greater inflammatory response in the early part of the reaction, but this is at least partly nonspecific. A mixture of afferent and efferent cells produces a lesion which is simply the sum of the two expected reactions, suggesting that the afferent lymphocytes are not merely inhibited by a factor or cell in the preparation.

Selection of the T-cell repertoire in transgenic mice expressing a transplantation antigen in distinct thymus subsets.

Transgenic mice that expressed a transplantation antigen, H-2Kb, in an unusual tissue distribution have been developed. Gene-regulatory elements from the immunoglobulin heavy-chain locus (Emu enhancer and heavy chain promoter) were linked to the class I Kb gene and the construct microinjected into fertilized mouse eggs of a different haplotype. It was expected that such gene-regulatory elements would direct expression of the foreign class I molecules only to B and T lymphocytes. However, expression was also detected in a subset of thymus medullary epithelium. The Kb molecules expressed on this thymic subset were unable to positively select T cells for passage to the periphery. The mice were, however, tolerant of the cell types expressing the foreign Kb molecules and were also tolerant of Kb presented as skin grafts. These results suggest that not all components of thymic epithelium are involved in positive selection of T cells and that transplantation antigens expressed on non-dendritic cells can induce tolerance.

Differences in Ly 2- L3T4- thymocytes from foetal and adult murine thymus.

The thymus is the major site of mammalian T cell production. The exact steps which occur in the thymus and give rise to mature T cells have not been defined, but there is general agreement that the earliest T cells are included in the group of thymic lymphoid cells lacking Ly 2 (CD8) and L3T4 (CD4). This population represents 2-6% of adult thymocytes and the vast majority of thymocytes in the mouse embryo until about day 16 of gestation. It has often been assumed that the foetal and adult CD4- CD8- thymocytes are equivalent. This paper shows that there are significant differences between the CD4- CD8- cells from these sources, in that the adult includes at least two subsets which are undetectable in the embryo. These two subsets of CD4- CD8- cells are both Ly 1high, B2A2- and M1/69-; one is Thy 1+ and one is Thy 1-. Each represents 20-25% of adult CBA double negative thymocytes. Both these populations are excluded from analyses of CD4- CD8- thymocytes which have been further selected as Ly 1low, a procedure adopted in several studies of early thymocytes. Even those subpopulations of CD4- CD8- cells which appear to express similar markers in adult and embryo thymus are quite different when analysed for cell size (forward light scatter), with the embryonic forms being much larger.

Stem cell antigen 2 expression in adult and developing mice.

Stem cell antigen 2 (Sca-2) expression can distinguish the most immature T-lymphocyte precursors in the thymus from the hemopoietic stem cells. Sequence analysis of the Sca-2 protein showed that Sca-2 is a glycosylphosphatidylinositol (GPI) anchored molecule that shares some characteristics with the members of the Ly-6 multigene family, and that it is the same as the thymic shared antigen-1 (TSA-1). Here we extend these studies and critically reassess the expression of the Sca-2/TSA-1 antigen in hematopoietic tissues of adult and developing mice. With more sensitive methods we show that the distribution of Sca-2/TSA-1 differs from existing reports. We find especially high expression of Sca-2/TSA1 at day 14 of fetal development.

Gene therapy: a brief overview of the past, present, and future.

Gene therapy has only recently begun to make serious progress, beginning with two approved gene therapy trials in the United States in late 1990. The death of an 18-year-old man participating in a gene therapy trial delivered a major setback in terms of public concerns, but the resulting improvements in scrutiny of trial design and ethical standards will benefit the field in the long run. The three main issues for the coming decade will be public perceptions, scale-up and manufacturing, and commercial considerations. Focusing on single-gene applications, which tend to be rarer diseases, will produce successful results sooner than the current focus on the commoner, yet more complex, cancer and heart disease.